# Patent

## Title
HWBAO62 polypeptides

## Abstract

The present invention relates to human secreted polypeptides, and isolated nucleic acid molecules encoding said polypeptides, useful for diagnosing and treating diseases, disorders, and/or conditions (such as immune, cardiovascular, cancer, and other proliferative diseases, disorders, and/or conditions) related to said human secreted proteins. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Ser. No. 12/136,548, filed Jun. 10, 2008, which is a divisional of U.S. application Ser. No. 11/781,665, filed Jul. 23, 2007, which is a continuation of U.S. application Ser. No. 10/994,608, filed Nov. 23, 2004, which is a divisional of U.S. application Ser. No. 10/105,299, filed Mar. 26, 2002, which claims the benefit of U.S. Provisional Application No. 60/278,650, filed Mar. 27, 2001; U.S. application Ser. No. 10/105,299 is a continuation-in-part of U.S. application Ser. No. 09/950,082, filed Sep. 12, 2001; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06043, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/167,061, filed Nov. 23, 1999, and 60/124,146, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06012, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/166,989, filed Nov. 23, 1999, and 60/124,093, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06058, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,654, filed Dec. 3, 1999, and 60/124,145, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06044, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,661, filed Dec. 3, 1999, and 60/124,099, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06059, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,622, filed Dec. 3, 1999, and 60/124,096, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06042, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,663, filed Dec. 3, 1999, and 60/124,143, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06014, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,665, filed Dec. 3, 1999, 60/138,598, filed Jun. 11, 1999, and 60/124,095, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06013, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,662, filed Dec. 3, 1999, 60/138,626, filed Jun. 11, 1999, and 60/125,360, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06049, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,667, filed Dec. 3, 1999, 60/138,574, filed Jun. 11, 1999, and 60/124,144, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06057, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,666, filed Dec. 3, 1999, 60/138,597, filed Jun. 11, 1999, and 60/124,142, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06824, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,664, filed Dec. 3, 1999, and 60/125,359, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06765, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,906, filed Dec. 10, 1999, and 60/126,051, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06792, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,980, filed Dec. 10, 1999, and 60/125,362, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06830, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,910, filed Dec. 10, 1999, and 60/125,361, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06782, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,936, filed Dec. 10, 1999, and 60/125,812, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06822, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,916, filed Dec. 10, 1999, and 60/126,054, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06791, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,946, filed Dec. 10, 1999, and 60/125,815, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06828, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,616, filed Dec. 8, 1999, and 60/125,358, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06823, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,623, filed Dec. 8, 1999, and 60/125,364, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06781, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,617, filed Dec. 8, 1999, and 60/125,363, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07505, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,410, filed Dec. 17, 1999, and 60/126,502, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07440, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,409, filed Dec. 17, 1999, and 60/126,503, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07506, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,412, filed Dec. 17, 1999, and 60/126,505, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07507, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,408, filed Dec. 17, 1999, and 60/126,594, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07535, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,413, filed Dec. 17, 1999, and 60/126,511, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07525, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,549, filed Dec. 22, 1999, and 60/126,595, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07534, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,504, filed Dec. 22, 1999, and 60/126,598, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07483, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,552, filed Dec. 22, 1999, and 60/126,596, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07526, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,550, filed Dec. 22, 1999, and 60/126,600, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07527, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,551, filed Dec. 22, 1999, and 60/126,501, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07661, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,847, filed Jan. 7, 2000, and 60/126,504, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07579, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,853, filed Jan. 7, 2000, and 60/126,509, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07723, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,852, filed Jan. 7, 2000, and 60/126,506, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07724, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,850, filed Jan. 7, 2000, and 60/126,510, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14929, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,851, filed Jan. 7, 2000, and 60/138,573, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07722, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,871, filed Jan. 7, 2000, and 60/126,508, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07578, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,872, filed Jan. 7, 2000, and 60/126,507, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07726, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,877, filed Jan. 7, 2000, and 60/126,597, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07677, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,064, filed Jan. 14, 2000, 60/154,373, filed Sep. 17, 1999, and 60/126,601, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07725, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,063, filed Jan. 14, 2000, and 60/126,602, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09070, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,052, filed Jan. 14, 2000, and 60/128,695, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08982, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,069, filed Jan. 14, 2000, and 60/128,696, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08983, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,068, filed Jan. 14, 2000, and 60/128,703, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09067, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,929, filed Jan. 20, 2000, and 60/128,697, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09066, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,926, filed Jan. 20, 2000, and 60/128,698, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09068, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,050, filed Jan. 20, 2000, and 60/128,699, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08981, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,166, filed Jan. 20, 2000, and 60/128,701, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08980, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,930, filed Jan. 20, 2000, and 60/128,700, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09071, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,931, filed Jan. 20, 2000, and 60/128,694, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09069, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,049, filed Jan. 20, 2000, and 60/128,702, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/15136, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,629, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14926, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,628, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14963, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,631, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/15135, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,632, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14934, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,599, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14933, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,572, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/15137, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,625, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14928, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,633, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14973, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,630, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14964, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,627, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26376, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,808, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26371, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,804, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26324, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,807, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26323, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,805, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26337, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,806, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US01/13318, filed Apr. 26, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/212,142, filed Jun. 16, 2000, and 60/201,194, filed May 2, 2000; U.S. application Ser. No. 10/105,299 is a continuation-in-part of U.S. application Ser. No. 09/950,083, filed Sep. 12, 2001; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06043, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/167,061, filed Nov. 23, 1999, and 60/124,146, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06012, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/166,989, filed Nov. 23, 1999, and 60/124,093, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06058, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,654, filed Dec. 3, 1999, and 60/124,145, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06044, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,661, filed Dec. 3, 1999, and 60/124,099, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06059, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,622, filed Dec. 3, 1999, and 60/124,096, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06042, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,663, filed Dec. 3, 1999, and 60/124,143, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06014, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,665, filed Dec. 3, 1999, 60/138,598, filed Jun. 11, 1999, and 60/124,095, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06013, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,662, filed Dec. 3, 1999, 60/138,626, filed Jun. 11, 1999, and 60/125,360, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06049, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,667, filed Dec. 3, 1999, 60/138,574, filed Jun. 11, 1999, and 60/124,144, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06057, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,666, filed Dec. 3, 1999, 60/138,597, filed Jun. 11, 1999, and 60/124,142, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06824, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,664, filed Dec. 3, 1999, and 60/125,359, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06765, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,906, filed Dec. 10, 1999, and 60/126,051, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06792, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,980, filed Dec. 10, 1999, and 60/125,362, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06830, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,910, filed Dec. 10, 1999, and 60/125,361, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06782, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,936, filed Dec. 10, 1999, and 60/125,812, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06822, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,916, filed Dec. 10, 1999, and 60/126,054, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06791, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,946, filed Dec. 10, 1999, and 60/125,815, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06828, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,616, filed Dec. 8, 1999, and 60/125,358, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06823, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,623, filed Dec. 8, 1999, and 60/125,364, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06781, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,617, filed Dec. 8, 1999, and 60/125,363, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07505, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,410, filed Dec. 17, 1999, and 60/126,502, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07440, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,409, filed Dec. 17, 1999, and 60/126,503, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07506, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,412, filed Dec. 17, 1999, and 60/126,505, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07507, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,408, filed Dec. 17, 1999, and 60/126,594, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07535, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,413, filed Dec. 17, 1999, and 60/126,511, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07525, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,549, filed Dec. 22, 1999, and 60/126,595, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07534, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,504, filed Dec. 22, 1999, and 60/126,598, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07483, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,552, filed Dec. 22, 1999, and 60/126,596, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07526, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,550, filed Dec. 22, 1999, and 60/126,600, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07527, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,551, filed Dec. 22, 1999, and 60/126,501, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07661, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,847, filed Jan. 7, 2000, and 60/126,504, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07579, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,853, filed Jan. 7, 2000, and 60/126,509, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07723, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,852, filed Jan. 7, 2000, and 60/126,506, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07724, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,850, filed Jan. 7, 2000, and 60/126,510, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14929, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,851, filed Jan. 7, 2000, and 60/138,573, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07722, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,871, filed Jan. 7, 2000, and 60/126,508, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07578, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,872, filed Jan. 7, 2000, and 60/126,507, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07726, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,877, filed Jan. 7, 2000, and 60/126,597, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07677, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,064, filed Jan. 14, 2000, 60/154,373, filed Sep. 17, 1999, and 60/126,601, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07725, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,063, filed Jan. 14, 2000, and 60/126,602, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09070, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,052, filed Jan. 14, 2000, and 60/128,695, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08982, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,069, filed Jan. 14, 2000, and 60/128,696, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08983, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,068, filed Jan. 14, 2000, and 60/128,703, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09067, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,929, filed Jan. 20, 2000, and 60/128,697, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09066, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,926, filed Jan. 20, 2000, and 60/128,698, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09068, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,050, filed Jan. 20, 2000, and 60/128,699, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08981, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,166, filed Jan. 20, 2000, and 60/128,701, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08980, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,930, filed Jan. 20, 2000, and 60/128,700, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09071, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,931, filed Jan. 20, 2000, and 60/128,694, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09069, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,049, filed Jan. 20, 2000, and 60/128,702, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/15136, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,629, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14926, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,628, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14963, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,631, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/15135, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,632, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14934, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,599, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14933, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,572, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/15137, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,625, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14928, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,633, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14973, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/,138,630, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14964, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,627, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26376, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,808, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26371, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,804, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26324, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,807, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26323, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,805, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26337, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,806, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US01/13318, filed Apr. 26, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/212,142, filed Jun. 16, 2000, and 60/201,194, filed May 2, 2000. This application is a continuation-in-part of U.S. application Ser. No. 11/366,486, filed Mar. 3, 2006, which is a continuation-in-part of 10/664,358, filed Sep. 20, 2003, which is a continuation-in-part of International Application No. PCT/US02/09785, filed Sep. 20, 2003; International Application No. PCT/US02/09785 is a continuation-in-part of U.S. application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/882,171 is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,161, filed Mar. 7, 1997, 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct., 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. application Ser. No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31162, filed Nov. 15, 2000; U.S. Provisional Application No. 60/215,136 claims the benefit of U.S. Provisional Application Nos. 60/215,136, filed Jun. 30, 2000, which claims the benefit of U.S. Provisional Application No. 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01396, filed Jan. 17, 2001; U.S. Provisional Application No. 60/256,968 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, which claims the benefit of U.S. Provisional Application No. 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01387, filed Jan. 17, 2001; U.S. Provisional Application No. 60/259,803 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. PCMS01/01431 is a continuation-in-part of U.S. application Ser. No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 11/687,755, filed Mar. 19, 2007, which is a divisional of U.S. patent application Ser. No. 10/664,356, filed Sep. 20, 2003, which is a continuation-in-part of PCT/US02/08123, filed Mar. 19, 2002, which is a continuation-in-part of U.S. application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/882,171 is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,161, filed Mar. 7, 1997, 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct. 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. application Ser. No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/11S99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31162, filed Nov. 15, 2000; U.S. Provisional Application No. 60/215,136 claims the benefit of U.S. Provisional Application Nos. 60/215,136, filed Jun. 30, 2000, which claims the benefit of U.S. Provisional Application No. 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01396, filed Jan. 17, 2001; U.S. Provisional Application No. 60/256,968 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, which claims the benefit of U.S. Provisional Application No. 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01387, filed Jan. 17, 2001; U.S. Provisional Application No. 60/259,803 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. PCT/US01/01431 is a continuation-in-part of U.S. application Ser. No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 12/198,817, filed Aug. 26, 2008, which is a divisional of U.S. application Ser. No. 11/346,470, filed Feb. 3, 2006, which is a continuation-in-part of U.S. application Ser. No. 10/472,532, filed Sep. 20, 2003 and accorded a National Stage filing date of Jun. 23, 2004, now abandoned, which is the 35 U.S.C. §371 National Stage of PCT/US02/08278, filed Mar. 19, 2002, which in turn claims benefit of U.S. patent application Ser. No. 10/100,683 (now U.S. Pat. No. 7,368,531, issued May 6, 2008). U.S. application Ser. No. 11/346,470 is also a continuation-in-part of U.S. patent application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/882,171 is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,161, filed Mar. 7, 1997, 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct. 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. application Ser. No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31162, filed Nov. 15, 2000; U.S. Provisional Application No. 60/215,136 claims the benefit of U.S. Provisional Application Nos. 60/215,136, filed Jun. 30, 2000, which claims the benefit of U.S. Provisional Application No. 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01396, filed Jan. 17, 2001; U.S. Provisional Application No. 60/256,968 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, which claims the benefit of U.S. Provisional Application No. 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01387, filed Jan. 17, 2001; U.S. Provisional Application No. 60/259,803 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. PCT/US01/01431 is a continuation-in-part of U.S. application Ser. No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 11/689,173, filed Mar. 21, 2007, which is a continuation of U.S. application Ser. No. 11/001,793, filed Dec. 2, 2004, which is a divisional of U.S. application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/040,161, filed Mar. 7, 1997; International Application No. PCT/US98/04493 claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct. 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. application Ser. No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/31162, filed Nov. 15, 2000; U.S. Provisional Application No. 60/215,136 claims the benefit of U.S. Provisional Application Nos. 60/215,136, filed Jun. 30, 2000, which claims the benefit of U.S. Provisional Application No. 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01396, filed Jan. 17, 2001; U.S. Provisional Application No. 60/256,968 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, which claims the benefit of U.S. Provisional Application No. 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01387, filed Jan. 17, 2001; U.S. Provisional Application No. 60/259,803 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. US01/01431 is a continuation-in-part of U.S. application Ser. No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 11/968,925, filed Jan. 3, 2008, which is a divisional of 10/644,765, filed Aug. 21, 2003, which is a continuation of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which in turn claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application Nos. 60/270,625 and 60/304,417, filed Feb. 23, 2001 and Jul. 12, 2001, respectively. This application is a continuation-in-part of U.S. application Ser. No. 12/538,668, filed Aug. 10, 2009, which is a continuation of U.S. application Ser. No. 11/240,769, filed Oct. 3, 2005, which is a continuation application of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, now abandoned, which is a continuation application of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, now abandoned, which is a continuation-in-part of PCT International Application Serial No. PCT/US00/03062, filed Feb. 8, 2000, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application No. 60/119,468 filed Feb. 10, 1999. This application is a continuation-in-part of U.S. application Ser. No. 11/777,133, filed Jul. 12, 2007, which is a continuation of U.S. application Ser. No. 11/229,769, filed Sep. 20, 2005, which is a continuation of U.S. application Ser. No. 10/233,453, filed Sep. 4, 2002, which is a divisional of U.S. application Ser. No. 09/489,847 filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130 filed Jul. 29, 1999, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998. This application is a continuation-in-part of U.S. application Ser. No. 12/268,263, filed Nov. 10, 2008, which is a divisional of U.S. application Ser. No. 11/375,555, filed Mar. 15, 2006, which is a continuation-in-part of application Ser. No. 10/103,295, filed Mar. 22, 2002 (now U.S. Pat. No. 7,091,315, issued Aug. 15, 2006), which is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; application Ser. No. 10/103,295 is also a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001 (now abandoned); application Ser. No. 10/103,295 is also a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000 (now U.S. Pat. No. 6,534,631, issued Mar. 18, 2003), which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/092,921, 60/092,922, and 60/092,956, all of which were filed on Jul. 15, 1998. This application is a continuation-in-part of U.S. application Ser. No. 11/760,578, filed Jun. 8, 2007, which is a continuation of U.S. application Ser. No. 10/886,642, filed Jul. 9, 2004, which is a continuation of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/263,230, filed on Jan. 23, 2001, and 60/263,681, filed on Jan. 24, 2001; said U.S. application Ser. No. 10/050,873 is also a continuation-in-part of U.S. patent application Ser. No. 09/461,325, filed on Dec. 14, 1999, now U.S. Pat. No. 6,475,753, which is a continuation-in-part of International Patent Application No: PCT/US99/13418, filed on Jun. 15, 1999, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/089,507, 60/089,508, 60/089,509, 60/089,510, each of which was filed on Jun. 16, 1998, and 60/090,112 and 60/090,113, each of which was filed on Jun. 22, 1998. This application is a continuation-in-part of U.S. application Ser. No. 12/274,626, filed Nov. 20, 2008, which is a divisional of U.S. application Ser. No. 11/608,978, filed Dec. 11, 2006, which is a continuation of U.S. application Ser. No. 10/918,446, filed Aug. 16, 2004, which is a divisional of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002 (now U.S. Pat. No. 6,924,356, issued Aug. 2, 2005), which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999 (now U.S. Pat. No. 6,569,992, issued May 27, 2003), which is a continuation-in-part of International Application PCT/US99/02293, filed Feb. 4, 1999, which is a non-provisional of, and claims benefit under 35 U.S.C. §119(e) to, U.S. Provisional Applications 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998. This application is a continuation-in-part of U.S. application Ser. No. 11/780,874, filed Jul. 20, 2007, which is a continuation of U.S. patent application Ser. No. 10/935,098, filed Sep. 8, 2004, which is a continuation of U.S. patent application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. patent application Ser. No. 09/739,907, filed Dec. 20, 2000 (now abandoned), which is a continuation of U.S. patent application Ser. No. 09/348,457, filed Jul. 7, 1999 (now abandoned), which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998; 60/070,658, filed Jan. 7, 1998; 60/070,692, filed Jan. 7, 1998; and 60/070,657, filed Jan. 7, 1998. This application is a continuation-in-part of U.S. application Ser. No. 12/325,800, filed Dec. 1, 2008, which is a divisional of U.S. application Ser. No. 11/565,909, filed Dec. 1, 2006, which is a divisional of U.S. application Ser. No. 10/970,493, filed on Oct. 22, 2004 (now U.S. Pat. No. 7,163,797, issued Jan. 16, 2007), which is a continuation of U.S. application Ser. No. 10/047,021, filed on Jan. 17, 2002 (now abandoned), which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/262,066, filed on Jan. 18, 2001; U.S. application Ser. No. 10/047,021 is also a continuation-in-part of U.S. application Ser. No. 09/722,329, filed on Nov. 28, 2000 (now abandoned), which is a continuation of U.S. application Ser. No. 09/262,109, filed on Mar. 4, 1999 (now abandoned), which is a continuation-in-part of International Application PCT/US98/18360, filed on Sep. 3, 1998, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/057,626; 60/057,663; 60/057,669, filed on Sep. 5, 1997; 60/058,666; 60/058,667; 60/058,973; 60/058,974, filed on Sep. 12, 1997; and, 60/090,112, filed on Jun. 22, 1998. This application is a continuation-in-part of U.S. application Ser. No. 11/735,351, filed Apr. 13, 2007, which is a continuation of U.S. application Ser. No. 10/866,878, filed Jun. 15, 2004, which is a divisional of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is also a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications 60/051,926; 60/052,793; 60/051,925; 60/051,929; 60/052,803; 60/052,732; 60/051,931; 60/051,932; 60/051,916; 60/051,930; 60/051,918; 60/051,920; 60/052,733; 60/052,795; 60/051,919; 60/051,928 (each of which was filed Jul. 8, 1997); and; 60/055,722; 60/055,723; 60/055,948; 60/055,949; 60/055,953; 60/055,950; 60/055,947; 60/055,964; 60/056,360; 60/055,684; 60/055,984; 60/055,954 (each of which was filed Aug. 18, 1997); and; 60/058,785; 60/058,664; 60/058,660; 60/058,661 (each of which was filed Sep. 12, 1997). This application is a continuation-in-part of U.S. application Ser. No. 11/759,448, filed Jun. 7, 2007, which is a continuation of U.S. application Ser. No. 11/229,770, filed Sep. 20, 2005 (abandoned), which is a continuation of U.S. application Ser. No. 09/933,767, filed Aug. 22, 2001 (abandoned), which is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Patent Application Ser. Nos. 60/184,836, filed Feb. 24, 2000 and 60/193,170, filed Mar. 29, 2000. application Ser. No. 09/933,767 is a continuation-in-part of U.S. patent application Ser. No. 09/205,258, filed Dec. 4, 1998 (now U.S. Pat. No. 6,525,174), which is a continuation-in-part of International Patent Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications 60/048,885; 60/049,375; 60/048,881; 60/048,880; 60/048,896; 60/049,020; 60/048,876; 60/048,895; 60/048,884; 60/048,894; 60/048,971; 60/048,964; 60/048,882; 60/048,899; 60/048,893; 60/048,900; 60/048,901; 60/048,892; 60/048,915; 60/049,019; 60/048,970; 60/048,972; 60/048,916; 60/049,373; 60/048,875; 60/049,374; 60/048,917; 60/048,949; 60/048,974; 60/048,883; 60/048,897; 60/048,898; 60/048,962; 60/048,963; 60/048,877; 60/048,878, all filed Jun. 6, 1997, and 60/057,645; 60/057,642; 60/057,668; 60/057,635; 60/057,627; 60/057,667; 60/057,666; 60/057,764; 60/057,643; 60/057,769; 60/057,763; 60/057,650; 60/057,584; 60/057,647; 60/057,661; 60/057,662; 60/057,646; 60/057,654; 60/057,651; 60/057,644; 60/057,765; 60/057,762; 60/057,775; 60/057,648; 60/057,774; 60/057,649; 60/057,770; 60/057,771; 60/057,761; 60/057,760; 60/057,776; 60/057,778; 60/057,629; 60/057,628; 60/057,777; 60/057,634, all filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997. This application in a continuation-in-part of U.S. application Ser. No. 12/324,825, filed Nov. 26, 2008, which is a continuation of U.S. application Ser. No. 11/226,657, filed Sep. 15, 2005, which is a divisional of U.S. application Ser. No. 10/062,831, filed Feb. 5, 2002, (now U.S. Pat. No. 7,001,992, issued Feb. 21, 2006), which is a divisional of U.S. application Ser. No. 09/690,454, filed Oct. 18, 2000 (now U.S. Pat. No. 6,531,447, issued Mar. 11, 2003), which is a continuation of U.S. application Ser. No. 09/189,144, filed Nov. 10, 1998 (now abandoned), which is a continuation-in-part of international Application No. PCT/US98/10868, filed May 28, 1998, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Applications: 60/044,039; 60/048,093; 60/048,190; 60/050,935; 60/048,101; 60/048,356 (each filed May 30, 1997); and 60/056,250; 60/056,296; 60/056,293 (each filed Aug. 29, 1997). This application in a continuation-in-part of U.S. application Ser. No. 11/801,040, filed May 7, 2007, which is a divisional of U.S. application Ser. No. 10/062,831, filed Feb. 5, 2002, which is a divisional of U.S. application Ser. No. 09/690,454, filed Oct. 18, 2000 (now U.S. Pat. No. 6,531,447, issued Mar. 11, 2003), which is a continuation of U.S. application Ser. No. 09/189,144 filed Nov. 10, 1998 (now abandoned), which is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Applications: 60/044,039; 60/048,093; 60/048,190; 60/050,935; 60/048,101; 60/048,356 (each filed May 30, 1997); and, 60/056,250; 60/056,296; 60/056,293 (each filed Aug. 29, 1997). This application in a continuation-in-part of U.S. application Ser. No. 11/744,695, filed May 4, 2007, which is a continuation of U.S. application Ser. No. 10/951,993 filed on Sep. 29, 2004, which is a divisional of U.S. application Ser. No. 10/058,993 filed on Jan. 30, 2002, which is a non-provisional of, and claims benefit under 35 U.S.C. §119(e) based on, U.S. Provisional Application No. 60/265,583 filed on Feb. 2, 2001; which is also a continuation-in-part, and claims priority under 35 U.S.C. §120, of U.S. application Ser. Nos. 09/852,659, 09/852,797, and 09/853,161 filed on May 11, 2001, each of which claim benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application No. 60/265,583 filed on Feb. 2, 2001, and each of which is a continuation-in-part, and claim priority under 35 U.S.C. §120, of U.S. application Ser. No. 09/152,060 filed on Sep. 11, 1998, which is a continuation-in-part, and claims priority under 35 U.S.C. §120, of International Application PCT/US98/04858 filed on Mar. 12, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications 60/040,710 and 60/040,762 (filed on Mar. 14, 1997), 60/050,934, 60/048,100, 60/048,189 and 60/048,357 (filed on May 30, 1997), 60/048,970 (filed on Jun. 6, 1997), 60/057,765 (filed on Sep. 5, 1997), and 60/068,368 (filed on Dec. 19, 1997); and U.S. application Ser. No. 10/951,993 is also a continuation-in-part, and claims priority under 35 U.S.C. §120, of U.S. application Ser. No. 09/152,060 filed on Sep. 11, 1998, which is a continuation-in-part, and claims priority under 35 U.S.C. §120, of International Application PCT/US98/04858 filed on Mar. 12, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications 60/040,710 and 60/040,762 (filed on Mar. 14, 1997), 60/050,934, 60/048,100, 60/048,189 and 60/048,357 (filed on May 30, 1997), 60/048,970 (filed on Jun. 6, 1997), 60/057,765 (filed on Sep. 5, 1997), and 60/068,368 (filed on Dec. 19, 1997). This application is a continuation-in-part of U.S. application Ser. No. 11/745,580, filed May 8, 2007, which is a continuation of U.S. application Ser. No. 11/144,947, filed Jun. 6, 2005, which is a continuation of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which is a continuation application of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, now abandoned, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 11/144,947 is a continuation-in-part of U.S. application Ser. No. 10/164,861, filed Jun. 10, 2002, which is a divisional of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, (now issued U.S. Pat. No. 6,420,526, issued on Jul. 16, 2002), which is a continuation-in-part of PCT International Application No. PCT/US98/04493, filed Mar. 6, 1998 which claims benefit under 35 U.S.C. §119(e) based on the following U.S. Provisional Applications 60/040,162; 60/040,333; 60/038,621; 60/040,626; 60/040,334; 60/040,336; 60/040,163; (each filed Mar. 7, 1997); and, 60/047,600; 60/047,615; 60/047,597; 60/047,502; 60/047,633; 60/047,583; 60/047,617; 60/047,618; 60/047,503; 60/047,592; 60/047,581; 60/047,584; 60/047,500; 60/047,587; 60/047,492; 60/047,598; 60/047,613; 60/047,582; 60/047,596; 60/047,612; 60/047,632; 60/047,601, (each filed May 23, 1997); and, 60/043,580; 60/043,568; 60/043,314; 60/043,569; 60/043,311; 60/043,671; 60/043,674; 60/043,669; 60/043,312; 60/043,313; 60/043,672; 60/043,315, (each filed Apr. 11, 1997); and, 60/048,974, (filed Jun. 6, 1997); and, 60/056,886; 60/056,877; 60/056,889; 60/056,893; 60/056,630; 60/056,878; 60/056,662; 60/056,872; 60/056,882; 60/056,637; 60/056,903; 60/056,888; 60/056,879; 60/056,880; 60/056,894; 60/056,911; 60/056,636; 60/056,874; 60/056,910; 60/056,864; 60/056,631; 60/056,845, 60/056,892, (each filed Aug. 22, 1997); and 60/057,761, (filed Sep. 5, 1997); and, 60/047,595; 60/047,599; 60/047,588; 60/047,585; 60/047,586; 60/047,590; 60/047,594; 60/047,589; 60/047,593; 60/047,614, (each filed May 23, 1997); and, 60/043,578, 60/043,576 (each filed Apr. 11, 1997); and, 60/047,501, (filed May 23, 1997); and, 60/043,670, (filed Apr. 11, 1997); and, 60/056,632; 60/056,664; 60/056,876; 60/056,881; 60/056,909; 60/056,875; 60/056,862; 60/056,887; 60/056,908, (each filed Aug. 22, 1997); and, 60/048,964, (filed Jun. 6, 1997); and, 60/057,650, (filed Sep. 5, 1997); and, 60/056,884, (filed Aug. 22, 1997); and, 60/057,669, (filed Sep. 5, 1997); and, 60/049,610, (filed Jun. 13, 1997); and, 60/061,060, (filed Oct. 2, 1997); and, 60/051,926, (filed Jul. 8, 1997); and, 60/052,874, (filed Jul. 16, 1997); and, 60/058,785, (filed Sep. 12, 1997); and, 60/055,724, (filed Aug. 18, 1997); and, 60/040,161, (filed Mar. 7, 1997). This application is a continuation-in-part of U.S. application Ser. No. 12/264,040, filed Nov. 3, 2008, which is a divisional of U.S. application Ser. No. 11/246,999, filed Oct. 11, 2005, which is a divisional of U.S. application Ser. No. 09/984,130, filed Oct. 29, 2001 (now abandoned), which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/243,792, filed on Oct. 30, 2000, and which is also a continuation-in-part of U.S. application Ser. No. 09/836,353, filed Apr. 18, 2001, (now abandoned), which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/198,407, filed Apr. 19, 2000, and which is a continuation-in-part of PCT International Application No. PCT/US99/25031 (published in English), filed Oct. 27, 1999, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/105,971, filed Oct. 28, 1998. Each of the above referenced patents and patent applications is hereby incorporated by reference herein in its entirety.


REFERENCE TO SEQUENCE LISTING AS TEXT FILE
This application refers to a “Sequence Listing” listed below, which is provided as a text file. The text file contains a document entitled “PS960_SeqList.txt” (473,508 bytes, created Apr. 2, 2010), which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to novel proteins/polypeptides (such as human secreted proteins/polypeptides), and isolated nucleic acid molecules encoding said proteins/polypeptides, useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders related to said proteins/polypeptides (relatedness may be by direct or indirect association, by cause, by consequence, or by effect on said diseases and disorders), such as immune, cardiovascular, cancer, and other proliferative disorders and diseases. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.
BACKGROUND OF THE INVENTION
Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eukaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses “sorting signals,” which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.
One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles.
Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space—a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a “linker” holding the protein to the membrane.
Thus there exists a clear need for identifying and using novel secreted polynucleotides and polypeptides. Identification and sequencing of human genes is a major goal of modern scientific research. For example, by identifying genes and determining their sequences, scientists have been able to make large quantities of valuable human “gene products.” These include human insulin, interferon, Factor VIII, tumor necrosis factor, human growth hormone, tissue plasminogen activator, erythropoietin, and numerous other compounds. Thus, in light of the pervasive role of secreted proteins in human physiology, a need exists for identifying and characterizing novel human secreted proteins and the genes that encode them. This knowledge will allow one to detect, to treat, and to prevent medical diseases, disorders, and/or conditions by using secreted proteins or the genes that encode them. Additionally, knowledge of gene sequences can provide the key to treatment or cure of genetic diseases (such as muscular dystrophy and cystic fibrosis).
Immune-Related Polynucleotides and Polypeptides
The immune system is an intricate network of cells, tissues and soluble molecules that function to protect the body from invasion by foreign substances and pathogens. The major cells of the immune system are lymphocytes, including B cells and T cells, and myeloid cells, including basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages and dendritic cells. In addition to these cellular components of the immune system, soluble molecules—such as antibodies, complement proteins, and cytokines—circulate in lymph and blood plasma, and play important roles in immunity.
The immune system can be subdivided into the acquired and innate immune systems. The cells of the innate immune system (e.g., neutrophils, eosinophils, basophils, mast cells) are not antigen specific and their action is not enhanced by repeated exposure to the same antigen. The cells of the acquired immune system (B and T cells) are antigen specific. Repeated exposure of B and T cells to an antigen results in improved immune responses (memory responses) produced by these cell types. The cells and products of the acquired immune system can recruit components of the innate system to mount a focused immune response. For a more extensive review of the immune system, see Fundamental Immunology, 4th edition, Ed. William Paul, Lippincott-Raven Pub. (1998).
An immune response is seldom carried out by a single cell type, but rather requires the coordinated efforts of several cell types. In order to coordinate an immune response, it is necessary that cells of the immune system communicate with each other and with other cells of the body. Communication between cells may be made by cell-cell contact, between membrane bound molecules on each cell, or by the interaction of soluble components of the immune system with cellular receptors. Signaling between cell types may have one or more of a variety of consequences, including activation, proliferation, differentiation, and apoptosis. Activation and differentiation of immune cells may result in the expression or secretion of polypeptides, or other molecules, which in turn affect the function of other cells and/or molecules of the immune system.
Molecules which stimulate or suppress immune system function are known as immunomodulators. These molecules, which include endogenous proteins (e.g., cytokines, cytokine receptors, and intracellular signal transduction molecules), molecules derived from microorganisms, and synthetic agents, may exert their modulatory effects at one or more stages of the immune response, such as antigen recognition, stimulation of cytokine production and release, and/or activation/differentiation of lymphocytes and myeloid cells. Immunomodulators may enhance (immunoprophylaxis, immunostimulation), restore (immunosubstitution, immunorestoration) or suppress (immunosuppression, immunodeviation) immunological functions or activities.
Immunomodulatory compounds have many important applications in clinical practice. For example, immunosuppressing agents (which attenuate or prevent unwanted immune responses) can be used to prevent tissue rejection during organ transplantation, to prevent Rh hemolytic disease of the newborn, or to treat autoimmune disorders. A mechanism of action common to many immunosuppressants is the inhibition of T cell activation and/or differentiation. Antilymphocyte antibodies have also been used to attenuate immune system functions. Currently-used immunosuppressive agents can produce a number of side effects which limit their use. Among the most serious secondary effects include kidney and liver toxicity, increased risk of infection, hyperglycemia, neoplasia, and osteoporosis (see, e.g., Freeman, Clin. Biochem. 24(1):9-14 (1991); Mitchison, Dig. Dis. 11(2):78-101 (1993)).
Immunostimulants, which enhance the activity of immune cells and molecules, comprise another class of immunomodulatory agents with important clinical applications. Such applications include, for example, the treatment of immunodeficiency disorders (e.g. AIDS and severe combined immunodeficiency), chronic infectious diseases (e.g. viral hepatitis, papillomavirus, and herpesvirus), and cancer. An important class of endogenous immunostimulants is the cytokines. These soluble signaling molecules are produced by a number of cell types, and are critical to the regulation of the immune response. Immunostimulatory mechanisms can include proliferation, differentiation and/or activation of immune cells or progenitors of immune cells. For example, interleukin-2 (IL-2) binds to IL-2 receptors on T lymphocytes and induces proliferation and differentiation. Another cytokine, interferon alpha, stimulates the immune system through a variety of mechanisms, including activation of macrophages, T lymphocytes, and natural killer cells. Interferon alpha also induces the expression of antiviral proteins (see Chapter 50, The Pharmacological Basis of Therapeutics, 9th Edition, Eds. Hardman, Limbird, Molinoff, Ruddon, and Gilman, McGraw Hill (1996)). Limitations of current immunostimulant therapies include anaphylaxis, pulmonary edema, and renal toxicity, to name a few.
The discovery of new human immune related polynucleotides, the polypeptides encoded by them, and antibodies that immunospecifically bind these polypeptides, satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, prevention and/or prognosis of disorders of the immune system, including, but not limited to, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura and multiple sclerosis), immunodeficiencies (e.g., X-linked agammaglobulinemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, and ataxia telangiectasia), chronic infections (e.g., HIV, viral hepatitis, and herpesvirus), and neoplastic disorders. See, e.g. “Immune Activity” section infra. Additionally, immune related molecules would be useful as agents to boost immune responsiveness to pathogens or to suppress immune reactions, for example as is necessary in conjunction with organ transplantation.
Cardiovascular-Related Polynucleotides and Polypeptides
The cardiovascular system is a component of a complex physiological network involved in maintaining the oxygen and nutrient supply to tissues of the body. The heart is the anatomical and functional centerpiece of the cardiovascular system. Weighing only 250-350 grams (less than a pound), the heart is one of our strongest and hardest working organs. It is composed of innervated muscle tissue with unique properties; e.g., it can pace itself in contraction. The main center of rhythm regulation is the sinoatrial (SA) node. Certain cardiac cells repeatedly fire impulses that trigger heart contractions. These autorhythmic cells have two important functions. One is to act as a pacemaker (set the pace for the entire heart), and the other is to form a conduction system, the route for conducting impulses throughout the heart muscle. This conduction system controls the pattern of blood flow through the heart.
The heart pumps at least five quarts of blood through a full circuit of the body every minute. The heart consists of two pumps, side by side. The pump on the right side moves blood to the lungs, where waste gases, such as carbon dioxide, are removed and oxygen is added. Freshly oxygenated blood returns to the pump on the left side, which moves it out into the rest of the body. Blood flows away from the heart to the lungs or to the rest of your body, though blood vessels called arteries. Arteries branch extensively, each branch become smaller, forming blood vessels called arterioles. Arterioles also become repeatedly smaller and smaller until they are tiny vessels called capillaries. Throughout the arteries and smaller vessels that stem from them, the blood delivers nutrients and oxygen to the tissues and picks up waste. This task is completed in the capillaries. As the blood moves on through the capillaries the blood vessels gradually become larger, eventually becoming veins. Veins ultimately carry blood back to the heart. The cycle then begins again.
Disorders of the cardiovascular system are many and varied, killing more Americans each year than any other category of disorders. For example, damage to the conduction system leads to arrhythmia, an irregular beating of the heart. If left untreated, the heart becomes unable to effectively pump blood, frequently leading to permanent heart damage and/or cardiac arrest.
One of the most prevalent conditions in industrialized countries today is atherosclerosis. Atherosclerosis is the buildup of fatty deposits in the intima of large and medium-sized arteries. The buildup of deposits narrowing of the arteries, reducing or potentially blocking the ability of blood to flow through the arteries. Untreated, atherosclerosis typically results in cardiac arrest and, frequently, death.
Clearly, the discovery of new human cardiovascular-associated polynucleotides, the polypeptides encoded by them, and antibodies that immunospecifically bind these polypeptides, satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, prevention and/or prognosis of cardiovascular disorders.
Cardiovascular disorders include, but are not limited to, stroke, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
Cancer and Hyperproliferative-Related Polynucleotides and Polypeptides
Cancer and other hyperproliferative disorders are a diverse group of disorders and diseases sharing one characteristic in common; all result from uncontrolled cell proliferation. The human body is composed of many different cell types, e.g. liver cells, muscle cells, brain cells, etc. Normally, these cells grow and divide to produce more cells only as the body needs them (e.g. to regenerate blood cells or replace epithelial cells lining the stomach). Sometimes, however, cells begin to divide unchecked even though new cells are not needed. These extra cells accumulate and form a mass of tissue, called a tumor. Although each of the over 200 cell types in the body can potentially become cancerous, some cell types become cancerous at relatively high rates while many other cell types rarely become cancerous.
Tumors are either benign or malignant. Benign tumors are not cancerous; they can usually be removed, they do not spread to other parts of the body and, they rarely threaten life. Malignant tumors, however, are cancerous. Cells in malignant tumors can invade and damage nearby or distant tissues and organs. The spread of cancerous cells is called metastasis. Malignant (or metastatic) cells can invade adjacent organs by proliferating directly from the primary tumor. Additionally, malignant cells can also metastasize to distant organs by breaking away from the primary tumor, entering the bloodstream or lymphatic system, and settling down in a new organ or tissue to produce a secondary tumor. The origin of secondary tumors is established by comparing cells comprising these tumors to cells in the original (primary) tumor.
In contrast to solid organ cancers (such as cancer in the liver, lung, and brain) cancer can also develop in blood-forming cells. These cancers are referred to as leukemias or lymphomas. Leukemia refers to cancer of blood forming cells such as red blood cells, platelets, and plasma cells. Lymphomas are a subset of leukemias, primarily involving white blood cells, in which the cancerous cells originated in, or are associated with, the lymph system and lymph organs (e.g. T-lymphocytes in the lymph nodes, spleen, or thymus).
In 1999 over 1.1 million people were newly diagnosed with 23 different types of cancer. The vast majority of these cases ( ̃75%) involved cancers of the prostate, breast, lung, colon, or urinary tract, or non-Hodgkin's lymphoma. Among the most fatal cancers are pancreatic, liver, esophageal, lung, stomach, and brain cancers, having up to 96% mortality rates depending on the specific cancer. In all, some 23 different types of cancer are expected to kill over 86,000 people each year.
Most cancers are treated with one or a combination therapies consisting of surgery, radiation therapy, chemotherapy, hormone therapy, and/or biological therapy. These five therapeutic modes are either local or systemic treatment strategies. Local treatments affect cancer cells in the tumor and immediately adjacent areas (for example, surgical tumor removal is a local treatment as are most radiation treatments). In contrast, systemic treatments travel through the bloodstream, and reach cancer and other cells all over the body. Chemotherapy, hormone therapy, and biological therapy are examples of systemic treatments.
Whether systemic or local, it is often difficult or impossible to protect healthy cells from the harmful effects of cancer treatment; healthy cells and tissues are inevitably damaged in the process of treating the cancerous cells. Damage and disruption of the normal functioning of healthy cells and tissues often produces the undesirable side effects experienced by patients undergoing cancer treatment.
Recombinant polypeptides and polynucleotides derived from naturally occurring molecules, as well as antibodies specifically targeted to these molecules, used alone or in conjunction with other existing therapies, hold great promise as improved therapeutic agents for the treatment of neoplastic disorders. Currently, most biological therapy can be classified as immunotherapy because these treatments often use naturally occurring molecules to assist the body's immune system in fighting the disease or in protecting the body from side effects of other cancer treatment(s). Among the most commonly used compounds in biological therapies are proteins called cytokines (e.g. interferons, interleukins, and colony stimulating factors) and monoclonal antibodies (targeted to particular cancer cells). Side effects caused by these commonly used biological therapies range from flu-like symptoms (chills, fever, muscle aches, weakness, loss of appetite, nausea, vomiting, and diarrhea) to rashes, swelling, easy bruising, or bleeding.
The discovery of human secreted proteins associated with initiation, progression, characterization, and/or distinction of neoplastic diseases (including antibodies that immunospecifically bind these polypeptides), satisfies a need in the art by providing new compositions useful in the detection, prevention, diagnosis, treatment, prevention, prognosis, and treatment of hyperproliferative disorders.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) corresponding to Gene No: 1.
FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.
The data presented in FIG. 2 are also represented in tabular form in Table 8. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 2, and Table 8: “Res”: amino acid residue of SEQ ID NO:2 and FIG. 1; “Position”: position of the corresponding residue within SEQ ID NO:2 and FIG. 1; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, Amphipathic Regions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.
FIGS. 3A-3B show the Gene No: 5 nucleotide (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) corresponding to this gene.
FIG. 4 shows an analysis of the amino acid sequence (SEQ ID NO:21). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides
FIG. 5 shows the nucleotide (SEQ ID NO:102) and deduced amino acid sequence (SEQ ID NO:103) corresponding to Gene No: 14.
FIG. 6 shows an analysis of the amino acid sequence (SEQ ID NO:103). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.
FIGS. 7A-C show the nucleotide sequence (SEQ ID NO:134) and the deduced amino acid sequence (SEQ ID NO:135) of the HCEJQ69 cDNA (Gene No: 20).
FIG. 8 shows an analysis of the amino acid sequence (SEQ ID NO:135). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.
The data presented in FIG. 8 are also represented in tabular form in Table 9. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 8, and Table 9: “Res”: amino acid residue of SEQ ID NO:135 and FIGS. 7A-7C; “Position”: position of the corresponding residue within SEQ ID NO:135 and FIGS. 7A-7C; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, Amphipathic Regions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.
FIGS. 9A-B show the nucleotide (SEQ ID NO:159) and deduced amino acid sequence (SEQ ID NO:160) of this polypeptide.
FIG. 10 shows an analysis of the amino acid sequence (SEQ ID NO:160). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained.
FIGS. 11A-F show the nucleotide (SEQ ID NO:186) and deduced amino acid sequence (SEQ ID NO:187) of all. Predicted amino acids from about 1 to about 22 constitute the predicted signal peptide (amino acid residues from about 1 to about 22 in SEQ ID NO:187) and are represented by the underlined amino acid regions; amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 constitute the predicted transmembrane domains (amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 in SEQ ID NO:187) and are represented by the double underlined amino acids; and amino acids from about 64 to about 96 constitute the predicted immunoglobulin and major histocompatibility complex protein domain (amino acids from about 64 to about 96 in SEQ ID NO:187) and are represented by the bold amino acids.
FIGS. 12A-E show the regions of similarity between the amino acid sequences of the integrin alpha 11 subunit (all) protein (SEQ ID NO:187) and the human integrin alpha 1 subunit (SEQ ID NO:189).
FIG. 13 shows an analysis of the integrin alpha 11 subunit (all) amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.



SUMMARY OF THE INVENTION
The present invention relates to novel secreted proteins/polypeptides (such as human proteins/polypeptides). More specifically, isolated nucleic acid molecules are provided encoding novel secreted polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides, polypeptides, and/or antibodies. The invention further relates to diagnostic and therapeutic methods useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders related to said proteins/polypeptides (relatedness may be by direct or indirect association, or by cause, consequence, or effect on said diseases and disorders), such as immune, cardiovascular, cancer, and other proliferative disorders and diseases. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
DETAILED DESCRIPTION
Polynucleotides and Polypeptides of the Invention
Features of Protein Encoded by Gene No: 1
For purposes of this application, this gene and its corresponding translation product are known as the B7-H5 gene and B7-H5 protein. This protein is believed to reside as a cell-surface molecule, and the transmembrane domain of this protein is believed to embody the following preferred amino acid residues:









(SEQ ID NO: 3)



IRVPVFNIVILLAGGF.






Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these peptides. The B7-H5 gene shares sequence homology with members of the B7 family of ligands (i.e., B7-1 (See Genbank Accession 507873)). These proteins and their corresponding receptors play vital roles in the growth, differentiation and death of T cells. For example, some members of this family (i.e., B7-H1) are involved in costimulation of the T cell response, as well as inducing increased cytokine production. Therefore, antagonists such as antibodies or small molecules directed against the B7-H5 gene are useful for treating T cell mediated immune system disorders. The gene encoding the disclosed cDNA is thought to reside on chromosome 6. Accordingly, polynucleotides related to this invention have uses, such as, for example, as a marker in linkage analysis for chromosome 6.

It has been discovered that this gene is expressed in activated neutrophils and activated T cells, and to a lesser extent in monocytes and heart tissue.
Preferred polypeptides of the present invention comprise, or alternatively consist of, one, two, three, four, five, six, seven or all seven of the immunogenic epitopes of the extracellular portion of the B7-H5 protein shown in SEQ ID NO:2 as residues: Leu-24 to Gln-35, Arg-59 to Pro-64, Glu-71 to His-78, Asp-89 to Gly-94, Pro-141 to Val-151, Thr-167 to Val-172, Ala-175 to Thr-180. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these peptides.
In additional nonexclusive embodiments, polypeptides of the invention comprise, or alternatively consist of, one or more of the following amino acid sequences: 1.) The extracellular domain of the B7-H5 protein:







MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFA


 


SSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRV


 


RMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENS


 


TQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVT


 


DI (SEQ ID NO: 4),






2.) The mature extracellular domain of the B7-H5 protein:








EKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSK


 


NSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHM


 


LFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVT


 


QAPPKSTADVSTPDSEINLTNVTDI (SEQ ID NO: 5),






and/or 3.) The leader sequence of the B7-H5 protein:










MRKTRLWGLLWMLFVSELRAATKLTE (SEQ ID NO: 6).






Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides.
Also preferred are polypeptides comprising, or alternatively consisting of, fragments of the mature extracellular portion of the B7-H5 protein demonstrating functional activity (SEQ ID NO:5). Polynucleotides encoding these polypeptides are also encompassed by the invention. By functional activity is meant, a polypeptide fragment capable of displaying one or more known functional activities associated with the full-length (complete) B7-H5 protein. Such functional activities include, but are not limited to, biological activity (e.g., T cell costimulatory activity, ability to bind ICOS, and ability to induce or inhibit cytokine production), antigenicity [ability to bind (or compete with a B7-H5 polypeptide for binding) to an anti-B7-H5 antibody], immunogenicity (ability to generate antibody which binds to a B7-H5 polypeptide), ability to form multimers with B7-H5 polypeptides of the invention, and ability to bind to a receptor or ligand for a B7-H5 polypeptide.
FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) corresponding to this gene.
FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.
The data presented in FIG. 2 are also represented in tabular form in Table 8. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 2, and Table 8: “Res”: amino acid residue of SEQ ID NO:2 and FIG. 1; “Position”: position of the corresponding residue within SEQ ID NO:2 and FIG. 1; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, Amphipathic Regions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.
Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 2 and/or Table 8, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 8 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in FIG. 2, but may, as shown in Table 8, be represented or identified by using tabular representations of the data presented in FIG. 2. The DNA*STAR computer algorithm used to generate FIG. 2 (set on the original default parameters) was used to present the data in FIG. 2 in a tabular format (See Table 8). The tabular format of the data in FIG. 2 is used to easily determine specific boundaries of a preferred region.
The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:1, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:1. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, and from about 801 to about 860, of SEQ ID NO:1, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.
Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m−234 where m is an integer from 2 to 228, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:2. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: R-2 to P-234; K-3 to P-234; T-4 to P-234; R-5 to P-234; L-6 to P-234; W-7 to P-234; G-8 to P-234; L-9 to P-234; L-10 to P-234; W-11 to P-234; M-12 to P-234; L-13 to P-234; F-14 to P-234; V-15 to P-234; S-16 to P-234; E-17 to P-234; L-18 to P-234; R-19 to P-234; A-20 to P-234; A-21 to P-234; T-22 to P-234; K-23 to P-234; L-24 to P-234; T-25 to P-234; E-26 to P-234; E-27 to P-234; K-28 to P-234; Y-29 to P-234; E-30 to P-234; L-31 to P-234; K-32 to P-234; E-33 to P-234; G-34 to P-234; Q-35 to P-234; T-36 to P-234; L-37 to P-234; D-38 to P-234; V-39 to P-234; K-40 to P-234; C-41 to P-234; D-42 to P-234; Y-43 to P-234; T-44 to P-234; L-45 to P-234; E-46 to P-234; K-47 to P-234; F-48 to P-234; A-49 to P-234; S-50 to P-234; S-51 to P-234; Q-52 to P-234; K-53 to P-234; A-54 to P-234; W-55 to P-234; Q-56 to P-234; I-57 to P-234; I-58 to P-234; R-59 to P-234; D-60 to P-234; G-61 to P-234; E-62 to P-234; M-63 to P-234; P-64 to P-234; K-65 to P-234; T-66 to P-234; L-67 to P-234; A-68 to P-234; C-69 to P-234; T-70 to P-234; E-71 to P-234; R-72 to P-234; P-73 to P-234; S-74 to P-234; K-75 to P-234; N-76 to P-234; S-77 to P-234; H-78 to P-234; P-79 to P-234; V-80 to P-234; Q-81 to P-234; V-82 to P-234; G-83 to P-234; R-84 to P-234; I-85 to P-234; I-86 to P-234; L-87 to P-234; E-88 to P-234; D-89 to P-234; Y-90 to P-234; H-91 to P-234; D-92 to P-234; H-93 to P-234; G-94 to P-234; L-95 to P-234; L-96 to P-234; R-97 to P-234; V-98 to P-234; R-99 to P-234; M-100 to P-234; V-101 to P-234; N-102 to P-234; L-103 to P-234; Q-104 to P-234; V-105 to P-234; E-106 to P-234; D-107 to P-234; S-108 to P-234; G-109 to P-234; L-110 to P-234; Y-111 to P-234; Q-112 to P-234; C-113 to P-234; V-114 to P-234; I-115 to P-234; Y-116 to P-234; Q-117 to P-234; P-118 to P-234; P-119 to P-234; K-120 to P-234; E-121 to P-234; P-122 to P-234; H-123 to P-234; M-124 to P-234; L-125 to P-234; F-126 to P-234; D-127 to P-234; R-128 to P-234; I-129 to P-234; R-130 to P-234; L-131 to P-234; V-132 to P-234; V-133 to P-234; T-134 to P-234; K-135 to P-234; G-136 to P-234; F-137 to P-234; S-138 to P-234; G-139 to P-234; T-140 to P-234; P-141 to P-234; G-142 to P-234; S-143 to P-234; N-144 to P-234; E-145 to P-234; N-146 to P-234; S-147 to P-234; T-148 to P-234; Q-149 to P-234; N-150 to P-234; V-151 to P-234; Y-152 to P-234; K-153 to P-234; I-154 to P-234; P-155 to P-234; P-156 to P-234; T-157 to P-234; T-158 to P-234; T-159 to P-234; K-160 to P-234; A-161 to P-234; L-162 to P-234; C-163 to P-234; P-164 to P-234; L-165 to P-234; Y-166 to P-234; T-167 to P-234; S-168 to P-234; P-169 to P-234; R-170 to P-234; T-171 to P-234; V-172 to P-234; T-173 to P-234; Q-174 to P-234; A-175 to P-234; P-176 to P-234; P-177 to P-234; K-178 to P-234; S-179 to P-234; T-180 to P-234; A-181 to P-234; D-182 to P-234; V-183 to P-234; S-184 to P-234; T-185 to P-234; P-186 to P-234; D-187 to P-234; S-188 to P-234; E-189 to P-234; I-190 to P-234; N-191 to P-234; L-192 to P-234; T-193 to P-234; N-194 to P-234; V-195 to P-234; T-196 to P-234; D-197 to P-234; I-198 to P-234; I-199 to P-234; R-200 to P-234; V-201 to P-234; P-202 to P-234; V-203 to P-234; F-204 to P-234; N-205 to P-234; I-206 to P-234; V-207 to P-234; I-208 to P-234; L-209 to P-234; L-210 to P-234; A-211 to P-234; G-212 to P-234; G-213 to P-234; F-214 to P-234; L-215 to P-234; S-216 to P-234; K-217 to P-234; S-218 to P-234; L-219 to P-234; V-220 to P-234; F-221 to P-234; S-222 to P-234; V-223 to P-234; L-224 to P-234; F-225 to P-234; A-226 to P-234; V-227 to P-234; T-228 to P-234; and/or L-229 to P-234 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
Additionally, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the following group of C-terminal deletions: M-1 to V-233; M-1 to F-232; M-1 to S-231; M-1 to R-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to A-226; M-1 to F-225; M-1 to L-224; M-1 to V-223; M-1 to S-222; M-1 to F-221; M-1 to V-220; M-1 to L-219; M-1 to S-218; M-1 to K-217; M-1 to S-216; M-1 to L-215; M-1 to F-214; M-1 to G-213; M-1 to G-212; M-1 to A-211; M-1 to L-210; M-1 to L-209; M-1 to I-208; M-1 to V-207; M-1 to I-206; M-1 to N-205; M-1 to F-204; M-1 to V-203; M-1 to P-202; M-1 to V-201; M-1 to R-200; M-1 to I-199; M-1 to I-198; M-1 to D-197; M-1 to T-196; M-1 to V-195; M-1 to N-194; M-1 to T-193; M-1 to L-192; M-1 to N-191; M-1 to I-190; M-1 to E-189; M-1 to S-188; M-1 to D-187; M-1 to P-186; M-1 to T-185; M-1 to S-184; M-1 to V-183; M-1 to D-182; M-1 to A-181; M-1 to T-180; M-1 to S-179; M-1 to K-178; M-1 to P-177; M-1 to P-176; M-1 to A-175; M-1 to Q-174; M-1 to T-173; M-1 to V-172; M-1 to T-171; M-1 to R-170; M-1 to P-169; M-1 to S-168; M-1 to T-167; M-1 to Y-166; M-1 to L-165; M-1 to P-164; M-1 to C-163; M-1 to L-162; M-1 to A-161; M-1 to K-160; M-1 to T-159; M-1 to T-158; M-1 to T-157; M-1 to P-156; M-1 to P-155; M-1 to I-154; M-1 to K-153; M-1 to Y-152; M-1 to V-151; M-1 to N-150; M-1 to Q-149; M-1 to T-148; M-1 to S-147; M-1 to N-146; M-1 to E-145; M-1 to N-144; M-1 to S-143; M-1 to G-142; M-1 to P-141; M-1 to T-140; M-1 to G-139; M-1 to S-138; M-1 to F-137; M-1 to G-136; M-1 to K-135; M-1 to T-134; M-1 to V-133; M-1 to V-132; M-1 to L-131; M-1 to R-130; M-1 to I-129; M-1 to R-128; M-1 to D-127; M-1 to F-126; M-1 to L-125; M-1 to M-124; M-1 to H-123; M-1 to P-122; M-1 to E-121; M-1 to K-120; M-1 to P-119; M-1 to P-118; M-1 to Q-117; M-1 to Y-116; M-1 to I-115; M-1 to V-114; M-1 to C-113; M-1 to Q-112; M-1 to Y-111; M-1 to L-110; M-1 to G-109; M-1 to S-108; M-1 to D-107; M-1 to E-106; M-1 to V-105; M-1 to Q-104; M-1 to L-103; M-1 to N-102; M-1 to V-101; M-1 to M-100; M-1 to R-99; M-1 to V-98; M-1 to R-97; M-1 to L-96; M-1 to L-95; M-1 to G-94; M-1 to H-93; M-1 to D-92; M-1 to H-91; M-1 to Y-90; M-1 to D-89; M-1 to E-88; M-1 to L-87; M-1 to I-86; M-1 to I-85; M-1 to R-84; M-1 to G-83; M-1 to V-82; M-1 to Q-81; M-1 to V-80; M-1 to P-79; M-1 to H-78; M-1 to S-77; M-1 to N-76; M-1 to K-75; M-1 to S-74; M-1 to P-73; M-1 to R-72; M-1 to E-71; M-1 to T-70; M-1 to C-69; M-1 to A-68; M-1 to L-67; M-1 to T-66; M-1 to K-65; M-1 to P-64; M-1 to M-63; M-1 to E-62; M-1 to G-61; M-1 to D-60; M-1 to R-59; M-1 to I-58; M-1 to I-57; M-1 to Q-56; M-1 to W-55; M-1 to A-54; M-1 to K-53; M-1 to Q-52; M-1 to S-51; M-1 to S-50; M-1 to A-49; M-1 to F-48; M-1 to K-47; M-1 to E-46; M-1 to L-45; M-1 to T-44; M-1 to Y-43; M-1 to D-42; M-1 to C-41; M-1 to K-40; M-1 to V-39; M-1 to D-38; M-1 to L-37; M-1 to T-36; M-1 to Q-35; M-1 to G-34; M-1 to E-33; M-1 to K-32; M-1 to L-31; M-1 to E-30; M-1 to Y-29; M-1 to K-28; M-1 to E-27; M-1 to E-26; M-1 to T-25; M-1 to L-24; M-1 to K-23; M-1 to T-22; M-1 to A-21; M-1 to A-20; M-1 to R-19; M-1 to L-18; M-1 to E-17; M-1 to S-16; M-1 to V-15; M-1 to F-14; M-1 to L-13; M-1 to M-12; M-1 to W-11; M-1 to L-10; M-1 to L-9; M-1 to G-8; and/or M-1 to W-7; of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein (e.g., ability to inhibit the Mixed Lymphocyte Reaction), other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example, the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIG. 1 (SEQ ID NO:2), as described by the general formula 1−n, where n is an integer from 6 to 228, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:2.
More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group of N-terminal deletions of the mature extracellular portion of the B7-H5 protein (SEQ ID NO:5): K-28 to I-198; Y-29 to I-198; E-30 to I-198; L-31 to I-198; K-32 to I-198; E-33 to I-198; G-34 to I-198; Q-35 to I-198; T-36 to I-198; L-37 to I-198; D-38 to I-198; V-39 to I-198; K-40 to I-198; C-41 to I-198; D-42 to I-198; Y-43 to I-198; T-44 to I-198; L-45 to I-198; E-46 to I-198; K-47 to I-198; F-48 to I-198; A-49 to I-198; S-50 to I-198; S-51 to I-198; Q-52 to I-198; K-53 to I-198; A-54 to I-198; W-55 to I-198; Q-56 to I-198; I-57 to I-198; I-58 to I-198; R-59 to I-198; D-60 to I-198; G-61 to I-198; E-62 to I-198; M-63 to I-198; P-64 to I-198; K-65 to I-198; T-66 to I-198; L-67 to I-198; A-68 to I-198; C-69 to I-198; T-70 to I-198; E-71 to I-198; R-72 to I-198; P-73 to I-198; S-74 to I-198; K-75 to I-198; N-76 to I-198; S-77 to I-198; H-78 to I-198; P-79 to I-198; V-80 to I-198; Q-81 to I-198; V-82 to I-198; G-83 to I-198; R-84 to I-198; I-85 to I-198; I-86 to I-198; L-87 to I-198; E-88 to I-198; D-89 to I-198; Y-90 to I-198; H-91 to I-198; D-92 to I-198; H-93 to I-198; G-94 to I-198; L-95 to I-198; L-96 to I-198; R-97 to I-198; V-98 to I-198; R-99 to I-198; M-100 to I-198; V-101 to I-198; N-102 to I-198; L-103 to I-198; Q-104 to I-198; V-105 to I-198; E-106 to I-198; D-107 to I-198; S-108 to I-198; G-109 to I-198; L-110 to I-198; Y-111 to I-198; Q-112 to I-198; C-113 to I-198; V-114 to I-198; I-115 to I-198; Y-116 to I-198; Q-117 to I-198; P-118 to I-198; P-119 to I-198; K-120 to I-198; E-121 to I-198; P-122 to I-198; H-123 to I-198; M-124 to I-198; L-125 to I-198; F-126 to I-198; D-127 to I-198; R-128 to I-198; I-129 to I-198; R-130 to I-198; L-131 to I-198; V-132 to I-198; V-133 to I-198; T-134 to I-198; K-135 to I-198; G-136 to I-198; F-137 to I-198; S-138 to I-198; G-139 to I-198; T-140 to I-198; P-141 to I-198; G-142 to I-198; S-143 to I-198; N-144 to I-198; E-145 to I-198; N-146 to I-198; S-147 to I-198; T-148 to I-198; Q-149 to I-198; N-150 to I-198; V-151 to I-198; Y-152 to I-198; K-153 to I-198; I-154 to I-198; P-155 to I-198; P-156 to I-198; T-157 to I-198; T-158 to I-198; T-159 to I-198; K-160 to I-198; A-161 to I-198; L-162 to I-198; C-163 to I-198; P-164 to I-198; L-165 to I-198; Y-166 to I-198; T-167 to I-198; S-168 to I-198; P-169 to I-198; R-170 to I-198; T-171 to I-198; V-172 to I-198; T-173 to I-198; Q-174 to I-198; A-175 to I-198; P-176 to I-198; P-177 to I-198; K-178 to I-198; S-179 to I-198; T-180 to I-198; A-181 to I-198; D-182 to I-198; V-183 to I-198; S-184 to I-198; T-185 to I-198; P-186 to I-198; D-187 to I-198; S-188 to I-198; E-189 to I-198; I-190 to I-198; N-191 to I-198; L-192 to I-198; T-193 to I-198; and/or K-23 to A-29 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
Additionally, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group of C-terminal deletions of the mature extracellular portion of the B7-H5 protein (SEQ ID NO:5): E-27 to D-197; E-27 to T-196; E-27 to V-195; E-27 to N-194; E-27 to T-193; E-27 to L-192; E-27 to N-191; E-27 to I-190; E-27 to E-189; E-27 to S-188; E-27 to D-187; E-27 to P-186; E-27 to T-185; E-27 to S-184; E-27 to V-183; E-27 to D-182; E-27 to A-181; E-27 to T-180; E-27 to S-179; E-27 to K-178; E-27 to P-177; E-27 to P-176; E-27 to A-175; E-27 to Q-174; E-27 to T-173; E-27 to V-172; E-27 to T-171; E-27 to R-170; E-27 to P-169; E-27 to S-168; E-27 to T-167; E-27 to Y-166; E-27 to L-165; E-27 to P-164; E-27 to C-163; E-27 to L-162; E-27 to A-161; E-27 to K-160; E-27 to T-159; E-27 to T-158; E-27 to T-157; E-27 to P-156; E-27 to P-155; E-27 to I-154; E-27 to K-153; E-27 to Y-152; E-27 to V-151; E-27 to N-150; E-27 to Q-149; E-27 to T-148; E-27 to S-147; E-27 to N-146; E-27 to E-145; E-27 to N-144; E-27 to S-143; E-27 to G-142; E-27 to P-141; E-27 to T-140; E-27 to G-139; E-27 to S-138; E-27 to F-137; E-27 to G-136; E-27 to K-135; E-27 to T-134; E-27 to V-133; E-27 to V-132; E-27 to L-131; E-27 to R-130; E-27 to I-129; E-27 to R-128; E-27 to D-127; E-27 to F-126; E-27 to L-125; E-27 to M-124; E-27 to H-123; E-27 to P-122; E-27 to E-121; E-27 to K-120; E-27 to P-119; E-27 to P-118; E-27 to Q-117; E-27 to Y-116; E-27 to I-115; E-27 to V-114; E-27 to C-113; E-27 to Q-112; E-27 to Y-111; E-27 to L-110; E-27 to G-109; E-27 to S-108; E-27 to D-107; E-27 to E-106; E-27 to V-105; E-27 to Q-104; E-27 to L-103; E-27 to N-102; E-27 to V-101; E-27 to M-100; E-27 to R-99; E-27 to V-98; E-27 to R-97; E-27 to L-96; E-27 to L-95; E-27 to G-94; E-27 to H-93; E-27 to D-92; E-27 to H-91; E-27 to Y-90; E-27 to D-89; E-27 to E-88; E-27 to L-87; E-27 to I-86; E-27 to I-85; E-27 to R-84; E-27 to G-83; E-27 to V-82; E-27 to Q-81; E-27 to V-80; E-27 to P-79; E-27 to H-78; E-27 to S-77; E-27 to N-76; E-27 to K-75; E-27 to S-74; E-27 to P-73; E-27 to R-72; E-27 to E-71; E-27 to T-70; E-27 to C-69; E-27 to A-68; E-27 to L-67; E-27 to T-66; E-27 to K-65; E-27 to P-64; E-27 to M-63; E-27 to E-62; E-27 to G-61; E-27 to D-60; E-27 to R-59; E-27 to I-58; E-27 to I-57; E-27 to Q-56; E-27 to W-55; E-27 to A-54; E-27 to K-53; E-27 to Q-52; E-27 to S-51; E-27 to S-50; E-27 to A-49; E-27 to F-48; E-27 to K-47; E-27 to E-46; E-27 to L-45; E-27 to T-44; E-27 to Y-43; E-27 to D-42; E-27 to C-41; E-27 to K-40; E-27 to V-39; E-27 to D-38; E-27 to L-37; E-27 to T-36; E-27 to Q-35; E-27 to G-34; and/or E-27 to E-33 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:2, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present invention is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein as m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit Nos. 97903 and/or 209049, where this portion excludes any integer of amino acid residues from 1 to about 228 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit Nos. 97903 and/or 209049, or any integer of amino acid residues from 1 to about 228 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit Nos. 97903 and/or 209049. Polypeptides encoded by these polynucleotides also are encompassed by the invention.
As described herein or otherwise known in the art, the polynucleotides of the invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of immune system tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, diseases and/or disorders involving immune system activation, stimulation and/or surveillance, particularly involving T cells and/or neutrophils. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). Particularly contemplated are the use of antibodies directed against the extracellular portion of this protein which act as antagonists for the activity of the B7-H5 protein. Such antagonistic antibodies would be useful for the prevention and/or inhibition of such biological activities as are disclosed herein (e.g. T cell modulated activities).
For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution in immune cells (e.g., T-cells, neutrophils), and the homology to members of the B7 family of ligands, indicates that the polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, detection and/or treatment of diseases and/or disorders involving immune system activation, stimulation and/or surveillance, particularly as relating to T cells and/or neutrophils. In particular, the translation product of the B7-H5 gene may be involved in the costimulation of T cells, binding to ICOS, and/or may play a role in modulation of the expression of particular cytokines. Representative uses are also described in the “Immune Activity” section below and elsewhere herein.
More generally, the tissue distribution in immune system cells indicates that this gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 888 of SEQ ID NO:1, b is an integer of 15 to 902, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 2
This gene is expressed primarily in placenta and several tumors of various tissue origin and to a lesser extent in normal tissues including liver, lung, brain, and skin,
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of cancers of all kinds. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the central nervous system, respiratory system and skin, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g., liver, lung, brain and other tissues of the nervous system, and skin, and cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
The high expression of this gene in multiple tumors indicates that the protein product of the clone may be involved in cell growth control and therefore would be useful for treatment of certain cancers. Likewise molecules developed to block the activity of the protein product of this clone could be used to block its potential role in tumor growth promotion.
This gene is believed to reside on chromosome 6. Polynucleotides related to this gene are believed, therefore, to be useful in linkage analysis for chromosome 6.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO:8 as residues: Gln-37 to Gln-43, Cys-51 to Cys-65.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:7 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1909 of SEQ ID NO:7, b is an integer of 15 to 1923, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:7, and where the b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 3
The translation product of this gene shares sequence homology with CpG islands genes which are short stretches of DNA containing a high density of non-methylated CpG dinucleotides, predominantly associated with coding regions. As CpG islands overlap with approximately 60% of human genes, the CpG island library can be used to isolate full-length cDNAs and to place genes on genomic maps. The translation product also shares distant homology with the A33 protein, which is a transmembrane protein and a member of the immunoglobulin superfamily.
This gene is expressed primarily in the testes and to a lesser extent in the lung, tonsils, placenta, and rhabdomyosarcoma.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, diseases related to the testes, lung, tonsils, placenta, and tumors. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the diseases related to the testes, lung, tonsils, placenta, and tumors, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., testes and other reproductive tissue, lung, tonsils, placenta, and striated muscle, and cancerous and wounded tissues) or bodily fluids (e.g., seminal fluid, serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO:10 as residues: Met-1 to His-7.
The tissue distribution indicates that polynucleotides and polypeptides corresponding to the gene are useful for diagnosis and treatment of diseases related to the testes, lung, tonsils, placenta, and tumors. More specifically, the tissue distribution indicates that the protein product of this clone is useful for the treatment and diagnosis of conditions concerning proper testicular function (e.g. endocrine function, sperm maturation). Therefore, this gene product is useful in the treatment of male infertility and/or impotence. This gene product is also useful in assays designed to identify binding agents as such agents (antagonists) are useful as male contraceptive agents. Similarly, the protein is believed to by useful in the treatment and/or diagnosis of testicular cancer. The testes are also a site of active gene expression of transcripts that may be expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Features of Protein Encoded by Gene No: 4
When tested against Jurkat T cells, supernatants removed from cells containing this gene activated the GAS assay. Thus, it is likely that this gene activates T cells through the Jak-STAT signal transduction pathway. The gamma activating sequence (GAS) is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
A preferred polypeptide variant of the invention comprises the following amino acid sequence:







MARGSLRRLLRLLVLGLWLALLRSVAGEQAPGTAPCSRGSSWSADLDKC


 


MDCSTSCPLPAALAHPWGRSEPDLRAGAAFWLFGLETMPQEREVHHPHR


 


GDRRRGLPSCGADPVTMCPLPAGARPLIIHSSILEPVSASQTRREPSSS


 


NHKGGGGR (SEQ ID NO: 19).






Polynucleotides encoding these polypeptides are also provided.

The gene encoding the disclosed cDNA is believed to reside on chromosome 16. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 16. This gene is expressed primarily in tumor growth factor or lipopolysaccharide treated bone marrow stroma, epitheloid sarcoma, umbilical vein endothelial cells, in keratinocytes, and to a lesser extent, in other tissues including endothelial cells and placenta.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, hematopoiesis or immune disorders including integumentary or vascular disorders, particularly impaired wound healing and autoimmune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the skin and/or hematopoietic, integumentary, or immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g. hematopoietic, immune, integumentary, endothelial, and/or cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and/or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred polypeptides of the present invention comprise immunogenic epitopes including those comprising a sequence shown in SEQ ID NOs: 14 and 12 as residues: Pro-35 to Trp-42, Ala-53 to Asp-62, Pro-65 to Asp-72, Thr-86 to Phe-93, Ile-97 to Glu-103, and Arg-103 to Pro-113. Polynucleotides encoding said polypeptides are also provided.
The tissue distribution in tumor growth factor or lipopolysaccharide treated bone marrow stroma, epitheloid sarcoma, and umbilical vein endothelial cells; and activation of the Jak-Stat promoter element in immune cells, specifically Jurkat T-cells, indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, prevention, and/or treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues.
The tissue distribution in keratinocytes indicates that the protein products of this gene are useful for the treatment of wound healing deficiency and skin disorders, including congenital disorders (i.e. nevi, moles, freckles, Mongolian spots, hemangiomas, port-wine syndrome), integumentary tumors (i.e. keratoses, Bowen's disease, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, mycosis fungoides, and Kaposi's sarcoma), injuries and inflammation of the skin (i.e. wounds, rashes, prickly heat disorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema, photosensitivity, autoimmune disorders (i.e. lupus erythematosus, vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus), keloids, striae, erythema, petechiae, purpura, and xanthelasma. Moreover, such disorders may predispose increased susceptibility to viral and bacterial infections of the skin (i.e. cold sores, warts, chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis, erysipelas, impetigo, tinea, athletes foot, and ringworm). Moreover, expression within endothelial cells and pancreatic tissues indicates that the protein product of this gene may be useful in the treatment and/or prevention of a variety of vascular disorders, particularly those involving highly vascularized tissues, which include, but are not limited to, embolism, aneurysm, stroke, atherosclerosis, and microvascular disease. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The secreted protein can be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological activities although no evidence for any is provided in the specification. Typical of these are cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency-virus infection, cancer, autoimmune diseases and allergy); regulation of haematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation of growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumours); haemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative disease; for regulation of metabolism, behaviour, and many others. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NOs: 11 and 13 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1027 of SEQ ID NO:11 or 1 to 1038 of SEQ ID NO:13, b is an integer of 15 to 1041 of SEQ ID NO:11 or 15 to 1052 of SEQ ID NO:13, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NOs: 11 and 13, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 5
In a specific embodiment, polypeptides of the invention, comprise or alternatively consist of, one or more of the following amino acid sequences:







TTILRTCTIVCFYYWFNGVMVLLFFLDRNLLTFNQASIMPFSNTDFLHC


 


LSFKKKLMLLRYIFYVVLTGPTLSLKGDENQIKNLFT (SEQ ID NO:


 


24), IVCFYYWFNGVMVLLFFLDRNLL (SEQ ID NO: 25),


 


and/or LLRYIFYVVLTGPTLSLKGDENQI (SEQ ID NO: 26).






Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also preferred are polypeptides, comprising or alternatively consisting of, the mature polypeptide which is predicted to consist of residues:







PTCYSRMRALSQEITRDFNLLQVSEPSEPCVRYLPRLYLDIHNYCVLDK


 


LRDFVASPPCWKVAQVDSLKDKARKLYTIMNSFCRRDLVFLLDDCNALE


 


YPIPVTTVLPDRQR (SEQ ID NO: 27)






of the foregoing sequence (SEQ ID NO:21), and biologically active fragments of the mature polypeptide (e.g., fragments that induce hematopoiesis). Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

FIGS. 3A-B show the nucleotide (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) corresponding to this gene.
FIG. 4 shows an analysis of the amino acid sequence (SEQ ID NO:21). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.
The data presented in FIG. 4 are also represented in tabular form in Table 10. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 4, and Table 10: “Res”: amino acid residue of SEQ ID NO:21 and FIGS. 3A-B; “Position”: position of the corresponding residue within SEQ ID NO:21 and FIGS. 3A-B; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.
Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIGS. 3A-B and/or Table 10, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 10 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in FIGS. 3A-B, but may, as shown in Table 10, be represented or identified by using tabular representations of the data presented in FIG. 4 The DNA*STAR computer algorithm used to generate FIG. 4 set on the original default parameters) was used to present the data in FIG. 4 in a tabular format (See Table 10). The tabular format of the data in FIG. 4 is used to easily determine specific boundaries of a preferred region.
The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:20, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:20. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:20. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, or from about 951 to about 985 of SEQ ID NO:20, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.
Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxyl terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m−136 where m is an integer from 2 to 136, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:21. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: R-2 to R-136; T-3 to R-136; P-4 to R-136; G-5 to R-136; P-6 to R-136; L-7 to R-136; P-8 to R-136; V-9 to R-136; L-10 to R-136; L-11 to R-136; L-12 to R-136; L-13 to R-136; L-14 to R-136; A-15 to R-136; G-16 to R-136; A-17 to R-136; P-18 to R-136; A-19 to R-136; A-20 to R-136; R-21 to R-136; P-22 to R-136; T-23 to R-136; P-24 to R-136; P-25 to R-136; T-26 to R-136; C-27 to R-136; Y-28 to R-136; S-29 to R-136; R-30 to R-136; M-31 to R-136; R-32 to R-136; A-33 to R-136; L-34 to R-136; S-35 to R-136; Q-36 to R-136; E-37 to R-136; I-38 to R-136; T-39 to R-136; R-40 to R-136; D-41 to R-136; F-42 to R-136; N-43 to R-136; L-44 to R-136; L-45 to R-136; Q-46 to R-136; V-47 to R-136; S-48 to R-136; E-49 to R-136; P-50 to R-136; S-51 to R-136; E-52 to R-136; P-53 to R-136; C-54 to R-136; V-55 to R-136; R-56 to R-136; Y-57 to R-136; L-58 to R-136; P-59 to R-136; R-60 to R-136; L-61 to R-136; Y-62 to R-136; L-63 to R-136; D-64 to R-136; I-65 to R-136; H-66 to R-136; N-67 to R-136; Y-68 to R-136; C-69 to R-136; V-70 to R-136; L-71 to R-136; D-72 to R-136; K-73 to R-136; L-74 to R-136; R-75 to R-136; D-76 to R-136; F-77 to R-136; V-78 to R-136; A-79 to R-136; S-80 to R-136; P-81 to R-136; P-82 to R-136; C-83 to R-136; W-84 to R-136; K-85 to R-136; V-86 to R-136; A-87 to R-136; Q-88 to R-136; V-89 to R-136; D-90 to R-136; S-91 to R-136; L-92 to R-136; K-93 to R-136; D-94 to R-136; K-95 to R-136; A-96 to R-136; R-97 to R-136; K-98 to R-136; L-99 to R-136; Y-100 to R-136; T-101 to R-136; I-102 to R-136; M-103 to R-136; N-104 to R-136; S-105 to R-136; F-106 to R-136; C-107 to R-136; R-108 to R-136; R-109 to R-136; D-110 to R-136; L-111 to R-136; V-112 to R-136; F-113 to R-136; L-114 to R-136; L-115 to R-136; D-116 to R-136; D-117 to R-136; C-118 to R-136; N-119 to R-136; A-120 to R-136; L-121 to R-136; E-122 to R-136; Y-123 to R-136; P-124 to R-136; I-125 to R-136; P-126 to R-136; V-127 to R-136; T-128 to R-136; T-129 to R-136; V-130 to R-136; and L-131 to R-136 of SEQ ID NO:21. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification or loss of one or more biological functions of the protein (e.g., ability to induce hematopoiesis), other functional activities (e.g., biological activities, ability to multimerize, ability to bind receptors, ability to activate receptors, ability to bind and block receptor activation, ability to inhibit receptor activation without binding (e.g., as a dominant negative inhibitor of oligomeric complexes), ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxyl terminus of the amino acid sequence of the polypeptide shown in FIGS. 3A-B (SEQ ID NO:21), as described by the general formula 1−n, where n is an integer from 6 to 135, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:21. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to Q-135; M-1 to R-134; M-1 to D-133; M-1 to P-132; M-1 to L-131; M-1 to V-130; M-1 to T-129; M-1 to T-128; M-1 to V-127; M-1 to P-126; M-1 to I-125; M-1 to P-124; M-1 to Y-123; M-1 to E-122; M-1 to L-121; M-1 to A-120; M-1 to N-119; M-1 to C-118; M-1 to D-117; M-1 to D-116; M-1 to L-115; M-1 to L-114; M-1 to F-113; M-1 to V-112; M-1 to L-111; M-1 to D-110; M-1 to R-109; M-1 to R-108; M-1 to C-107; M-1 to F-106; M-1 to S-105; M-1 to N-104; M-1 to M-103; M-1 to I-102; M-1 to T-101; M-1 to Y-100; M-1 to L-99; M-1 to K-98; M-1 to R-97; M-1 to A-96; M-1 to K-95; M-1 to D-94; M-1 to K-93; M-1 to L-92; M-1 to S-91; M-1 to D-90; M-1 to V-89; M-1 to Q-88; M-1 to A-87; M-1 to V-86; M-1 to K-85; M-1 to W-84; M-1 to C-83; M-1 to P-82; M-1 to P-81; M-1 to S-80; M-1 to A-79; M-1 to V-78; M-1 to F-77; M-1 to D-76; M-1 to R-75; M-1 to L-74; M-1 to K-73; M-1 to D-72; M-1 to L-71; M-1 to V-70; M-1 to C-69; M-1 to Y-68; M-1 to N-67; M-1 to H-66; M-1 to I-65; M-1 to D-64; M-1 to L-63; M-1 to Y-62; M-1 to L-61; M-1 to R-60; M-1 to P-59; M-1 to L-58; M-1 to Y-57; M-1 to R-56; M-1 to V-55; M-1 to C-54; M-1 to P-53; M-1 to E-52; M-1 to S-51; M-1 to P-50; M-1 to E-49; M-1 to S-48; M-1 to V-47; M-1 to Q-46; M-1 to L-45; M-1 to L-44; M-1 to N-43; M-1 to F-42; M-1 to D-41; M-1 to R-40; M-1 to T-39; M-1 to I-38; M-1 to E-37; M-1 to Q-36; M-1 to S-35; M-1 to L-34; M-1 to A-33; M-1 to R-32; M-1 to M-31; M-1 to R-30; M-1 to S-29; M-1 to Y-28; M-1 to C-27; M-1 to T-26; M-1 to P-25; M-1 to P-24; M-1 to T-23; M-1 to P-22; M-1 to R-21; M-1 to A-20; M-1 to A-19; M-1 to P-18; M-1 to A-17; M-1 to G-16; M-1 to A-15; M-1 to L-14; M-1 to L-13; M-1 to L-12; M-1 to L-11; M-1 to L-10; M-1 to V-9; M-1 to P-8; and M-1 to L-7 of SEQ ID NO:21. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:21, where n and m are integers as described above. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to A-15; R-2 to G-16; T-3 to A-17; P-4 to P-18; G-5 to A-19; P-6 to A-20; L-7 to R-21; P-8 to P-22; V-9 to T-23; L-10 to P-24; L-11 to P-25; L-12 to T-26; L-13 to C-27; L-14 to Y-28; A-15 to S-29; G-16 to R-30; A-17 to M-31; P-18 to R-32; A-19 to A-33; A-20 to L-34; R-21 to S-35; P-22 to Q-36; T-23 to E-37; P-24 to I-38; P-25 to T-39; T-26 to R-40; C-27 to D-41; Y-28 to F-42; S-29 to N-43; R-30 to L-44; M-31 to L-45; R-32 to Q-46; A-33 to V-47; L-34 to S-48; S-35 to E-49; Q-36 to P-50; E-37 to S-51; I-38 to E-52; T-39 to P-53; R-40 to C-54; D-41 to V-55; F-42 to R-56; N-43 to Y-57; L-44 to L-58; L-45 to P-59; Q-46 to R-60; V-47 to L-61; S-48 to Y-62; E-49 to L-63; P-50 to D-64; S-51 to I-65; E-52 to H-66; P-53 to N-67; C-54 to Y-68; V-55 to C-69; R-56 to V-70; Y-57 to L-71; L-58 to D-72; P-59 to K-73; R-60 to L-74; L-61 to R-75; Y-62 to D-76; L-63 to F-77; D-64 to V-78; I-65 to A-79; H-66 to S-80; N-67 to P-81; Y-68 to P-82; C-69 to C-83; V-70 to W-84; L-71 to K-85; D-72 to V-86; K-73 to A-87; L-74 to Q-88; R-75 to V-89; D-76 to D-90; F-77 to S-91; V-78 to L-92; A-79 to K-93; S-80 to D-94; P-81 to K-95; P-82 to A-96; C-83 to R-97; W-84 to K-98; K-85 to L-99; V-86 to Y-100; A-87 to T-101; Q-88 to I-102; V-89 to M-103; D-90 to N-104; S-91 to S-105; L-92 to F-106; K-93 to C-107; D-94 to R-108; K-95 to R-109; A-96 to D-110; R-97 to L-111; K-98 to V-112; L-99 to F-113; Y-100 to L-114; T-101 to L-115; I-102 to D-116; M-103 to D-117; N-104 to C-118; S-105 to N-119; F-106 to A-120; C-107 to L-121; R-108 to E-122; R-109 to Y-123; D-110 to P-124; L-111 to I-125; V-112 to P-126; F-113 to V-127; L-114 to T-128; L-115 to T-129; D-116 to V-130; D-117 to L-131; C-118 to P-132; N-119 to D-133; A-120 to R-134; L-121 to Q-135; and E-122 to R-136 of SEQ ID NO:21. Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present invention is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein as m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N and C terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97975 (deposited Apr. 4, 1997) and ATCC Deposit No. 209081 (deposited May 29, 1997), where this portion excludes any integer of amino acid residues from 1 to about 606 (end of protein minus six) amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97975 and 209081, or any integer of amino acid residues from 6 to about 612 amino acids from the carboxyl terminus, or any combination of the above amino terminal and carboxyl terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97975 and 209081. Polypeptides encoded by these polynucleotides also are encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The gene encoding the disclosed cDNA is believed to reside on chromosome 4. Accordingly, polynucleotides related to this invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis for chromosome 4.
This gene is expressed primarily in fetal liver and fetal spleen.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, hematopoietic, immunological, developmental, and/or hepatic disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and hematopoetic systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., hematopoietic, immune, hepatic, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. For example, polynucleotides and polypeptides of the invention, polynucleotide and polypeptide fragments, and polynucleotide and polypeptide variants, and antibodies directed to these polypeptides are useful for identifying, selecting, targeting and/or stimulating proliferation of hematopoietic stem cells (a.k.a., hematopoietic progenitor cells).
Cytokines typically exert their respective biochemical and physiological effects by binding to specific receptor molecules. Receptor binding then stimulates specific signal transduction pathways (Kishimoto, T., et al., Cell 76:253 262 (1994)). The specific interactions of cytokines with their receptors are often the primary regulators of a wide variety of cellular processes including activation, proliferation, and differentiation (Arai, K. I, et al., Ann. Rev. Biochem. 59:783 836 (1990); Paul, W. E. and Seder, R. A., Cell 76:241 251 (1994)).
The polynucleotides and polypeptides of this invention may be useful for the diagnosis and treatment of a variety of immune system and hematopoietic disorders, pathologies, and/or deficiencies. For example, this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Furthermore, polypeptides of this invention may be involved in the regulation of cytokine production, antigen presentation, or other processes useful for treatment of cancer, particularly leukemia (e.g., by boosting immune responses, suppressing hyperproliferative activity, or enhancing recovery of healthy hematopoietic cell populations during or following chemotherapy). Moreover, the polynucleotides and polypeptides of this invention, as well as antibodies against the polypeptides of this invention, may be useful for treating immunological and hematopoietic disorders; such as for examples, arthritis, asthma, immunodeficiency diseases (e.g. AIDS), leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the polypeptide of this invention represents a secreted factor that is likely to have activity in stimulating the differentiation of blood cells, or recruiting immune and hematopoietic cells to sites of injury. Thus, this polypeptide is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more of the immunogenic epitopes shown in SEQ ID NO:21 as residues: Met-1 to Leu-7, Pro-18 to Cys-27, Ser-29 to Ser-35, Glu-37 to Asp-41, Gln-46 to Cys-54, Asp-72 to Val-78, Pro-81 to Trp-84, Ser-91 to Lys-98, Asn-104 to Leu-111, Asp-116 to Leu-121, and Val-130 to Arg-136. Polynucleotides encoding said polypeptides are also encompassed by the invention. Antibodies that bind said epitopes or other polypeptides of the invention are also encompassed.
The tissue distribution of this gene in fetal liver and spleen indicates that the gene could be important for the treatment or detection of immune or hematopoietic disorders including arthritis, leukemia, and immunodeficiency diseases. Moreover, the protein product of this gene is useful for the treatment and diagnosis of hematopoetic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Moreover, expression within fetal tissue indicates that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus, this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 982 of SEQ ID NO:20, b is an integer of 15 to 996, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 6
The translation product of this gene is homologous to the Clostridium perfringens enterotoxin (CPE) receptor gene product and shares sequence homology with a human ORF specific to prostate and a glycoprotein specific to oligodendrocytes, both of which are tissue specific proteins. See e.g., Katahira et al. J. Cell Biol. 136(6):1239-1247 (1997). PMID: 9087440; UI: 97242441.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequences:







TMASMGLQV(SEQ ID NO: 30), KSWMMLWAVQDTGTITIRPANR


 


NTTPATIMVLALALSSSRQLVHLPPTTDSSTPRAATMMLMMTRARAAC


 


RSCGSASSESYTLHCIWPVLCTTQFIHRPSQMVCEVTMLLPMKAVTRH


 


MGSAQHSMTASQPRTASAMPITCSPMEAIVQRPRELRTWKAEGIRLWG


 


P(SEQ ID NO: 31), LQVMGIALAVLGWLAVMLCCALPMWRVT


 


(SEQ ID NO: 32), SNIVTSQTIWEGLWMNCVVQST(SEQ ID


 


NO: 33), QMQCKVYDSLLALPQDLQ (SEQ ID NO: 34), KCT


 


NCLEDESAKAKTMIV(SEQ ID NO: 35), GVVFLLAGLMVIVPVS 


 


WTAHNIIQDFYNPLVA (SEQ ID NO: 36), and/or CCNCPPR


 


TDKPY (SEQ ID NO: 37).






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 7. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 7.
This gene is expressed primarily in pancreas tumor and ulcerative colitis, and to a lesser extent in several tumors and normal tissues.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, metabolic, gastrointestinal, or proliferative disorders, such as pancreatic disorders, ulcerative colitis, tumors and food poisoning. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the digestive system or tumorigenic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., metabolic, gastrointestinal, pancreatic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Predicted epitopes include those comprising a sequence shown in SEQ ID NO:29 as residues: Gly-147 to Met-152, Cys-177 to Lys-188.
The tissue distribution in pancreas, combined with the homology to a prostate and oligodendrocyte-specific protein, indicates that the protein product of this gene is useful as a marker for the diagnosis or treatment of disorders in pancreas, ulcerative colitis, and tumors. Furthermore, identity to the human receptor for Clostridium perfringenes enterotoxin indicates that the soluble portion of this receptor could be used in the treatment of food poisoning associated with Clostridia perfringens by blocking the activity of the perfringens enterotoxin. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1691 of SEQ ID NO:28, b is an integer of 15 to 1705, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:28, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 7
The translation product of this gene shares sequence homology with alpha 3 type IX collagen, which is thought to be important in hyaline cartilage formation via its ability to uptake inorganic sulfate by cells (See Genbank Accession No. gi|975657).
In specific embodiments, polypeptides of the invention comprise, or alternatively consists of, an amino acid sequence selected from the group:







(SEQ ID NO: 40)


SLRRPRSAAXQTLTTFLSSVSSASSSALPGSREPCDPRAPPPPRSGSAAS


CCSCCCSCPRRRAPLRSPRGSKRRIRQREVVDLYNGMCLQGPAGVPGRDG


SPGANGIPGTPGIPGRDGFKGEKGECLRESFEESWTPNYKQCSWSSLNYG


IDLGKIAECTFTKMRSNSALRVLFSGSLRLKCRNACCQRWYFTFNGAECS


GPLPIEAIIYLDQGSPEMNSTINIHRTSSVEGLCEGIGAGLVDVAIWVGT


CSDYPKGDASTGWNSVSRIIIEELPK,


 


(SEQ ID NO: 41)


SLRRPRSAAXQTLTTFLSSVSSASSSALPGSREPCDPRAPPPPRSGSAAS


CCSCCCSCPRR,


 


(SEQ ID NO: 42)


RAPLRSPRGSKRRIRQREVVDLYNGMCLQGPAGVPGRDGSPGANGIPGTP


GI,


 


(SEQ ID NO: 43)


TPGIPGRDGFKGEKGECLRESFEESWTPNYKQCSWSSLNYGIDLGKIAEC


TF,


 


(SEQ ID NO: 44)


FTKMRSNSALRVLFSGSLRLKCRNACCQRWYFTFNGAECSGPLPIEAIIY


LDQGSPEMNSTINIHR,


and/or


 


(SEQ ID NO: 45)


RTSSVEGLCEGIGAGLVDVAIWVGTCSDYPKGDASTGWNSVSRIIIEELP


K.






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in smooth muscle, and to a lesser extent in synovial tissue.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias, i.e. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid and autoimmune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g. muscle, synovial tissues, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution in smooth muscle, and homology to alpha 3 type IX collagen indicates that the protein product of this gene is useful for the treatment and diagnosis of diseases associated with the mutation in this gene which leads to the many different types of chondrodysplasias. By the use of this product, the abnormal growth and development of bones of the limbs and spine could be detected or treated in utero, since the protein or polypeptides thereof could affect epithelial cells early in development, and later the chondrocytes of the developing craniofacial structure. In addition, the expression of this gene product in synovium would suggest a role in the detection and treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g. arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (i.e. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Moreover, the expression within smooth muscle indicates t that polynucleotides and polypeptides corresponding to this gene are useful for the treatment, detection, and/or prevention of a variety of vascular disorders, which include, but are not limited to, atherosclerosis, embolism, stroke, aneurysm, or microvascular disease. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:38 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1274 of SEQ ID NO:38, b is an integer of 15 to 1288, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:38, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 8
The translation product of this gene shares sequence homology with both the RIC and MAT8 proteins (mouse), which are thought to be important in regulating chloride conductance in cells by modulating the response mediated by cAMP and protein kinase C to extracellular signals.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequences:







GTSLDAAATAASLSPRGCRLRTPSSD (SEQ ID NO: 52), QIQRH


 


TRAPKQLIPLMTPRRSLRDHPQAQTSRQTPRPSSHLVFMRMTPSSMMNT


 


PSGNGGCWSQLCCSSQASSSSPVASAGSCPGYAGIIAGESIRNRS


 


(SEQ ID NO: 53), PRRSLRDHPQAQTSRQTPRPSSHLVFM (SEQ


 


ID NO: 54), THPPETGAVGRSCAVHHRHHHPHQWQVQAAVPVMPES


 


LQVSPSETGADNXLGTRRPSPLPAHRAQPPASPRRAWPEREDTDDEAGA


 


RAAGPSLLPPPTLPAPEGYLAPWGLSLKLSPLLRQKVKHCGLC (SEQ


 


ID NO: 55), PESLQVSPSETGADNXLGTRRPSPLPAHRAQPPASP


 


(SEQ ID NO: 56), GTAPKAPGSLQGRAGLGEVGDSDRQPWLQLHH


 


LCLPSLARLFEGMQEAGHGELAGGLVFGCPAGCQLLFLMDSPAMIPA


 


(SEQ ID NO: 57), GEVGDSDRQPWLQLHHLCLPSLARLFEGMQEA


 


GH (SEQ ID NO: 58), GSGGLSGRLCLGMVSQRASWCHQWDELLW


 


CSCVSLDLSLEAHPFLPVAGSGSGVVVFHQQARLGLERWAGVLCRLHLG


 


LVSGPECP (SEQ ID NO: 59), and/or QWDELLWCSCVSLDLS


 


LEAHPFLPVAGSGSGVVVFHQQARL  (SEQ ID NO: 60).






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 19. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 19.
This gene is expressed primarily in amniotic cells and hematopoietic cells including macrophages, neutrophils, T cells, TNF induced aortic endothelium, and to a lesser extent in testes, TNF induced epithelial cells, and smooth muscle.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoietic disorders, particularly inflammatory responses mediated by T cells, macrophages, and/or neutrophils, particularly those involving TNF, and also cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Predicted epitopes include those comprising a sequence shown in SEQ ID NO:47 as residues: Thr-19 to Ala-33, Leu-54 to Asp-82, Pro-89 to Ala-97, Pro-100 to Lys-125, Ser-127 to Phe-135, Gly-164 to Leu-169, Cys-173 to Arg-178.
The tissue distribution in hematopoietic cells, combined with the homology to the RIC and mat-8 genes, indicates that the protein product of this gene is useful for modifying inflammatory responses to cytokines such as TNF, and thus modifying the duration and/or severity of inflammation. Polynucleotides and polypeptides derived from this gene are thought to be useful in the diagnosis and treatment of cancer. The protein product of this gene is useful for the treatment and diagnosis of hematopoetic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:46 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 927 of SEQ ID NO:46, b is an integer of 15 to 941, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:46, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 9
In specific embodiments, polypeptides of the invention comprise, or alternatively consists of, an amino acid sequence selected from the group:







MRILQLILLALATGLVGGETRIIKGFECKLHSQPWQAALFEKTRLLCGA


 


TLIAPRWLLTAAHCLKPRYIVHLGQHNLQKEEGCEQTRTATESFPHPGF


 


NNSLPNKDHRNDIMLVKMASPVSITWAVRPLTLSSRCVTAGTSCSFPAG


 


AARPDPSYACLTPCDAPTSPSLSTRSVRTPTPATSQTPWCVPACRKGAR


 


TPARVTPGALWSVTSLFKALSPGARIRVRSPESLVSTRKSANMWTGSRR 


 


R (SEQ ID NO: 67); ETRIIKGFECKLHSQPWQAALFEKTRLLCG


 


ATLIAPRWLLTAAHCLKPRYIVHLGQHNLQKEEGCEQTRTATESFPHPG


 


FNNSLPNKDHRNDIMLVKMASPVSITWAVRPLTLSSRCVTAGTSCSFPA


 


GAARPDPSYACLTPCDAPTSPSLSTRSVRTPTPATSQTPWCVPACRKGA


 


RTPARVTPGALWSVTSLFKALSPGARIRVRSPESLVSTRKSANMWTGSR


 


RR (SEQ ID NO: 68); and/or CKLHSQPWQAALFEKTRLLCGA


 


TLIAPRWLLTAAHCLKPRYIVHLGQHNLQKEEGCEQTRTATESFPHPGF


 


NNS (SEQ ID NO: 69).






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The translation product of this gene shares sequence homology with neuropsin, a novel serine protease, which is thought to be important in modulating extracellular signaling pathways in the brain. Owing to the structural similarity to other serine proteases, the protein products of this gene are expected to have serine protease activity which may be assayed by methods known in the art and described elsewhere herein. Moreover, this protein has been shown to also have homology to PSA (prostate specific antigen). PSA is a serum marker for prostate cancer and it is a member of the kallikrein family. The members of the kallikrein family are secreted serine proteases and some of them are good tissue specific markers. This new member of the kallikrein family has been detected twice in endometrial tumor cDNA library and therefore is a good candidate as a serum marker for endometrial tumor.
This gene is expressed primarily in endometrial tumor, and to a lesser extent, in colon cancer, benign hypertrophic prostate, and thymus.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive, immune, or endocrine disorders, particularly cancers of the endometrium or colon and benign hypertrophy of the prostate. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the urogenital or reproductive systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, immune, endocrine, gastrointestinal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, seminal fluid, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Predicted epitopes include those comprising a sequence shown in SEQ ID NO:62 as residues: Glu-27 to Trp-35, Leu-77 to Ala-89, Pro-96 to Asn-109, Ser-149 to Arg-156, Gln-172 to Ile-182, Glu-193 to Gly-204, Glu-245 to Asn-250.
The tissue distribution in proliferative reproductive tissues, combined with the homology to serine proteases indicates that the protein product of this gene is useful for diagnosing, treating, and/or preventing hyperproliferative disorders such as cancer of the endometrium or colon and hyperplasia of the prostate. Similarly, expression within cellular sources marked by proliferating cells indicates that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:61 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1278 of SEQ ID NO:61, b is an integer of 15 to 1292, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:61, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 10
The translation product of this gene shares sequence homology with tenascin, which is thought to be important in development. The translation product of this gene is believed to be a ligand of the fibroblast growth factor family. FGF ligand activity is known in the art and can be assayed by methods known in the art and disclosed elsewhere herein.
Northern analysis indicates that a 2.5 kb band is expressed in brain and lung. It has also been discovered that this gene is expressed in endometrial tumor, synovial sarcoma, pancreas tumor, fetal lung, retinal, and immune tissues (e.g., bone marrow)
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, cancers, growth disorders of the brain and lung. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the cancer tissues, brain, lung, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g. brain, lung, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Predicted epitopes include those comprising a sequence shown in SEQ ID NO:71 as residues: Gly-29 to Glu-34, Arg-71 to Arg-76, Thr-176 to Cys-182, Gly-184 to Glu-199. As a preferred embodiment, antibodies that bind said epitopes are encompassed by the invention and may be useful as a cancer diagnostic and/or an agonist/antagonist of the polypeptides of the invention.
Fragments and variants of the polypeptide encoded by this gene (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention). Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention. Antibodies that bind polypeptides of the invention would be useful as a cancer diagnostic.
Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m−379 where m is an integer from 2 to 371, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:71. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: A-2 to W-379; R-3 to W-379; R-4 to W-379; S-5 to W-379; A-6 to W-379; F-7 to W-379; P-8 to W-379; A-9 to W-379; A-10 to W-379; A-11 to W-379; L-12 to W-379; W-13 to W-379; L-14 to W-379; W-15 to W-379; S-16 to W-379; I-17 to W-379; L-18 to W-379; L-19 to W-379; C-20 to W-379; L-21 to W-379; L-22 to W-379; A-23 to W-379; L-24 to W-379; R-25 to W-379; A-26 to W-379; E-27 to W-379; A-28 to W-379; G-29 to W-379; P-30 to W-379; P-31 to W-379; Q-32 to W-379; E-33 to W-379; E-34 to W-379; S-35 to W-379; L-36 to W-379; Y-37 to W-379; L-38 to W-379; W-39 to W-379; I-40 to W-379; D-41 to W-379; A-42 to W-379; H-43 to W-379; Q-44 to W-379; A-45 to W-379; R-46 to W-379; V-47 to W-379; L-48 to W-379; I-49 to W-379; G-50 to W-379; F-51 to W-379; E-52 to W-379; E-53 to W-379; D-54 to W-379; I-55 to W-379; L-56 to W-379; I-57 to W-379; V-58 to W-379; S-59 to W-379; E-60 to W-379; G-61 to W-379; K-62 to W-379; M-63 to W-379; A-64 to W-379; P-65 to W-379; F-66 to W-379; T-67 to W-379; H-68 to W-379; D-69 to W-379; F-70 to W-379; R-71 to W-379; K-72 to W-379; A-73 to W-379; Q-74 to W-379; Q-75 to W-379; R-76 to W-379; M-77 to W-379; P-78 to W-379; A-79 to W-379; I-80 to W-379; P-81 to W-379; V-82 to W-379; N-83 to W-379; I-84 to W-379; H-85 to W-379; S-86 to W-379; M-87 to W-379; N-88 to W-379; F-89 to W-379; T-90 to W-379; W-91 to W-379; Q-92 to W-379; A-93 to W-379; A-94 to W-379; G-95 to W-379; Q-96 to W-379; A-97 to W-379; E-98 to W-379; Y-99 to W-379; F-100 to W-379; Y-101 to W-379; E-102 to W-379; F-103 to W-379; L-104 to W-379; S-105 to W-379; L-106 to W-379; R-107 to W-379; S-108 to W-379; L-109 to W-379; D-110 to W-379; K-111 to W-379; G-112 to W-379; I-113 to W-379; M-114 to W-379; A-115 to W-379; D-116 to W-379; P-117 to W-379; T-118 to W-379; V-119 to W-379; N-120 to W-379; V-121 to W-379; P-122 to W-379; L-123 to W-379; L-124 to W-379; G-125 to W-379; T-126 to W-379; V-127 to W-379; P-128 to W-379; H-129 to W-379; K-130 to W-379; A-131 to W-379; S-132 to W-379; V-133 to W-379; V-134 to W-379; Q-135 to W-379; V-136 to W-379; G-137 to W-379; F-138 to W-379; P-139 to W-379; C-140 to W-379; L-141 to W-379; G-142 to W-379; K-143 to W-379; Q-144 to W-379; D-145 to W-379; G-146 to W-379; V-147 to W-379; A-148 to W-379; A-149 to W-379; F-150 to W-379; E-151 to W-379; V-152 to W-379; D-153 to W-379; V-154 to W-379; I-155 to W-379; V-156 to W-379; M-157 to W-379; N-158 to W-379; S-159 to W-379; E-160 to W-379; G-161 to W-379; N-162 to W-379; T-163 to W-379; I-164 to W-379; L-165 to W-379; Q-166 to W-379; T-167 to W-379; P-168 to W-379; Q-169 to W-379; N-170 to W-379; A-171 to W-379; I-172 to W-379; F-173 to W-379; F-174 to W-379; K-175 to W-379; T-176 to W-379; C-177 to W-379; Q-178 to W-379; Q-179 to W-379; A-180 to W-379; E-181 to W-379; C-182 to W-379; P-183 to W-379; G-184 to W-379; G-185 to W-379; C-186 to W-379; R-187 to W-379; N-188 to W-379; G-189 to W-379; G-190 to W-379; F-191 to W-379; C-192 to W-379; N-193 to W-379; E-194 to W-379; R-195 to W-379; R-196 to W-379; I-197 to W-379; C-198 to W-379; E-199 to W-379; C-200 to W-379; P-201 to W-379; D-202 to W-379; G-203 to W-379; F-204 to W-379; H-205 to W-379; G-206 to W-379; P-207 to W-379; H-208 to W-379; C-209 to W-379; E-210 to W-379; K-211 to W-379; A-212 to W-379; L-213 to W-379; C-214 to W-379; T-215 to W-379; P-216 to W-379; R-217 to W-379; C-218 to W-379; M-219 to W-379; N-220 to W-379; G-221 to W-379; G-222 to W-379; L-223 to W-379; C-224 to W-379; V-225 to W-379; T-226 to W-379; P-227 to W-379; G-228 to W-379; F-229 to W-379; C-230 to W-379; I-231 to W-379; C-232 to W-379; P-233 to W-379; P-234 to W-379; G-235 to W-379; F-236 to W-379; Y-237 to W-379; G-238 to W-379; V-239 to W-379; N-240 to W-379; C-241 to W-379; D-242 to W-379; K-243 to W-379; A-244 to W-379; N-245 to W-379; C-246 to W-379; S-247 to W-379; T-248 to W-379; T-249 to W-379; C-250 to W-379; F-251 to W-379; N-252 to W-379; G-253 to W-379; G-254 to W-379; T-255 to W-379; C-256 to W-379; F-257 to W-379; Y-258 to W-379; P-259 to W-379; G-260 to W-379; K-261 to W-379; C-262 to W-379; I-263 to W-379; C-264 to W-379; P-265 to W-379; P-266 to W-379; G-267 to W-379; L-268 to W-379; E-269 to W-379; G-270 to W-379; E-271 to W-379; Q-272 to W-379; C-273 to W-379; E-274 to W-379; I-275 to W-379; S-276 to W-379; K-277 to W-379; C-278 to W-379; P-279 to W-379; Q-280 to W-379; P-281 to W-379; C-282 to W-379; R-283 to W-379; N-284 to W-379; G-285 to W-379; G-286 to W-379; K-287 to W-379; C-288 to W-379; I-289 to W-379; G-290 to W-379; K-291 to W-379; S-292 to W-379; K-293 to W-379; C-294 to W-379; K-295 to W-379; C-296 to W-379; S-297 to W-379; K-298 to W-379; G-299 to W-379; Y-300 to W-379; Q-301 to W-379; G-302 to W-379; D-303 to W-379; L-304 to W-379; C-305 to W-379; S-306 to W-379; K-307 to W-379; P-308 to W-379; V-309 to W-379; C-310 to W-379; E-311 to W-379; P-312 to W-379; G-313 to W-379; C-314 to W-379; G-315 to W-379; A-316 to W-379; H-317 to W-379; G-318 to W-379; T-319 to W-379; C-320 to W-379; H-321 to W-379; E-322 to W-379; P-323 to W-379; N-324 to W-379; K-325 to W-379; C-326 to W-379; Q-327 to W-379; C-328 to W-379; Q-329 to W-379; E-330 to W-379; G-331 to W-379; W-332 to W-379; H-333 to W-379; G-334 to W-379; R-335 to W-379; H-336 to W-379; C-337 to W-379; N-338 to W-379; K-339 to W-379; R-340 to W-379; Y-341 to W-379; E-342 to W-379; A-343 to W-379; S-344 to W-379; L-345 to W-379; I-346 to W-379; H-347 to W-379; A-348 to W-379; L-349 to W-379; R-350 to W-379; P-351 to W-379; A-352 to W-379; G-353 to W-379; A-354 to W-379; Q-355 to W-379; L-356 to W-379; R-357 to W-379; Q-358 to W-379; H-359 to W-379; T-360 to W-379; P-361 to W-379; S-362 to W-379; L-363 to W-379; K-364 to W-379; K-365 to W-379; A-366 to W-379; E-367 to W-379; E-368 to W-379; R-369 to W-379; R-370 to W-379; D-371 to W-379; P-372 to W-379; P-373 to W-379; and E-374 to W-379 of SEQ ID NO:71. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in SEQ ID NO:71, as described by the general formula 1−n, where n is an integer from 6 to 378, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:71. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to I-378; M-1 to Y-377; M-1 to N-376; M-1 to S-375; M-1 to E-374; M-1 to P-373; M-1 to P-372; M-1 to D-371; M-1 to R-370; M-1 to R-369; M-1 to E-368; M-1 to E-367; M-1 to A-366; M-1 to K-365; M-1 to K-364; M-1 to L-363; M-1 to S-362; M-1 to P-361; M-1 to T-360; M-1 to H-359; M-1 to Q-358; M-1 to R-357; M-1 to L-356; M-1 to Q-355; M-1 to A-354; M-1 to G-353; M-1 to A-352; M-1 to P-351; M-1 to R-350; M-1 to L-349; M-1 to A-348; M-1 to H-347; M-1 to I-346; M-1 to L-345; M-1 to S-344; M-1 to A-343; M-1 to E-342; M-1 to Y-341; M-1 to R-340; M-1 to K-339; M-1 to N-338; M-1 to C-337; M-1 to H-336; M-1 to R-335; M-1 to G-334; M-1 to H-333; M-1 to W-332; M-1 to G-331; M-1 to E-330; M-1 to Q-329; M-1 to C-328; M-1 to Q-327; M-1 to C-326; M-1 to K-325; M-1 to N-324; M-1 to P-323; M-1 to E-322; M-1 to H-321; M-1 to C-320; M-1 to T-319; M-1 to G-318; M-1 to H-317; M-1 to A-316; M-1 to G-315; M-1 to C-314; M-1 to G-313; M-1 to P-312; M-1 to E-311; M-1 to C-310; M-1 to V-309; M-1 to P-308; M-1 to K-307; M-1 to S-306; M-1 to C-305; M-1 to L-304; M-1 to D-303; M-1 to G-302; M-1 to Q-301; M-1 to Y-300; M-1 to G-299; M-1 to K-298; M-1 to S-297; M-1 to C-296; M-1 to K-295; M-1 to C-294; M-1 to K-293; M-1 to S-292; M-1 to K-291; M-1 to G-290; M-1 to I-289; M-1 to C-288; M-1 to K-287; M-1 to G-286; M-1 to G-285; M-1 to N-284; M-1 to R-283; M-1 to C-282; M-1 to P-281; M-1 to Q-280; M-1 to P-279; M-1 to C-278; M-1 to K-277; M-1 to S-276; M-1 to I-275; M-1 to E-274; M-1 to C-273; M-1 to Q-272; M-1 to E-271; M-1 to G-270; M-1 to E-269; M-1 to L-268; M-1 to G-267; M-1 to P-266; M-1 to P-265; M-1 to C-264; M-1 to I-263; M-1 to C-262; M-1 to K-261; M-1 to G-260; M-1 to P-259; M-1 to Y-258; M-1 to F-257; M-1 to C-256; M-1 to T-255; M-1 to G-254; M-1 to G-253; M-1 to N-252; M-1 to F-251; M-1 to C-250; M-1 to T-249; M-1 to T-248; M-1 to S-247; M-1 to C-246; M-1 to N-245; M-1 to A-244; M-1 to K-243; M-1 to D-242; M-1 to C-241; M-1 to N-240; M-1 to V-239; M-1 to G-238; M-1 to Y-237; M-1 to F-236; M-1 to G-235; M-1 to P-234; M-1 to P-233; M-1 to C-232; M-1 to I-231; M-1 to C-230; M-1 to F-229; M-1 to G-228; M-1 to P-227; M-1 to T-226; M-1 to V-225; M-1 to C-224; M-1 to L-223; M-1 to G-222; M-1 to G-221; M-1 to N-220; M-1 to M-219; M-1 to C-218; M-1 to R-217; M-1 to P-216; M-1 to T-215; M-1 to C-214; M-1 to L-213; M-1 to A-212; M-1 to K-211; M-1 to E-210; M-1 to C-209; M-1 to H-208; M-1 to P-207; M-1 to G-206; M-1 to H-205; M-1 to F-204; M-1 to G-203; M-1 to D-202; M-1 to P-201; M-1 to C-200; M-1 to E-199; M-1 to C-198; M-1 to I-197; M-1 to R-196; M-1 to R-195; M-1 to E-194; M-1 to N-193; M-1 to C-192; M-1 to F-191; M-1 to G-190; M-1 to G-189; M-1 to N-188; M-1 to R-187; M-1 to C-186; M-1 to G-185; M-1 to G-184; M-1 to P-183; M-1 to C-182; M-1 to E-181; M-1 to A-180; M-1 to Q-179; M-1 to Q-178; M-1 to C-177; M-1 to T-176; M-1 to K-175; M-1 to F-174; M-1 to F-173; M-1 to I-172; M-1 to A-171; M-1 to N-170; M-1 to Q-169; M-1 to P-168; M-1 to T-167; M-1 to Q-166; M-1 to L-165; M-1 to I-164; M-1 to T-163; M-1 to N-162; M-1 to G-161; M-1 to E-160; M-1 to S-159; M-1 to N-158; M-1 to M-157; M-1 to V-156; M-1 to I-155; M-1 to V-154; M-1 to D-153; M-1 to V-152; M-1 to E-151; M-1 to F-150; M-1 to A-149; M-1 to A-148; M-1 to V-147; M-1 to G-146; M-1 to D-145; M-1 to Q-144; M-1 to K-143; M-1 to G-142; M-1 to L-141; M-1 to C-140; M-1 to P-139; M-1 to F-138; M-1 to G-137; M-1 to V-136; M-1 to Q-135; M-1 to V-134; M-1 to V-133; M-1 to S-132; M-1 to A-131; M-1 to K-130; M-1 to H-129; M-1 to P-128; M-1 to V-127; M-1 to T-126; M-1 to G-125; M-1 to L-124; M-1 to L-123; M-1 to P-122; M-1 to V-121; M-1 to N-120; M-1 to V-119; M-1 to T-118; M-1 to P-117; M-1 to D-116; M-1 to A-115; M-1 to M-114; M-1 to I-113; M-1 to G-112; M-1 to K-111; M-1 to D-110; M-1 to L-109; M-1 to S-108; M-1 to R-107; M-1 to L-106; M-1 to S-105; M-1 to L-104; M-1 to F-103; M-1 to E-102; M-1 to Y-101; M-1 to F-100; M-1 to Y-99; M-1 to E-98; M-1 to A-97; M-1 to Q-96; M-1 to G-95; M-1 to A-94; M-1 to A-93; M-1 to Q-92; M-1 to W-91; M-1 to T-90; M-1 to F-89; M-1 to N-88; M-1 to M-87; M-1 to S-86; M-1 to H-85; M-1 to I-84; M-1 to N-83; M-1 to V-82; M-1 to P-81; M-1 to I-80; M-1 to A-79; M-1 to P-78; M-1 to M-77; M-1 to R-76; M-1 to Q-75; M-1 to Q-74; M-1 to A-73; M-1 to K-72; M-1 to R-71; M-1 to F-70; M-1 to D-69; M-1 to H-68; M-1 to T-67; M-1 to F-66; M-1 to P-65; M-1 to A-64; M-1 to M-63; M-1 to K-62; M-1 to G-61; M-1 to E-60; M-1 to S-59; M-1 to V-58; M-1 to I-57; M-1 to L-56; M-1 to I-55; M-1 to D-54; M-1 to E-53; M-1 to E-52; M-1 to F-51; M-1 to G-50; M-1 to I-49; M-1 to L-48; M-1 to V-47; M-1 to R-46; M-1 to A-45; M-1 to Q-44; M-1 to H-43; M-1 to A-42; M-1 to D-41; M-1 to I-40; M-1 to W-39; M-1 to L-38; M-1 to Y-37; M-1 to L-36; M-1 to S-35; M-1 to E-34; M-1 to E-33; M-1 to Q-32; M-1 to P-31; M-1 to P-30; M-1 to G-29; M-1 to A-28; M-1 to E-27; M-1 to A-26; M-1 to R-25; M-1 to L-24; M-1 to A-23; M-1 to L-22; M-1 to L-21; M-1 to C-20; M-1 to L-19; M-1 to L-18; M-1 to I-17; M-1 to S-16; M-1 to W-15; M-1 to L-14; M-1 to W-13; M-1 to L-12; M-1 to A-11; M-1 to A-10; M-1 to A-9; M-1 to P-8; M-1 to F-7; and M-1 to A-6 of SEQ ID NO:71. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The tissue distribution in brain and lung, combined with the homology to tenascin indicates that the protein product of this gene is useful for diagnosis and treatment of cancers. Alternatively, given the tissue distribution indicated by Northern analysis, the translation product of this gene is thought to be a growth factor functioning in the brain and lung that may be useful in treating neurodegeneration and lung disorder. For example, the protein product of this gene is useful for the detection and treatment of disorders associated with developing lungs, particularly in premature infants where the lungs are the last tissues to develop. Furthermore, the tissue distribution indicates that the protein product of this gene is useful for the diagnosis and intervention of lung tumors, since the gene may be involved in the regulation of cell division. Additionally, expression in the brain indicates that it may be involved in neuronal survival; synapse formation; conductance; neural differentiation, etc. Such involvement may impact many processes, such as learning and cognition. It may also be useful in the treatment of such neurodegenerative disorders as schizophrenia; ALS; or Alzheimer's.
Polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
Protein, as well as, antibodies directed against the protein may show utility as a tissue-specific marker and/or immunotherapy target for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:70 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1960 of SEQ ID NO:70, b is an integer of 15 to 1974, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:70, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 11
The translation product of this gene shares sequence homology with human squamous cell E48 antigen which is thought to be important in self-recognition and immune function.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:







(SEQ ID NO: 80)


MMATPSTRPPPPAASTTSATAPALPPRPPWPWPPSSWPPSGVSSKAPEAD


PLKNKALID;


 


(SEQ ID NO: 81)


LLLTSPLPRCPPACSHDAPAHPDPGGPHGLTSGPGLGLPRVCLQRRQLLQ


PHALPGYGCLLHDHAHLLHPHQDEGQ;


and/or


 


(SEQ ID NO: 82)


WLLQARVHHLLLPVRPLQRHRPCHPGHPGPGPHPPGHPLGSPLKPPRQTH


SRTKLS.






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention and polynucleotides encoding these polypeptides are also encompassed by the invention.
When tested against K562 leukemia cell lines, supernatants removed from cells containing this gene activated the ISRE assay. Thus, it is likely that this gene activates leukemia cells through the Jak-STAT signal transduction pathway. The interferon-sensitive response element is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
This gene is expressed primarily in adult brain, and to a lesser extent in fetal lung.
Polynucleotides and polypeptides of the invention would be useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, autoimmune disorders. Similarly, polypeptides and antibodies directed to these polypeptides would be useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise, or alternatively consist of one, two or all three of the immunogenic epitopes shown in SEQ ID NO:77 as residues: Tyr-28 to Phe-34, Thr-54 to Val-60, Tyr-73 to Thr-82. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution and homology to human squamous cell E48 antigen indicates that polynucleotides and polypeptides corresponding to this gene would be useful for study, diagnosis, detection, prevention and/or treatment of autoimmune diseases and disorders, such as lupus, transplant rejection, allergic reactions, and arthritis. Protein, as well as, antibodies directed against the protein may show utility as a tissue-specific marker and/or immunotherapy target for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:76 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 599 of SEQ ID NO:76, b is an integer of 15 to 613, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:76, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 12
The gene encoding the disclosed cDNA is thought to reside on chromosome 19. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 19.
The translation product of this gene is a transmembrane protein that forms disulfide-bonded homodimers and contains a motif in its cytoplasmic domain (located at the carboxy terminus of the protein relative to the transmembrane domain) that functions as an adaptor for associating protein complexes involved in triggering cellular activation. The transmembrane domain is predicted to consist of the amino acid sequence:









VLAGIVMGDLVLTVLIALAVYFLG (SEQ ID NO: 85).






In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:







QAQSDCSCSTVSPG (SEQ ID NO: 86), VLAGIVMGDLVLTVLIA


 


LAVYFLG (SEQ ID NO: 87), VPRGRGAAEATRKQRITETESPYQ


 


ELQGQRSDVYSDL(SEQ ID NO: 88), and/or ETESPYQELQGQ


 


RSDVYSDLNT (SEQ ID NO: 89).






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention and polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in macrophage, and to a lesser extent in primary dendritic cells and neutrophils.
Polynucleotides and polypeptides of the invention would be useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immunologically mediated disorders. Similarly, polypeptides and antibodies directed to these polypeptides would be useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types and cell types (e.g., blood cells, and cells and tissue of the immune system, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise, or alternatively consist of one or both of the immunogenic epitopes shown in SEQ ID NO:84 as residues: Ala-28 to Ser-33, Ala-76 to Lys-111. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in immune tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g., AIDS), immuno-suppressive conditions (transplantation) and hematopoietic disorders. Furthermore, expression of this gene product in macrophage and primary dendritic cells also strongly indicates a role for this protein in immune function and immune surveillance. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:83 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 683 of SEQ ID NO:83, b is an integer of 15 to 697, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:83, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 13
The protein product of this gene was found to have homology to the human CD84 protein which, as a novel member of the Ig superfamily, is thought to play an important role in the modulation of the immune response. The present invention appears to encode a novel full-length CD84 homolog and is highly enriched, if not specific, for activated T cells.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:







ITPLGLGAAD (SEQ ID NO: 96), TLRVLGKVPAVCPWCALWRKA


 


GMDMTYSWLSRGDSTYTFHEGPVLSTSWRPGDSALSYTCRANNPISNVS


 


SCPIPDGPFYADPNYASEKPSTAFCLLAKGLLIFLLLVILAMGLWVIRV


 


QKRHKMPRMKKLMRNRMKLRKEAKPGSSPA (SEQ ID NO: 97), A


 


VCPWCALWRKAGMDMTYSWL (SEQ ID NO: 98), PGDSALSYTCR


 


ANNPISNVSSCPI (SEQ ID NO: 99), YASEKPSTAFCLLAKGLL


 


IFLLLV (SEQ ID NO: 100), and/or QKRHKMPRMKKLMRNRM


 


KLRKEAKPG (SEQ ID NO: 101).






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention and polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in human activated T-Cells.
Polynucleotides and polypeptides of the invention would be useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immunodeficiencies, inflammatory conditions, infections, and other immune or hematopoietic disorders. Similarly, polypeptides and antibodies directed to these polypeptides would be useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the disorders of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise, or alternatively consist of one or both of the immunogenic epitopes shown in SEQ ID NO:91 as residues: Glu-15 to Arg-23, Asn-79 to Gly-84. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in activated T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, treatment, and/or prevention of a variety of immune system disorders. Expression of this gene product in T-cells indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also indicate a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, and leukemia. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tumors and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:90 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1126 of SEQ ID NO:90, b is an integer of 15 to 1140, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:90, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 14
Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more of the immunogenic epitopes shown in SEQ ID NO:103 as residues: Val-54 to Asp-59, Thr-55 to Leu-60 and Trp-98 to Cys-104. Polynucleotides encoding said polypeptides are also encompassed by the invention. In a specific embodiment, antibodies that bind said epitopes or other polypeptides of the invention are also encompassed by the invention.
In a specific embodiment, polypeptides of the invention, comprise or alternatively consist of, the following amino acid sequences:









LSPPRGACR (SEQ ID NO: 106).






Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also preferred are polypeptides, comprising or alternatively consisting of, the mature polypeptide which is predicted to consist of residues 23-108 of the foregoing sequence (SEQ ID NO:103), and biologically active fragments of the mature polypeptide. Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
FIG. 5 show the nucleotide (SEQ ID NO:102) and deduced amino acid sequence (SEQ ID NO:103) corresponding to this gene.
FIG. 6 shows an analysis of the amino acid sequence (SEQ ID NO:103). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.
The data presented in FIG. 6 are also represented in tabular form in Table 11. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 6, and Table 11: “Res”: amino acid residue of SEQ ID NO:103 and FIG. 5; “Position”: position of the corresponding residue within SEQ ID NO:103 and FIG. 5; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.
Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 6 and/or Table 11, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 11 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in FIG. 6, but may, as shown in Table 11, be represented or identified by using tabular representations of the data presented in FIG. 6. The DNA*STAR computer algorithm used to generate FIG. 6 (set on the original default parameters) was used to present the data in FIG. 6 in a tabular format (See Table 11). The tabular format of the data in FIG. 6 is used to easily determine specific boundaries of a preferred region.
The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:102, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:102. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:102. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 553 of SEQ ID NO:102, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.
Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m−108 where m is an integer from 2 to 102, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:103. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: K-2 to P-108; A-3 to P-108; L-4 to P-108; C-5 to P-108; L-6 to P-108; L-7 to P-108; L-8 to P-108; L-9 to P-108; P-10 to P-108; V-11 to P-108; L-12 to P-108; G-13 to P-108; L-14 to P-108; L-15 to P-108; V-16 to P-108; S-17 to P-108; S-18 to P-108; K-19 to P-108; T-20 to P-108; L-21 to P-108; C-22 to P-108; S-23 to P-108; M-24 to P-108; E-25 to P-108; E-26 to P-108; A-27 to P-108; I-28 to P-108; N-29 to P-108; E-30 to P-108; R-31 to P-108; I-32 to P-108; Q-33 to P-108; E-34 to P-108; V-35 to P-108; A-36 to P-108; G-37 to P-108; S-38 to P-108; L-39 to P-108; I-40 to P-108; F-41 to P-108; R-42 to P-108; A-43 to P-108; I-44 to P-108; S-45 to P-108; S-46 to P-108; I-47 to P-108; G-48 to P-108; L-49 to P-108; E-50 to P-108; C-51 to P-108; Q-52 to P-108; S-53 to P-108; V-54 to P-108; T-55 to P-108; S-56 to P-108; R-57 to P-108; G-58 to P-108; D-59 to P-108; L-60 to P-108; A-61 to P-108; T-62 to P-108; C-63 to P-108; P-64 to P-108; R-65 to P-108; G-66 to P-108; F-67 to P-108; A-68 to P-108; V-69 to P-108; T-70 to P-108; G-71 to P-108; C-72 to P-108; T-73 to P-108; C-74 to P-108; G-75 to P-108; S-76 to P-108; A-77 to P-108; C-78 to P-108; G-79 to P-108; S-80 to P-108; W-81 to P-108; D-82 to P-108; V-83 to P-108; R-84 to P-108; A-85 to P-108; E-86 to P-108; T-87 to P-108; T-88 to P-108; C-89 to P-108; H-90 to P-108; C-91 to P-108; Q-92 to P-108; C-93 to P-108; A-94 to P-108; G-95 to P-108; M-96 to P-108; D-97 to P-108; W-98 to P-108; T-99 to P-108; G-100 to P-108; A-101 to P-108; R-102 to P-108; and C-103 to P-108 of SEQ ID NO:103. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIG. 5 (SEQ ID NO:103), as described by the general formula 1−n, where n is an integer from 6 to 107, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:103. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to Q-107; M-1 to V-106; M-1 to R-105; M-1 to C-104; M-1 to C-103; M-1 to R-102; M-1 to A-101; M-1 to G-100; M-1 to T-99; M-1 to W-98; M-1 to D-97; M-1 to M-96; M-1 to G-95; M-1 to A-94; M-1 to C-93; M-1 to Q-92; M-1 to C-91; M-1 to H-90; M-1 to C-89; M-1 to T-88; M-1 to T-87; M-1 to E-86; M-1 to A-85; M-1 to R-84; M-1 to V-83; M-1 to D-82; M-1 to W-81; M-1 to S-80; M-1 to G-79; M-1 to C-78; M-1 to A-77; M-1 to S-76; M-1 to G-75; M-1 to C-74; M-1 to T-73; M-1 to C-72; M-1 to G-71; M-1 to T-70; M-1 to V-69; M-1 to A-68; M-1 to F-67; M-1 to G-66; M-1 to R-65; M-1 to P-64; M-1 to C-63; M-1 to T-62; M-1 to A-61; M-1 to L-60; M-1 to D-59; M-1 to G-58; M-1 to R-57; M-1 to S-56; M-1 to T-55; M-1 to V-54; M-1 to S-53; M-1 to Q-52; M-1 to C-51; M-1 to E-50; M-1 to L-49; M-1 to G-48; M-1 to I-47; M-1 to S-46; M-1 to S-45; M-1 to I-44; M-1 to A-43; M-1 to R-42; M-1 to F-41; M-1 to I-40; M-1 to L-39; M-1 to S-38; M-1 to G-37; M-1 to A-36; M-1 to V-35; M-1 to E-34; M-1 to Q-33; M-1 to I-32; M-1 to R-31; M-1 to E-30; M-1 to N-29; M-1 to I-28; M-1 to A-27; M-1 to E-26; M-1 to E-25; M-1 to M-24; M-1 to S-23; M-1 to C-22; M-1 to L-21; M-1 to T-20; M-1 to K-19; M-1 to S-18; M-1 to S-17; M-1 to V-16; M-1 to L-15; M-1 to L-14; M-1 to G-13; M-1 to L-12; M-1 to V-11; M-1 to P-10; M-1 to L-9; M-1 to L-8; M-1 to L-7; and M-1 to L-6; of SEQ ID NO:103. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:103, where n and m are integers as described above.
Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCCTM Deposit No. 209215, where this portion excludes any integer of amino acid residues from 1 to about 102 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCCTM Deposit No. 209215, or any integer of amino acid residues from 6 to about 108 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCCTM Deposit No. 209215. Polypeptides encoded by these polynucleotides also are encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Additional preferred polypeptide fragments of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: M-1 to L-15; K-2 to V-16; A-3 to S-17; L-4 to S-18; C-5 to K-19; L-6 to T-20; L-7 to L-21; L-8 to C-22; L-9 to S-23; P-10 to M-24; V-11 to E-25; L-12 to E-26; G-13 to A-27; L-14 to I-28; L-15 to N-29; V-16 to E-30; S-17 to R-31; S-18 to I-32; K-19 to Q-33; T-20 to E-34; L-21 to V-35; C-22 to A-36; S-23 to G-37; M-24 to S-38; E-25 to L-39; E-26 to I-40; A-27 to F-41; I-28 to R-42; N-29 to A-43; E-30 to I-44; R-31 to S-45; I-32 to S-46; Q-33 to I-47; E-34 to G-48; V-35 to L-49; A-36 to E-50; G-37 to C-51; S-38 to Q-52; L-39 to S-53; I-40 to V-54; F-41 to T-55; R-42 to S-56; A-43 to R-57; I-44 to G-58; S-45 to D-59; S-46 to L-60; I-47 to A-61; G-48 to T-62; L-49 to C-63; E-50 to P-64; C-51 to R-65; Q-52 to G-66; S-53 to F-67; V-54 to A-68; T-55 to V-69; S-56 to T-70; R-57 to G-71; G-58 to C-72; D-59 to T-73; L-60 to C-74; A-61 to G-75; T-62 to S-76; C-63 to A-77; P-64 to C-78; R-65 to G-79; G-66 to S-80; F-67 to W-81; A-68 to D-82; V-69 to V-83; T-70 to R-84; G-71 to A-85; C-72 to E-86; T-73 to T-87; C-74 to T-88; G-75 to C-89; S-76 to H-90; A-77 to C-91; C-78 to Q-92; G-79 to C-93; S-80 to A-94; W-81 to G-95; D-82 to M-96; V-83 to D-97; R-84 to W-98; A-85 to T-99; E-86 to G-100; T-87 to A-101; T-88 to R-102; C-89 to C-103; H-90 to C-104; C-91 to R-105; Q-92 to V-106; C-93 to Q-107; and A-94 to P-108 of SEQ ID NO:103. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
As described herein or otherwise known in the art, the polynucleotides of the invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis.
This gene is expressed primarily in placenta and in some immune tissues and cells of the immune system (e.g., Jurkat T cell lines, and normal bone marrow).
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, fetal deficiencies, pre-natal disorders, and vascular diseases and conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developmental, proliferating, vascular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution in placenta indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of developmental anomalies, fetal deficiencies, reproductive disfunction or pre-natal disorders. Moreover, the protein is useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to microvascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
The tissue distribution of the expression of this gene indicates that polynucleotides and polypeptides corresponding to this gene (as well as antibodies raised against those polypeptides) are useful for the diagnosis and treatment of diseases and disorders associated with the immune system, including, but not limited to, allergy, asthma, graft rejection, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and other autoimmune conditions, infections, AIDS, chronic variable immune deficiency (CVID) and other immune deficiency syndromes, respiratory distress syndrome and inflammation, neoplasms of the immune/hematopoetic system including leukemias, lymphomas and other proliferative disorders such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, and myelodysplastic syndromes. The polynucleotides and/or polypeptides corresponding to this gene (and/or antibodies raised against those polypeptides) may also be useful for stimulating the immune response to bolster the immune response to diseases such as cancer or infection.
Furthermore, the protein may also be used to determine unknown biological activities, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose diseases and disorders of the endocrine system.
In specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor vascular diseases. In other specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to treat, prevent, or ameliorate vascular diseases
In other preferred embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor diseases and disorders associated with aberrant glucose metabolism or glucose uptake into cells. In other specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to treat, prevent, or ameliorate diseases and disorders associated with aberrant glucose metabolism or glucose uptake into cells.
It is believed that increased expression of this gene, at either the RNA or protein level, is increased in individuals (or a subset of individuals) that either have a predisposition to develop or have already developed type II diabetes mellitus (non-insulin dependent diabetes). Thus, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose, and/or monitor individuals with type II diabetes mellitus or individuals with a predisposition to develop type II diabetes mellitus.
By “agonist,” is meant any substance that enhances the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as agonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs), and peptidomimetics. By “antagonist,” is meant any substance that diminishes or abolishes the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as antagonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs) antisense polynucleotides, ribozymes, and peptidomimetics.
A biological sample of persons afflicted with type II diabetes mellitus is believed to be characterized by high levels of expression of this gene when compared to that observed in individuals not having type II diabetes mellitus. Thus, polynucleotides and/or polypeptides of the invention, and/or agonists or antagonists thereof, may be used according to the methods of the invention in the diagnosis and/or prognosis of individuals with type II diabetes mellitus, a subset of individuals with type II diabetes mellitus, and/or individuals or a subset of individuals with a predisposition to develop type II diabetes mellitus. For example, a biological sample obtained from a person suspected of being afflicted with type II diabetes mellitus, “the subject,” may be analyzed for the relative expression level(s) of polynucleotides and/or polypeptides of the invention. The expression level(s) of one or more of these molecules of the invention is (are) then compared to the expression level(s) of the same molecules of the invention as expressed in a person known not to be afflicted with rheumatoid arthritis. An increase in the expression level(s) of this gene in samples obtained from the subject compared to the control suggests that the subject is afflicted with type II diabetes mellitus or a subset thereof.
In another embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, and/or ameliorate type II diabetes. Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or anatgonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, or ameliorate conditions associated with type II diabetes mellitus, including, but not limited to, seizures, mental confusion, drowsiness, nonketotic hyperglycemic-hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, kidney disease (e.g., renal failure, nephropathy other diseases and disorders as described in the “Renal Disorders” section below), nerve damage, neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infections (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below, especially of the urinary tract and skin), carpal tunnel syndrome and Dupuytren's contracture.
In other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to regulate the animal's weight. In specific embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin. In still other embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin-like growth factor.
In a preferred embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to treat weight disorders, including but not limited to, obesity, cachexia, wasting disease, anorexia, and bulimia.
In other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are useful for the treatment, prevention or amelioration of neurodegenerative disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations, and other neurological diseases and disorders as described in the “Neural Activity and Neurological Activity diseases” section below.
In another embodiment, compositions of the invention (comprising polynucleotides, polypeptides of the invention, agonists and/or antagonists thereof (including antibodies) as well as fragments and variants of the polynucleotides, polypeptides of the invention, agonists and/or antagonists of the invention) are used in combination with anti-diabetic drugs. In a specific embodiment, compositions of the invention are administered in combination with thiazolidinediones (TZDs) including, but not limited to, rosiglitazone, pioglitazone, and troglitazone. In another specific embodiment, compositions of the invention are used in combination with oral hypoglycemic sulfonylurea drugs including, but not limited to, acarbose, acetohexamide, chlorpropamide, glimepiride, glipizide, glyburide, metformin, tolazamide, and/or tolbutamide. In still other embodiments, compositions of the invention are administered in combination with one or more of the following: a biguanide antidiabetic agent, a glitazone antidiabetic agent, and a sulfonylurea antidiabetic agent.
Features of Protein Encoded by Gene No: 15
The translation product of this gene shares sequence homology with drosophila peroxidasin which is thought to be important in extracellular matrix architecture. Moreover, the protein has homology to receptor-linked protein tyrosine phosphatases, which play important roles in inflammatory diseases and immune disorders. When tested against Jurkat T-cell lines, supernatants removed from cells containing this gene activated the GAS pathway. Thus, it is likely that this gene activates T-cells through the Jaks-STAT signal transduction pathway. GAS is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
In specific embodiments, polypeptides of the invention comprise the following amino acid sequence:







(SEQ ID NO: 111)


GRPTRPLRVA,


 


(SEQ ID NO: 112)


AWCPQTHTTSCLMGPFCCYSPLPGDMPTMARPCPQTWVSTHVRPATGLARQ


 


SAEALGCLWLSSGRISRSSLGTWWLWWVSSLLWNVGRPGATQSPQSHGGKM


 


GNPWPSSPEGTQCPGGPC,


 


(SEQ ID NO: 113)


CCYSPLPGDMPTMARPCPQTWVSTH,


 


(SEQ ID NO: 114)


ALGCLWLSSGRISRSSLG,


and/or


 


(SEQ ID NO: 115)


WNVGRPGATQSPQSHGGKMGNPWPSSPE.






Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in umbilical vein and to a lesser extent in endothelial and brain cells.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune, developmental and growth diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of fetal tissues, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, neural, cancerous and wounded tissues) or bodily fluids (e.g. lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO:108 as residues: Ala-55 to Thr-62, His-164 to Gly-175, Ala-197 to Glu-202.
The tissue distribution in umbilical vein cells, and homology to peroxidasin and receptor-linked protein tyrosine phosphatases indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of various fetal developmental and growth disorders involving the formation of extracellular matrix. Alternatively, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Activation of the GAS pathway by the gene product indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells.
This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene product demonstrates activity with regard to the GAS pathway, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis, and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein is useful in modulating the immune response to proliferative and vascular cells and tissues, particularly those having aberrant phenotypes. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Features of Protein Encoded by Gene No: 16
The translation product of this gene shares sequence homology with the kidney injury molecule-1 of Rattus norvegicus which is thought to play an important role in the restoration of the morphological integrity and function to postischemic kidney (See Genbank Accession No. gi|2665892 (AF035963)). Polynucleotides and polypeptides of the present invention are useful to promote growth of new tissue and survival of damaged tissue. Recombinant polypeptides of the present invention can be expressed in prokaryotic and eukaryotic host cells using a claimed process. Soluble variants fused to a toxin, imagable compound or radionuclide, and IgG fusion proteins are also claimed.
Polynucleotides and polypeptides of the present invention or an agonist, can be used to treat renal disease and to promote the growth of new tissue or the survival of damaged tissue, generally in conditions where the binding of specific ligand to the present invention stimulates cell growth, maintains cellular differentiation or reduces apoptosis, e.g. in cases of renal failure, nephritis, kidney transplants, toxic or hypoxic injury. A monoclonal antibody specific for polynucleotides and polypeptides of the present invention can be used to treat renal disease, e.g. where binding of the invention to a ligand results in neoplasia, loss of cellular function, susceptibility to apoptosis or promotion of inflammation, deliver imaging agents to the cells expressing the present invention in vivo or in vitro and measure the concentration of the present invention by immunoassay. Damage/regeneration of renal cells can be determined by measuring the present invention, particularly to diagnose or monitor the progress of disease or therapy. The tumour cells expressing the present invention can be inhibited by treatment with a fusion protein comprising a ligand of the present invention or MAb with a toxin or radionuclide, and tumour cells that express the present invention ligand can be inhibited with similarly tagged polypeptides of the present invention or anti-present invention ligand antibody.
Preferred polypeptides of the invention comprise the following amino acid sequence:









(SEQ ID NO: 122)



HESTVK.






Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in infant brain and fetal liver, and to a lesser extent in neoplastic cell lines and endocrine organs.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune, neural, endocrine, and growth disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and endocrine systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, neural, endocrine, growth, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, bile, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:117 as residues: Ser-44 to Ser-51, Cys-53 to Cys-64, Val-76 to Lys-83, Pro-102 to Gly-108, Arg-133 to Thr-162, Thr-169 to Lys-183. Polynucleotides encoding said polypeptides are also provided.
The tissue distribution in fetal liver and infant brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of immune and developmental conditions. Moreover, the expression within infant and fetal tissues and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Alternatively, the protein product of this gene could be used in the treatment and/or detection of kidney diseases including renal failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:116 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 557 of SEQ ID NO:116, b is an integer of 15 to 571, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:116, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 17
The translation product of this gene shares some sequence homology with various chains of the T-cell receptor, which are important in signalling between different cells of the immune system.
The gene encoding the disclosed cDNA is thought to reside on the X chromosome. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for the X chromosome.
This gene is expressed primarily in placental tissue, and to a lesser extent in activated monocytes and dendritic cells.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders and reproductive disorders, particularly pregnancy-associated disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system and female reproductive system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:124 as residues: Val-29 to Val-37, Asp-71 to His-76, Gln-78 to Gly-84, Met-105 to His-110, Trp-117 to Gly-122, Gln-136 to Lys-141, Leu-143 to Ala-149, Thr-162 to Asp-174, Ser-181 to Lys-186, Arg-214 to Glu-220, Glu-232 to Glu-238, Cys-249 to Asp-265. Polynucleotides encoding said polypeptides are also provided.
The tissue distribution in dendritic cells, activated monocytes and placental tissue (a tissue rich in hematopoeitic cells), and its homology to the T-cell receptor, indicates that polynucleotides and polypeptides corresponding to this gene are useful in the treatment, prophylaxis and/or diagnosis of immune and autoimmune diseases, such as lupus, transplant rejection, allergic reactions, arthritis, asthma, immunodeficiency diseases, leukemia, and AIDS. Its expression predominantly in hematopoietic cells also indicates that the gene could be important for the treatment and/or detection of hematopoietic disorders such as graft versus host reaction, graft versus host disease, transplant rejection, myelogenous leukemia, bone marrow fibrosis, and myeloproliferative disease. The protein could also be used to enhance or protect the proliferation, differentiation, and functional activation of hematopoietic progenitor cells such as bone marrow cells, which could be useful for cancer patients undergoing chemotherapy or patients undergoing bone marrow transplantation.
The protein may also be useful as a means to increase the proliferation of peripheral blood leukocytes, which could be useful in the combat of a range of hematopoietic disorders including immunodeficiency diseases, leukemia, and septicemia. In addition, expression in placenta indicates the gene or the protein encoded by this gene could be useful in the treatment, prophylaxis and/or diagnosis of placentitis, placenta previa, pregnancy disease, and miscarriage. Specific expression within the placenta indicates that this gene product may play a role in the proper establishment and maintenance of placental function. Alternately, this gene product is produced by the placenta and then transported to the embryo, where it may play a crucial role in the development and/or survival of the developing embryo or fetus. Expression of this gene product in a vascular-rich tissue such as the placenta also indicates that this gene product is produced more generally in endothelial cells or within the circulation. In such instances, it may play more generalized roles in vascular function, such as in angiogenesis. It may also be produced in the vasculature and have effects on other cells within the circulation, such as hematopoietic cells. It may serve to promote the proliferation, survival, activation, and/or differentiation of hematopoietic cells, as well as other cells throughout the body. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:123 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1576 of SEQ ID NO:123, b is an integer of 15 to 1590, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:123, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 18
This gene is expressed primarily in adipose tissue and dendritic cells.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, metabolic and immune disorders or diseases, particularly obesity. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune, metabolic and digestive systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, metabolic, digestive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:128 as residues: Ile-40 to Glu-45, Cys-63 to Val-69, Glu-83 to Asn-94, Pro-107 to Cys-115, Phe-137 to Ser-143, Ser-159 to Thr-167, Glu-200 to Tyr-210. Polynucleotides encoding said polypeptides are also provided.
The tissue distribution in primarily adipose tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment, diagnosis and/or prophylaxis of obesity related disorders. In addition, expression in dendritic cells indicates a potential role in the treatment, diagnosis and/or prophylaxis of immune and autoimmune disorders such as lupus, transplant rejection, allergic reactions, arthritis, asthma, immunodeficiency diseases, leukemia, and AIDS. The tissue distribution in adipose tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of obesity and other metabolic and endocrine conditions or disorders. Furthermore, the protein product of this gene may show utility in ameliorating conditions which occur secondary to aberrant fatty-acid metabolism (e.g. aberrant myelin sheath development), either directly or indirectly. Expression of this gene product in dendritic cells indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Expression of this gene product in dendritic cells also strongly indicates a role for this protein in immune function and immune surveillance. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:127 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1889 of SEQ ID NO:127, b is an integer of 15 to 1903, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:127, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 19
The translation product of this gene shares sequence homology with alpha-1 antitrypsin (See Genebank accession no. gnl|PID|d1021080 and BAA20264; all references available through this accession are hereby incorporated by reference herein). Alpha-1-antitrypsin is an important plasma protease inhibitor affecting a wide variety of serine proteases involved in coagulation, fibrinolysis and kinen generation.
In specific embodiments, polypeptides of the invention comprise, or alternatively consists of, the following amino acid sequence:









(SEQ ID NO: 133)



GERRNWGGEVYYSTGYSSRK.






Moreover, fragments and variants of this polypeptide (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding said polypeptides are also encompassed by the invention.

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 22-414 of the amino acid sequence referenced in Table 1A for this gene. Moreover, a cytoplasmic tail encompassing amino acids 5-21 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.
This gene is expressed in healing groin wound and to a lesser extent in some other tissues.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, wound healing disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the healing groin wound, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., healing, regenerative, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO:132 as residues: Phe-25 to Tyr-30, Gln-37 to Arg-42, Lys-106 to Leu-112, Leu-123 to Leu-130, Gln-142 to Phe-150, Gln-183 to Lys-188, Asp-219 to Glu-226, Lys-359 to Glu-366. Polynucleotides encoding said polypeptides are also encompassed by the invention.
The tissue distribution in healing groin wound and homology to alpha-1 antitrypsin indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and therapeutic treatment of wound healing disorders. In addition, since healing wounds have transcriptional environments similar to developing tissues, the translation product of this gene may be useful for the diagnosis and treatment of cancer and other proliferative disorders.
Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Features of Protein Encoded by Gene No: 20
This gene is expressed in cerebellum and ovarian cancer.
Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more of the immunogenic epitopes shown in SEQ ID NO:135 as residues: Thr-41 to Gly-47, Pro-170 to Asp-176, Leu-257 to Trp-262, Gln-276 to Ser-283, Arg-323 to Leu-330, Pro-409 to Ser-427, Gly 440 to Ala-449, Arg-323 to Gly-331, Glu-348 to Ser 354, Arg-256 to Trp-262, Phe 278 to Val 285, Arg 362 to Gly 385. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:140 as residues: Thr-41 to Gly-47, Pro-170 to Asp-176, Leu-257 to Trp-262, Gln-276 to Ser-283, Arg-323 to Leu-330, Pro-362 to Val-374. Polynucleotides encoding said polypeptides are also encompassed by the invention. Antibodies that bind said epitopes or other polypeptides of the invention are also encompassed.
In a specific embodiment, polypeptides of the invention, comprise or alternatively consist of, an amino acid sequence selected from the group:









(SEQ ID NO: 141)



FHGLGRLHTVHL,


 



(SEQ ID NO: 142)



AAFTGLALLEQLDLSDNAQLR,


 



(SEQ ID NO: 143)



HEVPDAPRPTPT,



and/or


 



  (SEQ ID NO: 144)



AFRGLHSLD.






Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also preferred are polypeptides, comprising or alternatively consisting of, the mature polypeptide which is predicted to consist of residues 27-473 of the foregoing sequence (SEQ ID NO:135), and biologically active fragments of the mature polypeptide (e.g., fragments that prevent neural/neuronal damage and/or spinal cord injury). Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In a nonexclusive specific embodiment, polypeptides of the invention, comprise or alternatively consist of, one or more of the following amino acid sequences and biologically active fragments thereof (e.g., fragments that prevent neural/neuronal damage and/or spinal cord injury):









(SEQ ID NO: 145)



SQRIFLHGNRISHVPAASFRAC,  


 



(SEQ ID NO: 146)



LTILWLHSNVLARIDAAAFTGL,


 



(SEQ ID NO: 147)



LEQLDLSDNAQLRSVDPATFHGL,


 



(SEQ ID NO: 148)



LHTLHLDRCGLQELGPGLFRGL,


 



(SEQ ID NO: 149)



LQYLYLQDNALQALPDDTFRDL,


 



(SEQ ID NO: 150)



LTHLFLHGNRISSVPERAFRGL,


 



(SEQ ID NO: 151)



LDRLLLHQNRVAHVHPHAFRDL,


 



(SEQ ID NO: 152)



LMTLYLFANNLSALPTEALAPL,


 



(SEQ ID NO: 153)



AHCSAARGLRATR,


 



(SEQ ID NO: 154)



PAHCSAARGLRATRF,


 



(SEQ ID NO: 155)



PSLTCSLTPLGLALVLWTVLGPC,


 



(SEQ ID NO: 156)



LPSLTCSLTPLGLALVLWTVL,


 



(SEQ ID NO: 157)



LPSLTCSLTPLGLALVLWTVLGPC,



and


 



(SEQ ID NO: 158)



CRNLTILWLHSNVL.






Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In a further embodiment of the invention, a Fc region of an immunoglobulin (e.g., IgG Fc) molecule is fused to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all fourteen of the polypeptide(s) selected from the group consisting of:









(SEQ ID NO: 145)



SQRIFLHGNRISHVPAASFRAC,


 



(SEQ ID NO: 146)



LTILWLHSNVLARIDAAAFTGL,


 



(SEQ ID NO: 147)



LEQLDLSDNAQLRSVDPATFHGL,


 



(SEQ ID NO: 148)



LHTLHLDRCGLQELGPGLFRGL,


 



(SEQ ID NO: 149)



LQYLYLQDNALQALPDDTFRDL,


 



(SEQ ID NO: 150)



LTHLFLHGNRISSVPERAFRGL,


 



(SEQ ID NO: 151)



LDRLLLHQNRVAHVHPHAFRDL,


 



(SEQ ID NO: 152)



LMTLYLFANNLSALPTEALAPL,


 



(SEQ ID NO: 153)



AHCSAARGLRATR,


 



(SEQ ID NO: 154)



PAHCSAARGLRATRF,


 



(SEQ ID NO: 155)



PSLTCSLTPLGLALVLWTVLGPC,


 



(SEQ ID NO: 156)



LPSLTCSLTPLGLALVLWTVL,


 



(SEQ ID NO: 157)



LPSLTCSLTPLGLALVLWTVLGPC,



and


 



(SEQ ID NO: 158)



CRNLTILWLHSNVL.






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In additional embodiments, proteins comprising fragments or variants of a polypeptide of SEQ ID NO:135 (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement there of) are fused to an Fc region of an immunoglobulin that bind the 66-amino acid extracellular loop of Nogo-A (See Genbank Accession CAB99248; GrandPre, T., et al., Nature, 403, pp. 439-444 (2000). Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides.
Thus, the fragment, derivative or analog of the polypeptide of FIGS. 7A-C (SEQ ID NO:135), or that encoded by the deposited cDNA plasmid, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the extracellular domain of the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).
As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides. By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1−z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (ìHAî) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
FIGS. 7A-C show the nucleotide (SEQ ID NO:134) and deduced amino acid sequence (SEQ ID NO:135) corresponding to this gene.
FIG. 8 shows an analysis of the amino acid sequence (SEQ ID NO:135). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.
The data presented in FIG. 8 are also represented in tabular form in Table 9. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 8, and Table 9: “Res”: amino acid residue of SEQ ID NO:135 and FIGS. 7A-C; “Position”: position of the corresponding residue within SEQ ID NO:135 and FIGS. 7A-C; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.
Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 8 and/or Table 9, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 9 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in FIG. 8, but may, as shown in Table 9, be represented or identified by using tabular representations of the data presented in FIG. 8. The DNA*STAR computer algorithm used to generate FIG. 8 (set on the original default parameters) was used to present the data in FIG. 8 in a tabular format (See Table 9). The tabular format of the data in FIG. 8 is used to easily determine specific boundaries of a preferred region.
The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:134, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:134. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:134. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150 from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, and from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650 from about 1651 to about 1700, from about 1701 to about 1750, from about 1751 to about 1777 of SEQ ID NO:134, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.
Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m−473 where m is an integer from 2 to 467, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:135. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: K-2 to C-473; R-3 to C-473; A-4 to C-473; S-5 to C-473; A-6 to C-473; G-7 to C-473; G-8 to C-473; S-9 to C-473; R-10 to C-473; L-11 to C-473; L-12 to C-473; A-13 to C-473; W-14 to C-473; V-15 to C-473; L-16 to C-473; W-17 to C-473; L-18 to C-473; Q-19 to C-473; A-20 to C-473; W-21 to C-473; Q-22 to C-473; V-23 to C-473; A-24 to C-473; A-25 to C-473; P-26 to C-473; C-27 to C-473; P-28 to C-473; G-29 to C-473; A-30 to C-473; C-31 to C-473; V-32 to C-473; C-33 to C-473; Y-34 to C-473; N-35 to C-473; E-36 to C-473; P-37 to C-473; K-38 to C-473; V-39 to C-473; T-40 to C-473; T-41 to C-473; S-42 to C-473; C-43 to C-473; P-44 to C-473; Q-45 to C-473; Q-46 to C-473; G-47 to C-473; L-48 to C-473; Q-49 to C-473; A-50 to C-473; V-51 to C-473; P-52 to C-473; V-53 to C-473; G-54 to C-473; I-55 to C-473; P-56 to C-473; A-57 to C-473; A-58 to C-473; S-59 to C-473; Q-60 to C-473; R-61 to C-473; I-62 to C-473; F-63 to C-473; L-64 to C-473; H-65 to C-473; G-66 to C-473; N-67 to C-473; R-68 to C-473; I-69 to C-473; S-70 to C-473; H-71 to C-473; V-72 to C-473; P-73 to C-473; A-74 to C-473; A-75 to C-473; S-76 to C-473; F-77 to C-473; R-78 to C-473; A-79 to C-473; C-80 to C-473; R-81 to C-473; N-82 to C-473; L-83 to C-473; T-84 to C-473; I-85 to C-473; L-86 to C-473; W-87 to C-473; L-88 to C-473; H-89 to C-473; S-90 to C-473; N-91 to C-473; V-92 to C-473; L-93 to C-473; A-94 to C-473; R-95 to C-473; I-96 to C-473; D-97 to C-473; A-98 to C-473; A-99 to C-473; A-100 to C-473; F-101 to C-473; T-102 to C-473; G-103 to C-473; L-104 to C-473; A-105 to C-473; L-106 to C-473; L-107 to C-473; E-108 to C-473; Q-109 to C-473; L-110 to C-473; D-111 to C-473; L-112 to C-473; S-113 to C-473; D-114 to C-473; N-115 to C-473; A-116 to C-473; Q-117 to C-473; L-118 to C-473; R-119 to C-473; S-120 to C-473; V-121 to C-473; D-122 to C-473; P-123 to C-473; A-124 to C-473; T-125 to C-473; F-126 to C-473; H-127 to C-473; G-128 to C-473; L-129 to C-473; G-130 to C-473; R-131 to C-473; L-132 to C-473; H-133 to C-473; T-134 to C-473; L-135 to C-473; H-136 to C-473; L-137 to C-473; D-138 to C-473; R-139 to C-473; C-140 to C-473; G-141 to C-473; L-142 to C-473; Q-143 to C-473; E-144 to C-473; L-145 to C-473; G-146 to C-473; P-147 to C-473; G-148 to C-473; L-149 to C-473; F-150 to C-473; R-151 to C-473; G-152 to C-473; L-153 to C-473; A-154 to C-473; A-155 to C-473; L-156 to C-473; Q-157 to C-473; Y-158 to C-473; L-159 to C-473; Y-160 to C-473; L-161 to C-473; Q-162 to C-473; D-163 to C-473; N-164 to C-473; A-165 to C-473; L-166 to C-473; Q-167 to C-473; A-168 to C-473; L-169 to C-473; P-170 to C-473; D-171 to C-473; D-172 to C-473; T-173 to C-473; F-174 to C-473; R-175 to C-473; D-176 to C-473; L-177 to C-473; G-178 to C-473; N-179 to C-473; L-180 to C-473; T-181 to C-473; H-182 to C-473; L-183 to C-473; F-184 to C-473; L-185 to C-473; H-186 to C-473; G-187 to C-473; N-188 to C-473; R-189 to C-473; I-190 to C-473; S-191 to C-473; S-192 to C-473; V-193 to C-473; P-194 to C-473; E-195 to C-473; R-196 to C-473; A-197 to C-473; F-198 to C-473; R-199 to C-473; G-200 to C-473; L-201 to C-473; H-202 to C-473; S-203 to C-473; L-204 to C-473; D-205 to C-473; R-206 to C-473; L-207 to C-473; L-208 to C-473; L-209 to C-473; H-210 to C-473; Q-211 to C-473; N-212 to C-473; R-213 to C-473; V-214 to C-473; A-215 to C-473; H-216 to C-473; V-217 to C-473; H-218 to C-473; P-219 to C-473; H-220 to C-473; A-221 to C-473; F-222 to C-473; R-223 to C-473; D-224 to C-473; L-225 to C-473; G-226 to C-473; R-227 to C-473; L-228 to C-473; M-229 to C-473; T-230 to C-473; L-231 to C-473; Y-232 to C-473; L-233 to C-473; F-234 to C-473; A-235 to C-473; N-236 to C-473; N-237 to C-473; L-238 to C-473; S-239 to C-473; A-240 to C-473; L-241 to C-473; P-242 to C-473; T-243 to C-473; E-244 to C-473; A-245 to C-473; L-246 to C-473; A-247 to C-473; P-248 to C-473; L-249 to C-473; R-250 to C-473; A-251 to C-473; L-252 to C-473; Q-253 to C-473; Y-254 to C-473; L-255 to C-473; R-256 to C-473; L-257 to C-473; N-258 to C-473; D-259 to C-473; N-260 to C-473; P-261 to C-473; W-262 to C-473; V-263 to C-473; C-264 to C-473; D-265 to C-473; C-266 to C-473; R-267 to C-473; A-268 to C-473; R-269 to C-473; P-270 to C-473; L-271 to C-473; W-272 to C-473; A-273 to C-473; W-274 to C-473; L-275 to C-473; Q-276 to C-473; K-277 to C-473; F-278 to C-473; R-279 to C-473; G-280 to C-473; S-281 to C-473; S-282 to C-473; S-283 to C-473; E-284 to C-473; V-285 to C-473; P-286 to C-473; C-287 to C-473; S-288 to C-473; L-289 to C-473; P-290 to C-473; Q-291 to C-473; R-292 to C-473; L-293 to C-473; A-294 to C-473; G-295 to C-473; R-296 to C-473; D-297 to C-473; L-298 to C-473; K-299 to C-473; R-300 to C-473; L-301 to C-473; A-302 to C-473; A-303 to C-473; N-304 to C-473; D-305 to C-473; L-306 to C-473; Q-307 to C-473; G-308 to C-473; C-309 to C-473; A-310 to C-473; V-311 to C-473; A-312 to C-473; T-313 to C-473; G-314 to C-473; P-315 to C-473; Y-316 to C-473; H-317 to C-473; P-318 to C-473; I-319 to C-473; W-320 to C-473; T-321 to C-473; G-322 to C-473; R-323 to C-473; A-324 to C-473; T-325 to C-473; D-326 to C-473; E-327 to C-473; E-328 to C-473; P-329 to C-473; L-330 to C-473; G-331 to C-473; L-332 to C-473; P-333 to C-473; K-334 to C-473; C-335 to C-473; C-336 to C-473; Q-337 to C-473; P-338 to C-473; D-339 to C-473; A-340 to C-473; A-341 to C-473; D-342 to C-473; K-343 to C-473; A-344 to C-473; S-345 to C-473; V-346 to C-473; L-347 to C-473; E-348 to C-473; P-349 to C-473; G-350 to C-473; R-351 to C-473; P-352 to C-473; A-353 to C-473; S-354 to C-473; A-355 to C-473; G-356 to C-473; N-357 to C-473; A-358 to C-473; L-359 to C-473; K-360 to C-473; G-361 to C-473; R-362 to C-473; V-363 to C-473; P-364 to C-473; P-365 to C-473; G-366 to C-473; D-367 to C-473; S-368 to C-473; P-369 to C-473; P-370 to C-473; G-371 to C-473; N-372 to C-473; G-373 to C-473; S-374 to C-473; G-375 to C-473; P-376 to C-473; R-377 to C-473; H-378 to C-473; I-379 to C-473; N-380 to C-473; D-381 to C-473; S-382 to C-473; P-383 to C-473; F-384 to C-473; G-385 to C-473; T-386 to C-473; L-387 to C-473; P-388 to C-473; G-389 to C-473; S-390 to C-473; A-391 to C-473; E-392 to C-473; P-393 to C-473; P-394 to C-473; A-395 to C-473; H-396 to C-473; C-397 to C-473; S-398 to C-473; A-399 to C-473; A-400 to C-473; R-401 to C-473; G-402 to C-473; L-403 to C-473; R-404 to C-473; A-405 to C-473; T-406 to C-473; R-407 to C-473; F-408 to C-473; P-409 to C-473; T-410 to C-473; S-411 to C-473; G-412 to C-473; P-413 to C-473; R-414 to C-473; R-415 to C-473; R-416 to C-473; P-417 to C-473; G-418 to C-473; C-419 to C-473; S-420 to C-473; R-421 to C-473; K-422 to C-473; N-423 to C-473; R-424 to C-473; T-425 to C-473; R-426 to C-473; S-427 to C-473; H-428 to C-473; C-429 to C-473; R-430 to C-473; L-431 to C-473; G-432 to C-473; Q-433 to C-473; A-434 to C-473; G-435 to C-473; S-436 to C-473; G-437 to C-473; G-438 to C-473; G-439 to C-473; G-440 to C-473; T-441 to C-473; G-442 to C-473; D-443 to C-473; S-444 to C-473; E-445 to C-473; G-446 to C-473; S-447 to C-473; G-448 to C-473; A-449 to C-473; L-450 to C-473; P-451 to C-473; S-452 to C-473; L-453 to C-473; T-454 to C-473; C-455 to C-473; S-456 to C-473; L-457 to C-473; T-458 to C-473; P-459 to C-473; L-460 to C-473; G-461 to C-473; L-462 to C-473; A-463 to C-473; L-464 to C-473; V-465 to C-473; L-466 to C-473; W-467 to C-473; and T-468 to C-473 of SEQ ID NO:135. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein (e.g., modulation of neurite growth, including, but not limited to, inhibitory activity and/or antiinflammatory activity), other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIGS. 7A-C (SEQ ID NO:135), as described by the general formula 1−n, where n is an integer from 6 to 472, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:135. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to P-472; M-1 to G-471; M-1 to L-470; M-1 to V-469; M-1 to T-468; M-1 to W-467; M-1 to L-466; M-1 to V-465; M-1 to L-464; M-1 to A-463; M-1 to L-462; M-1 to G-461; M-1 to L-460; M-1 to P-459; M-1 to T-458; M-1 to L-457; M-1 to S-456; M-1 to C-455; M-1 to T-454; M-1 to L-453; M-1 to S-452; M-1 to P-451; M-1 to L-450; M-1 to A-449; M-1 to G-448; M-1 to S-447; M-1 to G-446; M-1 to E-445; M-1 to S-444; M-1 to D-443; M-1 to G-442; M-1 to T-441; M-1 to G-440; M-1 to G-439; M-1 to G-438; M-1 to G-437; M-1 to S-436; M-1 to G-435; M-1 to A-434; M-1 to Q-433; M-1 to G-432; M-1 to L-431; M-1 to R-430; M-1 to C-429; M-1 to H-428; M-1 to S-427; M-1 to R-426; M-1 to T-425; M-1 to R-424; M-1 to N-423; M-1 to K-422; M-1 to R-421; M-1 to S-420; M-1 to C-419; M-1 to G-418; M-1 to P-417; M-1 to R-416; M-1 to R-415; M-1 to R-414; M-1 to P-413; M-1 to G-412; M-1 to S-411; M-1 to T-410; M-1 to P-409; M-1 to F-408; M-1 to R-407; M-1 to T-406; M-1 to A-405; M-1 to R-404; M-1 to L-403; M-1 to G-402; M-1 to R-401; M-1 to A-400; M-1 to A-399; M-1 to S-398; M-1 to C-397; M-1 to H-396; M-1 to A-395; M-1 to P-394; M-1 to P-393; M-1 to E-392; M-1 to A-391; M-1 to S-390; M-1 to G-389; M-1 to P-388; M-1 to L-387; M-1 to T-386; M-1 to G-385; M-1 to F-384; M-1 to P-383; M-1 to S-382; M-1 to D-381; M-1 to N-380; M-1 to I-379; M-1 to H-378; M-1 to R-377; M-1 to P-376; M-1 to G-375; M-1 to S-374; M-1 to G-373; M-1 to N-372; M-1 to G-371; M-1 to P-370; M-1 to P-369; M-1 to S-368; M-1 to D-367; M-1 to G-366; M-1 to P-365; M-1 to P-364; M-1 to V-363; M-1 to R-362; M-1 to G-361; M-1 to K-360; M-1 to L-359; M-1 to A-358; M-1 to N-357; M-1 to G-356; M-1 to A-355; M-1 to S-354; M-1 to A-353; M-1 to P-352; M-1 to R-351; M-1 to G-350; M-1 to P-349; M-1 to E-348; M-1 to L-347; M-1 to V-346; M-1 to S-345; M-1 to A-344; M-1 to K-343; M-1 to D-342; M-1 to A-341; M-1 to A-340; M-1 to D-339; M-1 to P-338; M-1 to Q-337; M-1 to C-336; M-1 to C-335; M-1 to K-334; M-1 to P-333; M-1 to L-332; M-1 to G-331; M-1 to L-330; M-1 to P-329; M-1 to E-328; M-1 to E-327; M-1 to D-326; M-1 to T-325; M-1 to A-324; M-1 to R-323; M-1 to G-322; M-1 to T-321; M-1 to W-320; M-1 to I-319; M-1 to P-318; M-1 to H-317; M-1 to Y-316; M-1 to P-315; M-1 to G-314; M-1 to T-313; M-1 to A-312; M-1 to V-311; M-1 to A-310; M-1 to C-309; M-1 to G-308; M-1 to Q-307; M-1 to L-306; M-1 to D-305; M-1 to N-304; M-1 to A-303; M-1 to A-302; M-1 to L-301; M-1 to R-300; M-1 to K-299; M-1 to L-298; M-1 to D-297; M-1 to R-296; M-1 to G-295; M-1 to A-294; M-1 to L-293; M-1 to R-292; M-1 to Q-291; M-1 to P-290; M-1 to L-289; M-1 to S-288; M-1 to C-287; M-1 to P-286; M-1 to V-285; M-1 to E-284; M-1 to S-283; M-1 to S-282; M-1 to S-281; M-1 to G-280; M-1 to R-279; M-1 to F-278; M-1 to K-277; M-1 to Q-276; M-1 to L-275; M-1 to W-274; M-1 to A-273; M-1 to W-272; M-1 to L-271; M-1 to P-270; M-1 to R-269; M-1 to A-268; M-1 to R-267; M-1 to C-266; M-1 to D-265; M-1 to C-264; M-1 to V-263; M-1 to W-262; M-1 to P-261; M-1 to N-260; M-1 to D-259; M-1 to N-258; M-1 to L-257; M-1 to R-256; M-1 to L-255; M-1 to Y-254; M-1 to Q-253; M-1 to L-252; M-1 to A-251; M-1 to R-250; M-1 to L-249; M-1 to P-248; M-1 to A-247; M-1 to L-246; M-1 to A-245; M-1 to E-244; M-1 to T-243; M-1 to P-242; M-1 to L-241; M-1 to A-240; M-1 to S-239; M-1 to L-238; M-1 to N-237; M-1 to N-236; M-1 to A-235; M-1 to F-234; M-1 to L-233; M-1 to Y-232; M-1 to L-231; M-1 to T-230; M-1 to M-229; M-1 to L-228; M-1 to R-227; M-1 to G-226; M-1 to L-225; M-1 to D-224; M-1 to R-223; M-1 to F-222; M-1 to A-221; M-1 to H-220; M-1 to P-219; M-1 to H-218; M-1 to V-217; M-1 to H-216; M-1 to A-215; M-1 to V-214; M-1 to R-213; M-1 to N-212; M-1 to Q-211; M-1 to H-210; M-1 to L-209; M-1 to L-208; M-1 to L-207; M-1 to R-206; M-1 to D-205; M-1 to L-204; M-1 to S-203; M-1 to H-202; M-1 to L-201; M-1 to G-200; M-1 to R-199; M-1 to F-198; M-1 to A-197; M-1 to R-196; M-1 to E-195; M-1 to P-194; M-1 to V-193; M-1 to S-192; M-1 to S-191; M-1 to I-190; M-1 to R-189; M-1 to N-188; M-1 to G-187; M-1 to H-186; M-1 to L-185; M-1 to F-184; M-1 to L-183; M-1 to H-182; M-1 to T-181; M-1 to L-180; M-1 to N-179; M-1 to G-178; M-1 to L-177; M-1 to D-176; M-1 to R-175; M-1 to F-174; M-1 to T-173; M-1 to D-172; M-1 to D-171; M-1 to P-170; M-1 to L-169; M-1 to A-168; M-1 to Q-167; M-1 to L-166; M-1 to A-165; M-1 to N-164; M-1 to D-163; M-1 to Q-162; M-1 to L-161; M-1 to Y-160; M-1 to L-159; M-1 to Y-158; M-1 to Q-157; M-1 to L-156; M-1 to A-155; M-1 to A-154; M-1 to L-153; M-1 to G-152; M-1 to R-151; M-1 to F-150; M-1 to L-149; M-1 to G-148; M-1 to P-147; M-1 to G-146; M-1 to L-145; M-1 to E-144; M-1 to Q-143; M-1 to L-142; M-1 to G-141; M-1 to C-140; M-1 to R-139; M-1 to D-138; M-1 to L-137; M-1 to H-136; M-1 to L-135; M-1 to T-134; M-1 to H-133; M-1 to L-132; M-1 to R-131; M-1 to G-130; M-1 to L-129; M-1 to G-128; M-1 to H-127; M-1 to F-126; M-1 to T-125; M-1 to A-124; M-1 to P-123; M-1 to D-122; M-1 to V-121; M-1 to S-120; M-1 to R-119; M-1 to L-118; M-1 to Q-117; M-1 to A-116; M-1 to N-115; M-1 to D-114; M-1 to S-113; M-1 to L-112; M-1 to D-111; M-1 to L-110; M-1 to Q-109; M-1 to E-108; M-1 to L-107; M-1 to L-106; M-1 to A-105; M-1 to L-104; M-1 to G-103; M-1 to T-102; M-1 to F-101; M-1 to A-100; M-1 to A-99; M-1 to A-98; M-1 to D-97; M-1 to I-96; M-1 to R-95; M-1 to A-94; M-1 to L-93; M-1 to V-92; M-1 to N-91; M-1 to S-90; M-1 to H-89; M-1 to L-88; M-1 to W-87; M-1 to L-86; M-1 to I-85; M-1 to T-84; M-1 to L-83; M-1 to N-82; M-1 to R-81; M-1 to C-80; M-1 to A-79; M-1 to R-78; M-1 to F-77; M-1 to S-76; M-1 to A-75; M-1 to A-74; M-1 to P-73; M-1 to V-72; M-1 to H-71; M-1 to S-70; M-1 to I-69; M-1 to R-68; M-1 to N-67; M-1 to G-66; M-1 to H-65; M-1 to L-64; M-1 to F-63; M-1 to I-62; M-1 to R-61; M-1 to Q-60; M-1 to S-59; M-1 to A-58; M-1 to A-57; M-1 to P-56; M-1 to I-55; M-1 to G-54; M-1 to V-53; M-1 to P-52; M-1 to V-51; M-1 to A-50; M-1 to Q-49; M-1 to L-48; M-1 to G-47; M-1 to Q-46; M-1 to Q-45; M-1 to P-44; M-1 to C-43; M-1 to S-42; M-1 to T-41; M-1 to T-40; M-1 to V-39; M-1 to K-38; M-1 to P-37; M-1 to E-36; M-1 to N-35; M-1 to Y-34; M-1 to C-33; M-1 to V-32; M-1 to C-31; M-1 to A-30; M-1 to G-29; M-1 to P-28; M-1 to C-27; M-1 to P-26; M-1 to A-25; M-1 to A-24; M-1 to V-23; M-1 to Q-22; M-1 to W-21; M-1 to A-20; M-1 to Q-19; M-1 to L-18; M-1 to W-17; M-1 to L-16; M-1 to V-15; M-1 to W-14; M-1 to A-13; M-1 to L-12; M-1 to L-11; M-1 to R-10; M-1 to S-9; M-1 to G-8; M-1 to G-7; and M-1 to A-6 of SEQ ID NO:135. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:135, where n and m are integers as described above. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to V-15; K-2 to L-16; R-3 to W-17; A-4 to L-18; S-5 to Q-19; A-6 to A-20; G-7 to W-21; G-8 to Q-22; S-9 to V-23; R-10 to A-24; L-11 to A-25; L-12 to P-26; A-13 to C-27; W-14 to P-28; V-15 to G-29; L-16 to A-30; W-17 to C-31; L-18 to V-32; Q-19 to C-33; A-20 to Y-34; W-21 to N-35; Q-22 to E-36; V-23 to P-37; A-24 to K-38; A-25 to V-39; P-26 to T-40; C-27 to T-41; P-28 to S-42; G-29 to C-43; A-30 to P-44; C-31 to Q-45; V-32 to Q-46; C-33 to G-47; Y-34 to L-48; N-35 to Q-49; E-36 to A-50; P-37 to V-51; K-38 to P-52; V-39 to V-53; T-40 to G-54; T-41 to I-55; S-42 to P-56; C-43 to A-57; P-44 to A-58; Q-45 to S-59; Q-46 to Q-60; G-47 to R-61; L-48 to I-62; Q-49 to F-63; A-50 to L-64; V-51 to H-65; P-52 to G-66; V-53 to N-67; G-54 to R-68; I-55 to I-69; P-56 to S-70; A-57 to H-71; A-58 to V-72; S-59 to P-73; Q-60 to A-74; R-61 to A-75; I-62 to S-76; F-63 to F-77; L-64 to R-78; H-65 to A-79; G-66 to C-80; N-67 to R-81; R-68 to N-82; I-69 to L-83; S-70 to T-84; H-71 to I-85; V-72 to L-86; P-73 to W-87; A-74 to L-88; A-75 to H-89; S-76 to S-90; F-77 to N-91; R-78 to V-92; A-79 to L-93; C-80 to A-94; R-81 to R-95; N-82 to I-96; L-83 to D-97; T-84 to A-98; I-85 to A-99; L-86 to A-100; W-87 to F-101; L-88 to T-102; H-89 to G-103; S-90 to L-104; N-91 to A-105; V-92 to L-106; L-93 to L-107; A-94 to E-108; R-95 to Q-109; I-96 to L-110; D-97 to D-111; A-98 to L-112; A-99 to S-113; A-100 to D-114; F-101 to N-115; T-102 to A-116; G-103 to Q-117; L-104 to L-118; A-105 to R-119; L-106 to S-120; L-107 to V-121; E-108 to D-122; Q-109 to P-123; L-110 to A-124; D-111 to T-125; L-112 to F-126; S-113 to H-127; D-114 to G-128; N-115 to L-129; A-116 to G-130; Q-117 to R-131; L-118 to L-132; R-119 to H-133; S-120 to T-134; V-121 to L-135; D-122 to H-136; P-123 to L-137; A-124 to D-138; T-125 to R-139; F-126 to C-140; H-127 to G-141; G-128 to L-142; L-129 to Q-143; G-130 to E-144; R-131 to L-145; L-132 to G-146; H-133 to P-147; T-134 to G-148; L-135 to L-149; H-136 to F-150; L-137 to R-151; D-138 to G-152; R-139 to L-153; C-140 to A-154; G-141 to A-155; L-142 to L-156; Q-143 to Q-157; E-144 to Y-158; L-145 to L-159; G-146 to Y-160; P-147 to L-161; G-148 to Q-162; L-149 to D-163; F-150 to N-164; R-151 to A-165; G-152 to L-166; L-153 to Q-167; A-154 to A-168; A-155 to L-169; L-156 to P-170; Q-157 to D-171; Y-158 to D-172; L-159 to T-173; Y-160 to F-174; L-161 to R-175; Q-162 to D-176; D-163 to L-177; N-164 to G-178; A-165 to N-179; L-166 to L-180; Q-167 to T-181; A-168 to H-182; L-169 to L-183; P-170 to F-184; D-171 to L-185; D-172 to H-186; T-173 to G-187; F-174 to N-188; R-175 to R-189; D-176 to I-190; L-177 to S-191; G-178 to S-192; N-179 to V-193; L-180 to P-194; T-181 to E-195; H-182 to R-196; L-183 to A-197; F-184 to F-198; L-185 to R-199; H-186 to G-200; G-187 to L-201; N-188 to H-202; R-189 to S-203; I-190 to L-204; S-191 to D-205; S-192 to R-206; V-193 to L-207; P-194 to L-208; E-195 to L-209; R-196 to H-210; A-197 to Q-211; F-198 to N-212; R-199 to R-213; G-200 to V-214; L-201 to A-215; H-202 to H-216; S-203 to V-217; L-204 to H-218; D-205 to P-219; R-206 to H-220; L-207 to A-221; L-208 to F-222; L-209 to R-223; H-210 to D-224; Q-211 to L-225; N-212 to G-226; R-213 to R-227; V-214 to L-228; A-215 to M-229; H-216 to T-230; V-217 to L-231; H-218 to Y-232; P-219 to L-233; H-220 to F-234; A-221 to A-235; F-222 to N-236; R-223 to N-237; D-224 to L-238; L-225 to S-239; G-226 to A-240; R-227 to L-241; L-228 to P-242; M-229 to T-243; T-230 to E-244; L-231 to A-245; Y-232 to L-246; L-233 to A-247; F-234 to P-248; A-235 to L-249; N-236 to R-250; N-237 to A-251; L-238 to L-252; S-239 to Q-253; A-240 to Y-254; L-241 to L-255; P-242 to R-256; T-243 to L-257; E-244 to N-258; A-245 to D-259; L-246 to N-260; A-247 to P-261; P-248 to W-262; L-249 to V-263; R-250 to C-264; A-251 to D-265; L-252 to C-266; Q-253 to R-267; Y-254 to A-268; L-255 to R-269; R-256 to P-270; L-257 to L-271; N-258 to W-272; D-259 to A-273; N-260 to W-274; P-261 to L-275; W-262 to Q-276; V-263 to K-277; C-264 to F-278; D-265 to R-279; C-266 to G-280; R-267 to S-281; A-268 to S-282; R-269 to S-283; P-270 to E-284; L-271 to V-285; W-272 to P-286; A-273 to C-287; W-274 to S-288; L-275 to L-289; Q-276 to P-290; K-277 to Q-291; F-278 to R-292; R-279 to L-293; G-280 to A-294; S-281 to G-295; S-282 to R-296; S-283 to D-297; E-284 to L-298; V-285 to K-299; P-286 to R-300; C-287 to L-301; S-288 to A-302; L-289 to A-303; P-290 to N-304; Q-291 to D-305; R-292 to L-306; L-293 to Q-307; A-294 to G-308; G-295 to C-309; R-296 to A-310; D-297 to V-311; L-298 to A-312; K-299 to T-313; R-300 to G-314; L-301 to P-315; A-302 to Y-316; A-303 to H-317; N-304 to P-318; D-305 to I-319; L-306 to W-320; Q-307 to T-321; G-308 to G-322; C-309 to R-323; A-310 to A-324; V-311 to T-325; A-312 to D-326; T-313 to E-327; G-314 to E-328; P-315 to P-329; Y-316 to L-330; H-317 to G-331; P-318 to L-332; I-319 to P-333; W-320 to K-334; T-321 to C-335; G-322 to C-336; R-323 to Q-337; A-324 to P-338; T-325 to D-339; D-326 to A-340; E-327 to A-341; E-328 to D-342; P-329 to K-343; L-330 to A-344; G-331 to S-345; L-332 to V-346; P-333 to L-347; K-334 to E-348; C-335 to P-349; C-336 to G-350; Q-337 to R-351; P-338 to P-352; D-339 to A-353; A-340 to S-354; A-341 to A-355; D-342 to G-356; K-343 to N-357; A-344 to A-358; S-345 to L-359; V-346 to K-360; L-347 to G-361; E-348 to R-362; P-349 to V-363; G-350 to P-364; R-351 to P-365; P-352 to G-366; A-353 to D-367; S-354 to S-368; A-355 to P-369; G-356 to P-370; N-357 to G-371; A-358 to N-372; L-359 to G-373; K-360 to S-374; G-361 to G-375; R-362 to P-376; V-363 to R-377; P-364 to H-378; P-365 to I-379; G-366 to N-380; D-367 to D-381; S-368 to S-382; P-369 to P-383; P-370 to F-384; G-371 to G-385; N-372 to T-386; G-373 to L-387; S-374 to P-388; G-375 to G-389; P-376 to S-390; R-377 to A-391; H-378 to E-392; I-379 to P-393; N-380 to P-394; D-381 to A-395; S-382 to H-396; P-383 to C-397; F-384 to S-398; G-385 to A-399; T-386 to A-400; L-387 to R-401; P-388 to G-402; G-389 to L-403; S-390 to R-404; A-391 to A-405; E-392 to T-406; P-393 to R-407; P-394 to F-408; A-395 to P-409; H-396 to T-410; C-397 to S-411; S-398 to G-412; A-399 to P-413; A-400 to R-414; R-401 to R-415; G-402 to R-416; L-403 to P-417; R-404 to G-418; A-405 to C-419; T-406 to S-420; R-407 to R-421; F-408 to K-422; P-409 to N-423; T-410 to R-424; S-411 to T-425; G-412 to R-426; P-413 to S-427; R-414 to H-428; R-415 to C-429; R-416 to R-430; P-417 to L-431; G-418 to G-432; C-419 to Q-433; S-420 to A-434; R-421 to G-435; K-422 to S-436; N-423 to G-437; R-424 to G-438; T-425 to G-439; R-426 to G-440; S-427 to T-441; H-428 to G-442; C-429 to D-443; R-430 to S-444; L-431 to E-445; G-432 to G-446; Q-433 to S-447; A-434 to G-448; G-435 to A-449; S-436 to L-450; G-437 to P-451; G-438 to S-452; G-439 to L-453; G-440 to T-454; T-441 to C-455; G-442 to S-456; D-443 to L-457; S-444 to T-458; E-445 to P-459; G-446 to L-460; S-447 to G-461; G-448 to L-462; A-449 to A-463; L-450 to L-464; P-451 to V-465; S-452 to L-466; L-453 to W-467; T-454 to T-468; C-455 to V-469; S-456 to L-470; L-457 to G-471; T-458 to P-472; and P-459 to C-473 of SEQ ID NO:135. Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present invention is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein as m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N and C terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 209782, where this portion excludes any integer of amino acid residues from 1 to about 467 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 209782, or any integer of amino acid residues from 6 to about 473 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209782. Polypeptides encoded by these polynucleotides also are encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The translation product of this gene shares sequence homology with ALS (Acid Labile Subunit of Insulin-Like Growth Factor) which is thought to be important in the regulation of IGF availability. As such, it is likely that the product of this gene is involved in the regulation of various proliferation-dependent cellular processes that may be attributable to cancer progression (See Genbank Accession No. gi|184808; all references available through this accession are hereby incorporated by reference herein).
The gene encoding the disclosed cDNA is believed to reside on chromosome 22. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 22.
As described herein or otherwise known in the art, the polynucleotides of the invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neurodegenerative diseases, growth deficiencies, osteoporosis, catabolic disorders, diabetes, ovarian cancer, neuronal injury spinal injury, post-spinal trauma neurite outgrowth, inflammation, and neuronal injury, and disorders of the central and peripheral nervous system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the nervous system and other peripheral tissues, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, neuronal, proliferating, and/or other tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution cerebellum and homology to ALS (Acid Labile Subunit of Insulin-Like Growth Factor) indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of a variety of metabolic disorders, growth deficiencies, osteoporosis, catabolic disorders (including AIDS) and diabetes. Nearly all of the insulin-like growth factor (IGF) in the circulation is bound in a heterotrimeric complex composed of IGF, IGF-binding protein-3, and the acid-labile subunit (ALS). The protein product of this gene therefore may afford the ability to potentiate the biological actions of IGF or similar growth factors and cytokines. Studies which demonstrate the beneficial effect of IGF-I in amyotrophic lateral-sclerosis, would suggest a role in this disease as well. Alternatively, the tissue distribution in cancerous ovarian tissue indicates polynucleotides and polypeptides corresponding to this gene are useful for the treatment, diagnosis, and/or prevention of various skin disorders. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose diseases and disorders of the nervous system.
In specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor neurological diseases, neural injury, and/or spinal cord injury. In other specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to treat, prevent, or ameliorate neurological disease, neural injury, and/or spinal cord injury.
In other preferred embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor diseases and disorders associated with aberrant neurite outgrowth.
By “agonist,” is meant any substance that enhances the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as agonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs), and peptidomimetics. By “antagonist,” is meant any substance that diminishes or abolishes the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as antagonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs) antisense polynucleotides, ribozymes, and peptidomimetics.
In another embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, and/or ameliorate neurological disease, neural injury, and/or spinal cord injury. Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or anatgonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, or ameliorate conditions associated with ameliorate neurological disease, neural injury, and/or spinal cord injury, including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations, and other neurological diseases and disorders as described in the “Neural Activity and Neurological Activity diseases” section below,
In a preferred embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, and/or ameliorate neural injury, and/or spinal cord injury following spinal cord and neural trauma. For example, neural outgrowth and inflammation. Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or anatgonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, or ameliorate conditions associated with ameliorate neurological disease, neural injury, and/or spinal cord injury, including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations, and other neurological diseases and disorders as described in the “Neural Activity and Neurological Activity diseases” section below.
In another embodiment, compositions of the invention i.e., Therapeutics (comprising polynucleotides, polypeptides of the invention, agonists and/or antagonists thereof (including antibodies) as well as fragments and variants of the polynucleotides, polypeptides of the invention, agonists and/or antagonists of the invention) are used in combination with antiinflammatory drugs (e.g., as described herein) and/or drugs used to treat spinal cord injury, neural injury, or neurological disease.
In certain embodiments, the Therapeutics of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the Therapeutics of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).
In other embodiments, the Therapeutics of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the Therapeutics of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenyloin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantadine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).
The polynucleotides, polypeptides and agonists or antagonists of the invention may also be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).
Additional diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include, but are not limited to neoplastic disease of the CNS e.g. glioma, glioblastoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, haemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma or retinoblastoma and degenerative nerve diseases e.g. Alzheimer's and Parkinson's diseases. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Examples 28, 30, 31, 32, 40, 42, and 46, and elsewhere herein. Therapeutics can be used to treat or prevent hyperproliferative or benign dysproliferative disorders e.g. psoriasis and tissue hypertrophy. Ribozymes or antisense nucleic acids can be used to inhibit production of the polypeptides of the invention, to induce regeneration of neurons or to promote structural plasticity of the CNS in disorders where neurite growth, regeneration or maintenance are deficient or desired. The animal models can be used in diagnostic and screening methods for predisposition to disorders and to screen for or test molecules which can treat or prevent disorders or diseases of the CNS.
Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Features of Protein Encoded by Gene No: 21
This gene is expressed primarily in bone marrow, CD34 positive cells, and immune cells, including, neutrophils, T-cells, B-cells, macrophages, monocytes, and dendritic cells and to a lesser extent in brain and tonsils.
In one embodiments, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention may comprise, or alternatively consist of the following amino acid sequence:







(SEQ ID NO: 163)


VDGIDKLDIEFLQQFLETHSRGPRLHSPGHASQEATPGANMSSGTELLWP


 


GAALLVLLGVAASLCVRCSRPGAKRSEKIYQQRSLREDQQSFTGSRTYSL


 


VGQAWPGPLADMAPTRKDKLLQFYPSLEDPASSRYQNFSKGSRHGSEEAY


 


IDPIAMEYYNWGRFSKPPEDDDANSYENVLICKQKTTETGAQQEGIGGLC


 


RGDLSLSLALKTGPTSGLCPSASPEEDEGI.






Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments may comprise or alternatively consist of one, two, three, four or more of the following amino acid sequence:







(SEQ ID NO: 164)


ASSRYQNFSKGSRHGSEEAYIDPIA,


 


(SEQ ID NO: 165)


MEYYNWGRFSKPPEDDDANSY,


 


(SEQ ID NO: 166)


ENVLICKQKTTETGAQQEGIGGLCRGD,


 


(SEQ ID NO: 167)


VRCSRPGAKRSEKIYQQRSLREDQQSFTGSRTYSLVGQAWPGPLADMAPTR


 


KDKLLQFYPSLEDPASS


and


 


(SEQ ID NO: 168)


LSLSLALKTGPTSGLCPSASPEEDEGI.






Polynucleotides encoding these polypeptide fragments (SEQ ID NOs: 164, 165, 166, 167, and/or 168), polynucleotides that hybridize to the complementary strand of these polynucleotides (e.g., under the hybridization conditions described herein) are encompassed by the invention, as are the polypeptides encoded by these hybridizing polynucleotides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptides of the present invention comprise, or alternatively consist of one, two, three, four, five, or more of the immunogenic epitopes shown in SEQ ID NO:160 as residues: Ser-29 to Thr-57, Pro-74 to Lys-79, Pro-85 to Glu-107, Tyr-118 to Tyr-136, Gln-144 to Gln-152, Ala-182 to Glu-188. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also preferred are polypeptides comprising the mature polypeptide which is predicted to consist of residues 26-190 of the foregoing sequence (SEQ ID NO:160), and biologically active fragments of the mature polypeptide (e.g., fragments that stimulate the proliferation of bone marrow CD34+ cells).
FIGS. 9A-B show the nucleotide (SEQ ID NO:159) and deduced amino acid sequence (SEQ ID NO:160) of this polypeptide.
FIG. 10 shows an analysis of the amino acid sequence (SEQ ID NO:160). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. The domains defined by these graphs are contemplated by the present invention.
The data presented in FIG. 10 are also represented in tabular form in Table 12. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 10, and Table 12: “Res”: amino acid residue of SEQ ID NO:160 and FIGS. 9A-B; “Position”: position of the corresponding residue within SEQ ID NO:160 and FIGS. 9A-B; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.
Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 10 and/or Table 12, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 12 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in FIG. 10, but may, as shown in Table 12, be represented or identified by using tabular representations of the data presented in FIG. 10. The DNA*STAR computer algorithm used to generate FIG. 10 (set on the original default parameters) was used to present the data in FIG. 10 in a tabular format (See Table 12). The tabular format of the data in FIG. 10 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in FIG. 10 and in Table 12 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in FIGS. 9A-B (SEQ ID NO:160). As set out in FIG. 10 and in Table 12, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:159 is intended DNA fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:159. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:159. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, and from about 601 to about 650, and from about 651 to about 700, and from about 701 to about 750, and from about 751 to about 800, and from about 801 to about 850, and from about 851 to about 900, and from about 901 to about 950, and from about 951 to about 1000, and from about 1001 to about 1050, and from about and from about 1051 to about 1100, and from about 1101 to about 1150, and from about 1151 to about 1200, and from about 1201 to about 1250, and from about 1251 to about 1300, and from about 1301 to about 1350, and from about 1351 to about 1400, and from about 1401 to about 1450, and from about 1451 to about 1500, and from about 1501 to about 1551, and from about 1551 to about 1600, and from about 1601 to about 1650, and from about 1651 to about 1700, and from about 1701 to about 1750, and from about 1751 to about 1797 of SEQ ID NO:159, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.
Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity. By a polypeptide demonstrating a “functional activity” is meant to be a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) or mature-form of the protein. Such functional activities include, but are not limited to, biological activity (e.g., ability to regulate (e.g., stimulate) hematopoiesis in vitro or in vivo), antigenicity, and immunogenicity. The functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods described herein.
In addition, assays described herein and otherwise known in the art may routinely be applied to measure biological activity of polypeptides and fragments of the invention, variants derivatives and analogs thereof (e.g., to regulate (e.g., to stimulate or inhibit) hematopoiesis in vitro or in vivo). For example, techniques known in the art (such as for example assaying for thymidine incorporation), may be applied or routinely modified to assay for the ability of the compositions of the invention to inhibit proliferation of hematopoietic cells. Other methods will be known to the skilled artisan and are within the scope of the invention.
Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in FIGS. 9A-B (i.e., SEQ ID NO:159), up to the Glu residue at position number 185 and polynucleotides encoding such polypeptides.
Particularly, N-terminal deletions of the polypeptide can be described by the general formula m−190, where m is an integer from 2 to 184, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:160. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: V-26 to I-190; R-27 to I-190; C-28 to I-190; S-29 to I-190; R-30 to I-190; P-31 to I-190; G-32 to I-190; A-33 to I-190; K-34 to I-190; R-35 to I-190; S-36 to I-190; E-37 to I-190; K-38 to I-190; I-39 to I-190; Y-40 to I-190; Q-41 to I-190; Q-42 to I-190; R-43 to I-190; S-44 to I-190; L-45 to I-190; R-46 to I-190; E-47 to I-190; D-48 to I-190; Q-49 to I-190; Q-50 to I-190; S-51 to I-190; F-52 to I-190; T-53 to I-190; G-54 to I-190; S-55 to I-190; R-56 to I-190; T-57 to I-190; Y-58 to I-190; S-59 to I-190; L-60 to I-190; V-61 to I-190; G-62 to I-190; Q-63 to I-190; A-64 to I-190; W-65 to I-190; P-66 to I-190; G-67 to I-190; P-68 to I-190; L-69 to I-190; A-70 to I-190; D-71 to I-190; M-72 to I-190; A-73 to I-190; P-74 to I-190; T-75 to I-190; R-76 to I-190; K-77 to I-190; D-78 to I-190; K-79 to I-190; L-80 to I-190; L-81 to I-190; Q-82 to I-190; F-83 to I-190; Y-84 to I-190; P-85 to I-190; S-86 to I-190; L-87 to I-190; E-88 to I-190; D-89 to I-190; P-90 to I-190; A-91 to I-190; S-92 to I-190; S-93 to I-190; R-94 to I-190; Y-95 to I-190; Q-96 to I-190; N-97 to I-190; F-98 to I-190; S-99 to I-190; K-100 to I-190; G-101 to I-190; S-102 to I-190; R-103 to I-190; H-104 to I-190; G-105 to I-190; S-106 to I-190; E-107 to I-190; E-108 to I-190; A-109 to I-190; Y-110 to I-190; I-111 to I-190; D-112 to I-190; P-113 to I-190; I-114 to I-190; A-115 to I-190; M-116 to I-190; E-117 to I-190; Y-118 to I-190; Y-119 to I-190; N-120 to I-190; W-121 to I-190; G-122 to I-190; R-123 to I-190; F-124 to I-190; S-125 to I-190; K-126 to I-190; P-127 to I-190; P-128 to I-190; E-129 to I-190; D-130 to I-190; D-131 to I-190; D-132 to I-190; A-133 to I-190; N-134 to I-190; S-135 to I-190; Y-136 to I-190; E-137 to I-190; N-138 to I-190; V-139 to I-190; L-140 to I-190; I-141 to I-190; C-142 to I-190; K-143 to I-190; Q-144 to I-190; K-145 to I-190; T-146 to I-190; T-147 to I-190; E-148 to I-190; T-149 to I-190; G-150 to I-190; A-151 to I-190; Q-152 to I-190; Q-153 to I-190; E-154 to I-190; G-155 to I-190; I-156 to I-190; G-157 to I-190; G-158 to I-190; L-159 to I-190; C-160 to I-190; R-161 to I-190; G-162 to I-190; D-163 to I-190; L-164 to I-190; S-165 to I-190; L-166 to I-190; S-167 to I-190; L-168 to I-190; A-169 to I-190; L-170 to I-190; K-171 to I-190; T-172 to I-190; G-173 to I-190; P-174 to I-190; T-175 to I-190; S-176 to I-190; G-177 to I-190; L-178 to I-190; C-179 to I-190; P-180 to I-190; S-181 to I-190; A-182 to I-190; S-183 to I-190; P-184 to I-190; and E-185 to I-190, of SEQ ID NO:160. Polypeptides encoding these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an mutein with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIGS. 9A-B (SEQ ID NO:160), as described by the general formula 1−n, where n is an integer from 6 to 184 where n corresponds to the position of amino acid residue identified in SEQ ID NO:160. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: V-26 to G-189; V-26 to E-188; V-26 to D-187; V-26 to E-186; V-26 to E-185; V-26 to P-184; V-26 to S-183; V-26 to A-182; V-26 to S-181; V-26 to P-180; V-26 to C-179; V-26 to L-178; V-26 to G-177; V-26 to S-176; V-26 to T-175; V-26 to P-174; V-26 to G-173; V-26 to T-172; V-26 to K-171; V-26 to L-170; V-26 to A-169; V-26 to L-168; V-26 to S-167; V-26 to L-166; V-26 to S-165; V-26 to L-164; V-26 to D-163; V-26 to G-162; V-26 to R-161; V-26 to C-160; V-26 to L-159; V-26 to G-158; V-26 to G-157; V-26 to I-156; V-26 to G-155; V-26 to E-154; V-26 to Q-153; V-26 to Q-152; V-26 to A-151; V-26 to G-150; V-26 to T-149; V-26 to E-148; V-26 to T-147; V-26 to T-146; V-26 to K-145; V-26 to Q-144; V-26 to K-143; V-26 to C-142; V-26 to I-141; V-26 to L-140; V-26 to V-139; V-26 to N-138; V-26 to E-137; V-26 to Y-136; V-26 to S-135; V-26 to N-134; V-26 to A-133; V-26 to D-132; V-26 to D-131; V-26 to D-130; V-26 to E-129; V-26 to P-128; V-26 to P-127; V-26 to K-126; V-26 to S-125; V-26 to F-124; V-26 to R-123; V-26 to G-122; V-26 to W-121; V-26 to N-120; V-26 to Y-119; V-26 to Y-118; V-26 to E-117; V-26 to M-116; V-26 to A-115; V-26 to I-114; V-26 to P-113; V-26 to D-112; V-26 to I-111; V-26 to Y-110; V-26 to A-109; V-26 to E-108; V-26 to E-107; V-26 to S-106; V-26 to G-105; V-26 to H-104; V-26 to R-103; V-26 to S-102; V-26 to G-101; V-26 to K-100; V-26 to S-99; V-26 to F-98; V-26 to N-97; V-26 to Q-96; V-26 to Y-95; V-26 to R-94; V-26 to S-93; V-26 to S-92; V-26 to A-91; V-26 to P-90; V-26 to D-89; V-26 to E-88; V-26 to L-87; V-26 to S-86; V-26 to P-85; V-26 to Y-84; V-26 to F-83; V-26 to Q-82; V-26 to L-81; V-26 to L-80; V-26 to K-79; V-26 to D-78; V-26 to K-77; V-26 to R-76; V-26 to T-75; V-26 to P-74; V-26 to A-73; V-26 to M-72; V-26 to D-71; V-26 to A-70; V-26 to L-69; V-26 to P-68; V-26 to G-67; V-26 to P-66; V-26 to W-65; V-26 to A-64; V-26 to Q-63; V-26 to G-62; V-26 to V-61; V-26 to L-60; V-26 to S-59; V-26 to Y-58; V-26 to T-57; V-26 to R-56; V-26 to S-55; V-26 to G-54; V-26 to T-53; V-26 to F-52; V-26 to S-51; V-26 to Q-50; V-26 to Q-49; V-26 to D-48; V-26 to E-47; V-26 to R-46; V-26 to L-45; V-26 to S-44; V-26 to R-43; V-26 to Q-42; V-26 to Q-41; V-26 to Y-40; V-26 to I-39; V-26 to K-38; V-26 to E-37; V-26 to S-36; V-26 to R-35; V-26 to K-34; V-26 to A-33; V-26 to G-32; and V-26 to P-31 of SEQ ID NO:160. Polypeptides encoding these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:160, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also included are a nucleotide sequence encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by the cDNA clone contained in ATCCTM Deposit No. 209889, where this portion excludes any integer of amino acid residues from 1 to about 184 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCCTM Deposit No. 209889, or any integer of amino acid residues from 1 to about 190 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCCTM Deposit No. 209889. Polynucleotides encoding all of the above deletion mutant polypeptide forms also are provided.
The present application is also directed to proteins containing polypeptides at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequence set forth herein m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N and C terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders affecting the immune and hematopoietic systems, particularly hematopoiesis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system and hematopoeitic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
This gene has been found to stimulate the proliferation of bone marrow CD34+ cells. This assay which is described in Example 52 herein is based on the ability of human CD34+ to proliferate in presence of hematopoietic growth factors and evaluates the ability of the polypeptides of the invention, and agonists and antagonists thereof, to stimulate or inhibit this proliferation.
The tissue distribution in immune and hematopoietic cells and tissues and the ability to stimulate the proliferation of bone marrow CD34+ cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51 and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Moreover, the protein represents a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
The polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof, can also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.
The polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof, may also be employed for the expansion of immature hematopoietic progenitor cells, for example, granulocytes, macrophages or monocytes, by temporarily preventing their differentiation. These bone marrow cells may be cultured in vitro. Thus, polynucleotides and/or polypeptides of the invention, or agonists or antagonists thereof, may be useful as a modulator of hematopoietic stem cells in vitro for the purpose of bone marrow transplantation and/or gene therapy. Since stem cells are rare and are most useful for introducing genes into for gene therapy, polynucleotides and/or polypeptides of the invention can be used to isolate enriched populations of stem cells. Stem cells can be enriched by culturing cells in the presence of cytotoxins, such as 5-Fu, which kills rapidly dividing cells, where as the stem cells will be protected by polynucleotides and/or polypeptides of the invention. These stem cells can be returned to a bone marrow transplant patient or can then be used for transfection of the desired gene for gene therapy. In addition, this gene can be injected into animals which results in the release of stem cells from the bone marrow of the animal into the peripheral blood. These stem cells can be isolated for the purpose of autologous bone marrow transplantation or manipulation for gene therapy. After the patient has finished chemotherapy or radiation treatment, the isolated stem cells can be returned to the patient.
Polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
This gene product may also be involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
This gene may also have a very wide range of biological activities. Representative uses are described in the “Chemotaxis” and “Binding Activity” sections below, in Examples 31, 42, 44, 45, 46, 47, 49, 50, and 51, and elsewhere herein. Briefly, the protein may possess the following activities: cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g. for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of hematopoiesis (e.g. for treating anemia or as adjunct to chemotherapy); stimulation or growth of bone, cartilage, tendons, ligaments and/or nerves (e.g. for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g. for treating infections, tumors); hemostatic or thrombolytic activity (e.g. for treating hemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g. for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative diseases; for regulation of metabolism, and behavior. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Based upon the proteins immune cell specific message distribution, it may be involved in many aspects of the immune response, especially its initial stages, inflammation, allograft rejection, infectious disease response etc. It is frequently found in the hematopoietic cell cDNA libraries. Thus, this factor could be involved in the control of hematopoietic cell proliferation, differentiation, and function. Based on this one can postulate its use in the management of anemias, leukemias, neutropenia, thrombocytopenia, autoimmune diseases, blood tissue engraftment, and poikilothromerythromatosis. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
The gene encoding the disclosed cDNA is believed to reside on chromosome 7. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 7.
Features of Protein Encoded by Gene No: 22
Preferred polypeptides of the invention comprise the following amino acid sequences:







(SEQ ID NO: 171)


FEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTF


 


SCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSLLPTLNRTFIWDVKA


 


HKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGT


 


VSRIKM,


 


(SEQ ID NO: 172)


MAGLNCGVSIALLGVLLLGAARLPRGAEAFEIALPRESNITVLIKLGTPTL


 


LAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSG


 


PCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGE


 


SCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKMQEGVKMALHLPWFHPR


 


NVSGFSIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQFV


 


VPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMA


 


GNFNLSLQGCDQDAQSPGILRLQFQVLVQHPQNESNKIYVVDLSNERAMSL


 


TIEPRPVKQSRKFVPGCFVCLESRTCSSNLTLTSGSKHKISFLCDDLTRLW


 


MNVEKP


and/or


 


(SEQ ID NO: 173)


GTRAAPGLGAWGRRSPPSFSPPRPRRPGVMAGLNCGVSIALLGVLLLGAAR


 


LPRGAEAFEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSG


 


ERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSLLPTLNRT


 


FIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIG


 


TFCSNGTVSRIKMQEGVKMALHLPWFHPRNVSGFSIANRSSIKRLCIIESV


 


FEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERK


 


EERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSLQGCDQDAQSPGILRL


 


QFQVLVQHPQNESNKIYVVDLSNERAMSLTIEPRPVKQSRKFVPGCFVCLE


 


SRTCSSNLTLTSGSKHKISFLCDDLTRLWMNVEKP.






Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in placenta, and to a lesser extent in, prostate and ovary.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, male and female infertility, and associated disorders of the reproductive system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, or cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution of this gene in the prostate, placenta and ovary indicates that polynucleotides and polypeptides corresponding to this gene are useful for treatment, prevention, and/or diagnosis of male or female infertility, endocrine disorders, fetal deficiencies, ovarian failure, amenorrhea, ovarian cancer, benign prostate hyperplasia and prostate cancer. Similarly, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of cancer and other proliferative disorders. Expression within placental tissue and other cellular sources marked by proliferating cells indicates that this protein may play a role in the regulation of cellular division. Similarly, embryonic development also involves decisions involving cell differentiation and/or apoptosis in pattern formation. Thus, this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:169 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2195 of SEQ ID NO:169, b is an integer of 15 to 2209, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:169, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 23
The translation product of this gene was found to be homologous to cell surface adhesion molecule (CAM) proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with CAM proteins. Such activities are known in the art, some of which are described elsewhere herein. A preferred polypeptide variant of the invention comprises the following amino acid sequence:







(SEQ ID NO: 178)


MLCPWRTANLGLLLILTIFLVAEAEGAAQPNNSLMLQTSKENHALASSSL


 


CMDEKQITQNYSKVLAEVNTSWPVKMATNAVLCCPPIALRNLIIITWEII


 


LRGQPSCTKAYKKETNETKETNCTDERITWVSRPDQNSDLQIRTVAITHD


 


GYYRCIMVTPDGNFHRGYHLQVLVTPEVTLFQNRNRTAVCKAVAGKPAAH


 


ISWIPEGDCATKQEYWSNGTVTVKSTCHWEVHNVSTVNCHVSHLTGNKSL


 


YIELLPVPGAKKSSKLYIPYIILTIIILTIVGXIWLLKVNGCXKYKLNKP


 


ESTPVVEEDEMQPYAFYTEKNNPLXXTTNKVKASEALQSEVDTDLHTL.






Polynucleotides encoding these polypeptides are also provided.

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 271-287 of the amino acid sequence referenced in Table 1A for this gene. Moreover, a cytoplasmic tail encompassing amino acids 288 to 348 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.
This gene is expressed primarily in dendritic cells.
Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immunodeficiency, tumor necrosis, infection, lymphomas, auto-immunities, cancer, metastasis, wound healing, inflammation, anemias (leukemia) and other hematopoeitic disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:175 as residues: Asp-53 to Tyr-61, Pro-105 to Ile-128, Arg-133 to Leu-140, Gln-182 to Ala-188, Pro-205 to Asn-218, Gly-259 to Ala-264, Asn-290 to Ser-302, Glu-307 to Tyr-314, Tyr-317 to Lys-332. Polynucleotides encoding said polypeptides are also provided.
The tissue distribution in dendritic cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g. AIDS), immuno-suppressive conditions (transplantation) and hematopoeitic disorders. In addition this gene product may be applicable in conditions of general microbial infection, inflammation or cancer. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:174 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3784 of SEQ ID NO:174, b is an integer of 15 to 3798, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:174, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 24
The translation product of this gene shares sequence homology with CMRF-35 antigen [Homo sapiens] (See, e.g., Genbank accession number AAD01646 and CAA46948; all references available through these accessions are hereby incorporated by reference herein), which is thought to be important as a cell membrane antigen present on the surface of monocytes, neutrophils, a proportion of peripheral blood T and B lymphocytes and lymphocytic cell lines.
The translation product of this gene also shares sequence homology with PIGR-1 protein (see, e.g., Genseq accession number W99070 which is a member of the Immunoglobulin (Ig) superfamily). All references available through this Genseq accession are hereby incorporated by reference herein. Based on the sequence similarity, the translation product of this clone is expected to share at least some biological activities with proteins of the Immunoglobulin superfamily such as, for example, PIGR-1 and CMRF-35 Ag. Such activities are known in the art, some of which are described elsewhere herein.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence:









(SEQ ID NO: 181)



EGGSSRARXSTSRRLGVCSLFLLPGSTEGNGDLSEEK.






Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 157-173 of the amino acid sequence referenced in Table 1A for this gene. Moreover, a cytoplasmic tail encompassing amino acids 174-290 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.
This gene is expressed primarily in eosinophils, and to a lesser extent in dendritic cells and activated monocytes.
Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred polypeptides of the present invention comprise, or alternatively consist of one, two, three, or all four, of the immunogenic epitopes shown in SEQ ID NO:180 as residues: Ser-69 to Arg-79, Ile-82 to Arg-89, Pro-129 to Ser-137, Leu-146 to Lys-151. Polynucleotides encoding said polypeptides are also encompassed by the invention.
The tissue distribution in eosinophils, monocytes, and dendritic cells, and the homology to CMRF-35 antigen, indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of immune disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Expression of this gene product in eosinophils, monocytes, and dendritic cells also strongly indicates a role for this protein in immune function and immune surveillance. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:179 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1782 of SEQ ID NO:179, b is an integer of 15 to 1796, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:179, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 25
This gene is expressed in the following tissues/cDNA libraries: B Cell lymphoma; pBMC stimulated w/ poly I/C; B-cells (unstimulated); NCI_CGAP_GCB1.
The tissue distribution indicates polynucleotides and polypeptides corresponding to this gene, as well as antibodies against those polypeptides, may be useful for the diagnosis, prevention, and/or treatment of immune system disorders; particularly immune cell proliferative disorders (e.g. leukemia), autoimmune disorders, and immunodeficiencies (including immunodeficiencies caused by genetic factors, microbial pathogens (e.g. HIV), chemotherapy, and radiation). See “Immune Activity” section, infra. The tissue distribution also indicates polynucleotides and polypeptides corresponding to this gene, as well as antibodies against those polypeptides, may be useful for the diagnosis, prevention, and/or treatment of cancer and other hyperproliferative disorders (e.g., see “Hyperproliferative Disorders” section, infra).
Features of Protein Encoded by Gene No: 26
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been putatively identified as a human integrin alpha 11 homolog derived from a human osteoblast II cDNA library. More particularly, the polypeptide of the present invention has been putatively identified as a human integrin alpha 11-subunit homolog, sometimes hereafter referred to as “integrin alpha 11”, “integrin alpha 11-subunit”, “a11”, “A11-subunit”, and/or “Integrin all-subunit”. The invention also relates to inhibiting the action of such polypeptides. The integrins are a large family of cell adhesion molecules consisting of noncovalently associated ab heterodimers.
We have cloned and sequenced a novel human integrin α-subunit cDNA, designated a11. The all cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120 residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced all protein shows the typical structural features of integrin a-subunits and is similar to a distinct group of a-subunits from collagen-binding integrins. However, it differs from most integrin a-chains by an incompletely preserved cytoplasmic GFFKR motif.
The human ITGA11 gene was located to bands q22.3-23 on chromosome 15, and its transcripts were found predominantly in bone, cartilage as well as in cardiac and skeletal muscle. Expression of the 5.5 kilobase all mRNA was also detectable in ovary and small intestine.
All vertebrate cells express members of the integrin family of cell adhesion molecules, which mediate cellular adhesion to other cells and extracellular subtratum, cell migration and participate in important physiologic processes from signal transduction to cell proliferation and differentiation (Hynes, 92; Springer, 92).
Integrins are structurally homologous heterodimeric type-I membrane glycoproteins formed by the noncovalent association of one of eight b-subunits with one of the 17 different a-subunits described to date, resulting in at least 22 different ab complexes. Their binding specificities for cellular and extracellular ligands are determined by both subunits and are dynamically regulated in a cell-type-specific mode by the cellular environment as well as by the developmental and activation state of the cell (Diamond and Springer, 94). In integrin a-subunits, the aminoterminal region of the large extracellular domain consists of a seven-fold repeated structure which is predicted to fold into a b-propeller domain (Corbi et al., 1987; Springer, 1997). The three or four C-terminal repeats contain putative divalent cation binding motifs that are thought to be important for ligand binding and subunit association (Diamond and Springer, 94). The a1, a2, a 10, aD, aE, aL, aM and aX-subunits contain an approximately 200 amino acid I-domain inserted between the second and third repeat that is not present in other a-chains (Larson et al., 1989). Several isolated I-domains have been shown to independently bind the ligands of the parent integrin heterodimer (Kamata and Takada, 1994; Randi and Hogg, 1994). The a3, a5-8, aIIb and aV-subunits are proteolytically processed at a conserved site into disulphide-linked heavy and light chains, while the a4-subunit is cleaved at a more aminoterminal site into two fragments that remain noncovalently associated (Hemler et al., 90). Additional a-subunit variants are generated by alternative splicing of primary transcripts (Ziober et al., 93; Delwel et al., 95; Leung et al., 98).
The extracellular domains of a-integrin subunits are connected by a single spanning transmembrane domain to short, diverse cytoplasmic domains whose only conserved feature is a membrane-proximal KXGFF(K/R)R motif (Sastry and Horwitz, 1993). The cytoplasmic domains have been implicated in the cell-type-specific modulation of integrin affinity states (Williams et al., 1994).
The polypeptide of the present invention has been putatively identified as a member of the integrin family and has been termed integrin alpha 11 subunit (“a11”). This identification has been made as a result of amino acid sequence homology to the human integrin alpha 1 subunit (See Genbank Accession No. gi|346210).
FIGS. 11A-F show the nucleotide (SEQ ID NO:186) and deduced amino acid sequence (SEQ ID NO:187) of a11. Predicted amino acids from about 1 to about 22 constitute the predicted signal peptide (amino acid residues from about 1 to about 22 in SEQ ID NO:187) and are represented by the underlined amino acid regions; amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 constitute the predicted transmembrane domains (amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 in SEQ ID NO:187) and are represented by the double underlined amino acids; and amino acids from about 64 to about 96 constitute the predicted immunoglobulin and major histocompatibility complex protein domain (amino acids from about 64 to about 96 in SEQ ID NO:187) and are represented by the bold amino acids.
FIGS. 12A-E shows the regions of similarity between the amino acid sequences of the integrin alpha 11 subunit (a11) protein (SEQ ID NO:187) and the human integrin alpha 1 subunit (SEQ ID NO:191).
FIG. 13 shows an analysis of the integrin alpha 11 subunit (a11) amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
Its translation product has homology to the characteristic immunoglobulin and major histocompatibility complex protein domain of integrin family members. As shown in FIGS. 11A-F, a11 has transmembrane domains (the transmembrane domains comprise amino acids 666-682 and/or 1145-1161 of SEQ ID NO:187; which correspond to amino acids 666-682 and/or 1145-1161 of FIGS. 11A-F) with strong conservation between other members of the integrin family. The polynucleotide contains an open reading frame encoding the a11 polypeptide of 1189 amino acids. The present invention exhibits a high degree of homology at the amino acid level to the human integrin alpha 1 subunit (as shown in FIGS. 12A-E).
Preferred polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence:









 (SEQ ID NO: 190)



TNGYQKTGDVYKCPVIHGNCTKLNLGRVTLSNV.






Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.

Preferred polypeptides of the present invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, or all thirty-three of the immunogenic epitopes shown in SEQ ID NO:187 as residues: Phe-23 to Arg-31, Leu-62 to Asp-72, Val-96 to Asp-101, Thr-111 to Asn-116, Glu-128 to Thr-135, Val-142 to Ser-149, Asn-217 to Val-222, Glu-233 to Arg-241, Gly-272 to Leu-280, Gln-286 to Thr-293, Tyr-303 to Ile-308, Gly-354 to Thr-360, Glu-408 to Lys-419, Glu-508 to Lys-514, Arg-521 to Val-526, Gly-529 to Phe-542, Asp-551 to Tyr-557, Thr-587 to Thr-593, His-656 to Asp-665, Met-697 to Arg-705, Asp-709 to Thr-716, Glu-755 to Gly-760, Asn-779 to His-786, Leu-810 to Asp-816, Leu-844 to Ala-851, Gln-871 to Gly-877, Glu-884 to Gln-889, Ser-931 to Asn-943, Ser-974 to Ile-982, Gly-1039 to Gln-1058, Arg-1121 to Arg-1127, Ser-1134 to Trp-1139, and/or Ser-1172 to Pro-1183. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the a11 polypeptide having the amino acid sequence shown in FIGS. 11A-F (SEQ ID NO:187). The nucleotide sequence shown in FIGS. 11A-F (SEQ ID NO:186) was obtained by sequencing a cloned cDNA (HOHBY69), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.
The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:186 is intended DNA fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:186. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:186. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of a11 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650, from about 1651 to about 1700, from about 1701 to about 1750, from about 1751 to about 1800, from about 1801 to about 1850, from about 1851 to about 1900, from about 1901 to about 1950, from about 1951 to about 2000, from about 2001 to about 2050, from about 2051 to about 2100, from about 2101 to about 2150, from about 2151 to about 2200, from about 2201 to about 2250, from about 2251 to about 2300, from about 2301 to about 2350, from about 2351 to about 2400, from about 2401 to about 2450, from about 2451 to about 2500, from about 2501 to about 2550, from about 2551 to about 2600, from about 2601 to about 2650, from about 2651 to about 2700, from about 2701 to about 2750, from about 2751 to about 2800, from about 2801 to about 2850, from about 2851 to about 2900, from about 2901 to about 2950, from about 2951 to about 3000, from about 3001 to about 3050, from about 3051 to about 3100, from about 3101 to about 3150, from about 3151 to about 3200, from about 3201 to about 3250, from about 3251 to about 3300, from about 3301 to about 3350, from about 3351 to about 3400, from about 3401 to about 3450, from about 3451 to about 3500, from about 3501 to about 3550, from about 3551 to about 3600, from about 3601 to about 3650, from about 3651 to about 3700, from about 3701 to about 3750, from about 3751 to about 3800, from about 3801 to about 3850, from about 3851 to about 3900, from about 3901 to about 3950, from about 3951 to about 4000, from about 4001 to about 4050, from about 4051 to about 4100, from about 4101 to about 4150, from about 4151 to about 4200, from about 4201 to about 4250, from about 4251 to about 4300, from about 4301 to about 4350, from about 4351 to about 4400, from about 4401 to about 4450, from about 4451 to about 4500, from about 4501 to about 4550, from about 4551 to about 4600, from about 4601 to about 4650, from about 4651 to about 4700, from about 4701 to about 4750, from about 4751 to about 4800, from about 4801 to about 4850, from about 4851 to about 4900, from about 4901 to about 4950, from about 4951 to about 4995, from about, from about 1 to about 236, from about 144 to about 188, from about 231 to about 276 of SEQ ID NO:186, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, any one of the transmembrane domains (amino acid residues from about 666 to about 682 and/or 1145 to about 1161 in FIGS. 11A-F (amino acids from about 666 to about 682 and/or 1145 to about 1161 in SEQ ID NO:187), in addition to the immunoglobulin and major histocompatibility complex protein domain (amino acid residues from about 64 to about 96 in FIGS. 11A-F (amino acids from about 64 to about 96 in SEQ ID NO:187). Since the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 amino acid residues) depending on the criteria used to define each domain. In additional embodiments, the polynucleotides of the invention encode functional attributes of a11.
Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of the present invention.
The data representing the structural or functional attributes of all set forth in FIG. 13 and/or Table 13, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 13 can be used to determine regions of all which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in FIG. 13, but may, as shown in Table 13, be represented or identified by using tabular representations of the data presented in FIG. 13. The DNA*STAR computer algorithm used to generate FIG. 13 (set on the original default parameters) was used to present the data in FIG. 13 in a tabular format (See Table 13). The tabular format of the data in FIG. 13 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in FIG. 13 and in Table 13 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in FIGS. 11A-F. As set out in FIG. 13 and in Table 13, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened all muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an all mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six all amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the all amino acid sequence shown in FIGS. 11A-F, up to the threonine residue at position number 1184 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues n1-1189 of FIGS. 11A-F, where n1 is an integer from 2 to 1184 corresponding to the position of the amino acid residue in FIGS. 11A-F (which is identical to the sequence shown as SEQ ID NO:187). In another embodiment, N-terminal deletions of the all polypeptide can be described by the general formula n2−1189, where n2 is a number from 2 to 1184, corresponding to the position of amino acid identified in FIGS. 11A-F. N-terminal deletions of the a11 polypeptide of the invention shown as SEQ ID NO:187 include polypeptides comprising the amino acid sequence of residues: N-terminal deletions of the a11 polypeptide of the invention shown as SEQ ID NO:187 include polypeptides comprising the amino acid sequence of residues: D-2 to E-1189; L-3 to E-1189; P-4 to E-1189; R-5 to E-1189; G-6 to E-1189; L-7 to E-1189; V-8 to E-1189; V-9 to E-1189; A-10 to E-1189; W-11 to E-1189; A-12 to E-1189; L-13 to E-1189; S-14 to E-1189; L-15 to E-1189; W-16 to E-1189; P-17 to E-1189; G-18 to E-1189; F-19 to E-1189; T-20 to E-1189; D-21 to E-1189; T-22 to E-1189; F-23 to E-1189; N-24 to E-1189; M-25 to E-1189; D-26 to E-1189; T-27 to E-1189; R-28 to E-1189; K-29 to E-1189; P-30 to E-1189; R-31 to E-1189; V-32 to E-1189; I-33 to E-1189; P-34 to E-1189; G-35 to E-1189; S-36 to E-1189; R-37 to E-1189; T-38 to E-1189; A-39 to E-1189; F-40 to E-1189; F-41 to E-1189; G-42 to E-1189; Y-43 to E-1189; T-44 to E-1189; V-45 to E-1189; Q-46 to E-1189; Q-47 to E-1189; H-48 to E-1189; D-49 to E-1189; I-50 to E-1189; S-51 to E-1189; G-52 to E-1189; N-53 to E-1189; K-54 to E-1189; W-55 to E-1189; L-56 to E-1189; V-57 to E-1189; V-58 to E-1189; G-59 to E-1189; A-60 to E-1189; P-61 to E-1189; L-62 to E-1189; E-63 to E-1189; T-64 to E-1189; N-65 to E-1189; G-66 to E-1189; Y-67 to E-1189; Q-68 to E-1189; K-69 to E-1189; T-70 to E-1189; G-71 to E-1189; D-72 to E-1189; V-73 to E-1189; Y-74 to E-1189; K-75 to E-1189; C-76 to E-1189; P-77 to E-1189; V-78 to E-1189; I-79 to E-1189; H-80 to E-1189; G-81 to E-1189; N-82 to E-1189; C-83 to E-1189; T-84 to E-1189; K-85 to E-1189; L-86 to E-1189; N-87 to E-1189; L-88 to E-1189; G-89 to E-1189; R-90 to E-1189; V-91 to E-1189; T-92 to E-1189; L-93 to E-1189; S-94 to E-1189; N-95 to E-1189; V-96 to E-1189; S-97 to E-1189; E-98 to E-1189; R-99 to E-1189; K-100 to E-1189; D-101 to E-1189; N-102 to E-1189; M-103 to E-1189; R-104 to E-1189; L-105 to E-1189; G-106 to E-1189; L-107 to E-1189; S-108 to E-1189; L-109 to E-1189; A-110 to E-1189; T-111 to E-1189; N-112 to E-1189; P-113 to E-1189; K-114 to E-1189; D-115 to E-1189; N-116 to E-1189; S-117 to E-1189; F-118 to E-1189; L-119 to E-1189; A-120 to E-1189; C-121 to E-1189; S-122 to E-1189; P-123 to E-1189; L-124 to E-1189; W-125 to E-1189; S-126 to E-1189; H-127 to E-1189; E-128 to E-1189; C-129 to E-1189; G-130 to E-1189; S-131 to E-1189; S-132 to E-1189; Y-133 to E-1189; Y-134 to E-1189; T-135 to E-1189; T-136 to E-1189; G-137 to E-1189; M-138 to E-1189; C-139 to E-1189; S-140 to E-1189; R-141 to E-1189; V-142 to E-1189; N-143 to E-1189; S-144 to E-1189; N-145 to E-1189; F-146 to E-1189; R-147 to E-1189; F-148 to E-1189; S-149 to E-1189; K-150 to E-1189; T-151 to E-1189; V-152 to E-1189; A-153 to E-1189; P-154 to E-1189; A-155 to E-1189; L-156 to E-1189; Q-157 to E-1189; R-158 to E-1189; C-159 to E-1189; Q-160 to E-1189; T-161 to E-1189; Y-162 to E-1189; M-163 to E-1189; D-164 to E-1189; I-165 to E-1189; V-166 to E-1189; I-167 to E-1189; V-168 to E-1189; L-169 to E-1189; D-170 to E-1189; G-171 to E-1189; S-172 to E-1189; N-173 to E-1189; S-174 to E-1189; I-175 to E-1189; Y-176 to E-1189; P-177 to E-1189; W-178 to E-1189; V-179 to E-1189; E-180 to E-1189; V-181 to E-1189; Q-182 to E-1189; H-183 to E-1189; F-184 to E-1189; L-185 to E-1189; I-186 to E-1189; N-187 to E-1189; I-188 to E-1189; L-189 to E-1189; K-190 to E-1189; K-191 to E-1189; F-192 to E-1189; Y-193 to E-1189; I-194 to E-1189; G-195 to E-1189; P-196 to E-1189; G-197 to E-1189; Q-198 to E-1189; I-199 to E-1189; Q-200 to E-1189; V-201 to E-1189; G-202 to E-1189; V-203 to E-1189; V-204 to E-1189; Q-205 to E-1189; Y-206 to E-1189; G-207 to E-1189; E-208 to E-1189; D-209 to E-1189; V-210 to E-1189; V-211 to E-1189; H-212 to E-1189; E-213 to E-1189; F-214 to E-1189; H-215 to E-1189; L-216 to E-1189; N-217 to E-1189; D-218 to E-1189; Y-219 to E-1189; R-220 to E-1189; S-221 to E-1189; V-222 to E-1189; K-223 to E-1189; D-224 to E-1189; V-225 to E-1189; V-226 to E-1189; E-227 to E-1189; A-228 to E-1189; A-229 to E-1189; S-230 to E-1189; H-231 to E-1189; I-232 to E-1189; E-233 to E-1189; Q-234 to E-1189; R-235 to E-1189; G-236 to E-1189; G-237 to E-1189; T-238 to E-1189; E-239 to E-1189; T-240 to E-1189; R-241 to E-1189; T-242 to E-1189; A-243 to E-1189; F-244 to E-1189; G-245 to E-1189; I-246 to E-1189; E-247 to E-1189; F-248 to E-1189; A-249 to E-1189; R-250 to E-1189; S-251 to E-1189; E-252 to E-1189; A-253 to E-1189; F-254 to E-1189; Q-255 to E-1189; K-256 to E-1189; G-257 to E-1189; G-258 to E-1189; R-259 to E-1189; K-260 to E-1189; G-261 to E-1189; A-262 to E-1189; K-263 to E-1189; K-264 to E-1189; V-265 to E-1189; M-266 to E-1189; I-267 to E-1189; V-268 to E-1189; I-269 to E-1189; T-270 to E-1189; D-271 to E-1189; G-272 to E-1189; E-273 to E-1189; S-274 to E-1189; H-275 to E-1189; D-276 to E-1189; S-277 to E-1189; P-278 to E-1189; D-279 to E-1189; L-280 to E-1189; E-281 to E-1189; K-282 to E-1189; V-283 to E-1189; I-284 to E-1189; Q-285 to E-1189; Q-286 to E-1189; S-287 to E-1189; E-288 to E-1189; R-289 to E-1189; D-290 to E-1189; N-291 to E-1189; V-292 to E-1189; T-293 to E-1189; R-294 to E-1189; Y-295 to E-1189; A-296 to E-1189; V-297 to E-1189; A-298 to E-1189; V-299 to E-1189; L-300 to E-1189; G-301 to E-1189; Y-302 to E-1189; Y-303 to E-1189; N-304 to E-1189; R-305 to E-1189; R-306 to E-1189; G-307 to E-1189; I-308 to E-1189; N-309 to E-1189; P-310 to E-1189; E-311 to E-1189; T-312 to E-1189; F-313 to E-1189; L-314 to E-1189; N-315 to E-1189; E-316 to E-1189; I-317 to E-1189; K-318 to E-1189; Y-319 to E-1189; I-320 to E-1189; A-321 to E-1189; S-322 to E-1189; D-323 to E-1189; P-324 to E-1189; D-325 to E-1189; D-326 to E-1189; K-327 to E-1189; H-328 to E-1189; F-329 to E-1189; F-330 to E-1189; N-331 to E-1189; V-332 to E-1189; T-333 to E-1189; D-334 to E-1189; E-335 to E-1189; A-336 to E-1189; A-337 to E-1189; L-338 to E-1189; K-339 to E-1189; D-340 to E-1189; I-341 to E-1189; V-342 to E-1189; D-343 to E-1189; A-344 to E-1189; L-345 to E-1189; G-346 to E-1189; D-347 to E-1189; R-348 to E-1189; I-349 to E-1189; F-350 to E-1189; S-351 to E-1189; L-352 to E-1189; E-353 to E-1189; G-354 to E-1189; T-355 to E-1189; N-356 to E-1189; K-357 to E-1189; N-358 to E-1189; E-359 to E-1189; T-360 to E-1189; S-361 to E-1189; F-362 to E-1189; G-363 to E-1189; L-364 to E-1189; E-365 to E-1189; M-366 to E-1189; S-367 to E-1189; Q-368 to E-1189; T-369 to E-1189; G-370 to E-1189; F-371 to E-1189; S-372 to E-1189; S-373 to E-1189; H-374 to E-1189; V-375 to E-1189; V-376 to E-1189; E-377 to E-1189; D-378 to E-1189; G-379 to E-1189; V-380 to E-1189; L-381 to E-1189; L-382 to E-1189; G-383 to E-1189; A-384 to E-1189; V-385 to E-1189; G-386 to E-1189; A-387 to E-1189; Y-388 to E-1189; D-389 to E-1189; W-390 to E-1189; N-391 to E-1189; G-392 to E-1189; A-393 to E-1189; V-394 to E-1189; L-395 to E-1189; K-396 to E-1189; E-397 to E-1189; T-398 to E-1189; S-399 to E-1189; A-400 to E-1189; G-401 to E-1189; K-402 to E-1189; V-403 to E-1189; I-404 to E-1189; P-405 to E-1189; L-406 to E-1189; R-407 to E-1189; E-408 to E-1189; S-409 to E-1189; Y-410 to E-1189; L-411 to E-1189; K-412 to E-1189; E-413 to E-1189; F-414 to E-1189; P-415 to E-1189; E-416 to E-1189; E-417 to E-1189; L-418 to E-1189; K-419 to E-1189; N-420 to E-1189; H-421 to E-1189; G-422 to E-1189; A-423 to E-1189; Y-424 to E-1189; L-425 to E-1189; G-426 to E-1189; Y-427 to E-1189; T-428 to E-1189; V-429 to E-1189; T-430 to E-1189; S-431 to E-1189; V-432 to E-1189; V-433 to E-1189; S-434 to E-1189; S-435 to E-1189; R-436 to E-1189; Q-437 to E-1189; G-438 to E-1189; R-439 to E-1189; V-440 to E-1189; Y-441 to E-1189; V-442 to E-1189; A-443 to E-1189; G-444 to E-1189; A-445 to E-1189; P-446 to E-1189; R-447 to E-1189; F-448 to E-1189; N-449 to E-1189; H-450 to E-1189; T-451 to E-1189; G-452 to E-1189; K-453 to E-1189; V-454 to E-1189; I-455 to E-1189; L-456 to E-1189; F-457 to E-1189; T-458 to E-1189; M-459 to E-1189; H-460 to E-1189; N-461 to E-1189; N-462 to E-1189; R-463 to E-1189; S-464 to E-1189; L-465 to E-1189; T-466 to E-1189; I-467 to E-1189; H-468 to E-1189; Q-469 to E-1189; A-470 to E-1189; M-471 to E-1189; R-472 to E-1189; G-473 to E-1189; Q-474 to E-1189; Q-475 to E-1189; I-476 to E-1189; G-477 to E-1189; S-478 to E-1189; Y-479 to E-1189; F-480 to E-1189; G-481 to E-1189; S-482 to E-1189; E-483 to E-1189; I-484 to E-1189; T-485 to E-1189; S-486 to E-1189; V-487 to E-1189; D-488 to E-1189; I-489 to E-1189; D-490 to E-1189; G-491 to E-1189; D-492 to E-1189; G-493 to E-1189; V-494 to E-1189; T-495 to E-1189; D-496 to E-1189; V-497 to E-1189; L-498 to E-1189; L-499 to E-1189; V-500 to E-1189; G-501 to E-1189; A-502 to E-1189; P-503 to E-1189; M-504 to E-1189; Y-505 to E-1189; F-506 to E-1189; N-507 to E-1189; E-508 to E-1189; G-509 to E-1189; R-510 to E-1189; E-511 to E-1189; R-512 to E-1189; G-513 to E-1189; K-514 to E-1189; V-515 to E-1189; Y-516 to E-1189; V-517 to E-1189; Y-518 to E-1189; E-519 to E-1189; L-520 to E-1189; R-521 to E-1189; Q-522 to E-1189; N-523 to E-1189; R-524 to E-1189; F-525 to E-1189; V-526 to E-1189; Y-527 to E-1189; N-528 to E-1189; G-529 to E-1189; T-530 to E-1189; L-531 to E-1189; K-532 to E-1189; D-533 to E-1189; S-534 to E-1189; H-535 to E-1189; S-536 to E-1189; Y-537 to E-1189; Q-538 to E-1189; N-539 to E-1189; A-540 to E-1189; R-541 to E-1189; F-542 to E-1189; G-543 to E-1189; S-544 to E-1189; S-545 to E-1189; I-546 to E-1189; A-547 to E-1189; S-548 to E-1189; V-549 to E-1189; R-550 to E-1189; D-551 to E-1189; L-552 to E-1189; N-553 to E-1189; Q-554 to E-1189; D-555 to E-1189; S-556 to E-1189; Y-557 to E-1189; N-558 to E-1189; D-559 to E-1189; V-560 to E-1189; V-561 to E-1189; V-562 to E-1189; G-563 to E-1189; A-564 to E-1189; P-565 to E-1189; L-566 to E-1189; E-567 to E-1189; D-568 to E-1189; N-569 to E-1189; H-570 to E-1189; A-571 to E-1189; G-572 to E-1189; A-573 to E-1189; I-574 to E-1189; Y-575 to E-1189; I-576 to E-1189; F-577 to E-1189; H-578 to E-1189; G-579 to E-1189; F-580 to E-1189; R-581 to E-1189; G-582 to E-1189; S-583 to E-1189; I-584 to E-1189; L-585 to E-1189; K-586 to E-1189; T-587 to E-1189; P-588 to E-1189; K-589 to E-1189; Q-590 to E-1189; R-591 to E-1189; I-592 to E-1189; T-593 to E-1189; A-594 to E-1189; S-595 to E-1189; E-596 to E-1189; L-597 to E-1189; A-598 to E-1189; T-599 to E-1189; G-600 to E-1189; L-601 to E-1189; Q-602 to E-1189; Y-603 to E-1189; F-604 to E-1189; G-605 to E-1189; C-606 to E-1189; S-607 to E-1189; I-608 to E-1189; H-609 to E-1189; G-610 to E-1189; Q-611 to E-1189; L-612 to E-1189; D-613 to E-1189; L-614 to E-1189; N-615 to E-1189; E-616 to E-1189; D-617 to E-1189; G-618 to E-1189; L-619 to E-1189; I-620 to E-1189; D-621 to E-1189; L-622 to E-1189; A-623 to E-1189; V-624 to E-1189; G-625 to E-1189; A-626 to E-1189; L-627 to E-1189; G-628 to E-1189; N-629 to E-1189; A-630 to E-1189; V-631 to E-1189; I-632 to E-1189; L-633 to E-1189; W-634 to E-1189; S-635 to E-1189; R-636 to E-1189; P-637 to E-1189; V-638 to E-1189; V-639 to E-1189; Q-640 to E-1189; I-641 to E-1189; N-642 to E-1189; A-643 to E-1189; S-644 to E-1189; L-645 to E-1189; H-646 to E-1189; F-647 to E-1189; E-648 to E-1189; P-649 to E-1189; S-650 to E-1189; K-651 to E-1189; I-652 to E-1189; N-653 to E-1189; I-654 to E-1189; F-655 to E-1189; H-656 to E-1189; R-657 to E-1189; D-658 to E-1189; C-659 to E-1189; K-660 to E-1189; R-661 to E-1189; S-662 to E-1189; G-663 to E-1189; R-664 to E-1189; D-665 to E-1189; A-666 to E-1189; T-667 to E-1189; C-668 to E-1189; L-669 to E-1189; A-670 to E-1189; A-671 to E-1189; F-672 to E-1189; L-673 to E-1189; C-674 to E-1189; F-675 to E-1189; T-676 to E-1189; P-677 to E-1189; I-678 to E-1189; F-679 to E-1189; L-680 to E-1189; A-681 to E-1189; P-682 to E-1189; H-683 to E-1189; F-684 to E-1189; Q-685 to E-1189; T-686 to E-1189; T-687 to E-1189; T-688 to E-1189; V-689 to E-1189; G-690 to E-1189; I-691 to E-1189; R-692 to E-1189; Y-693 to E-1189; N-694 to E-1189; A-695 to E-1189; T-696 to E-1189; M-697 to E-1189; D-698 to E-1189; E-699 to E-1189; R-700 to E-1189; R-701 to E-1189; Y-702 to E-1189; T-703 to E-1189; P-704 to E-1189; R-705 to E-1189; A-706 to E-1189; H-707 to E-1189; L-708 to E-1189; D-709 to E-1189; E-710 to E-1189; G-711 to E-1189; G-712 to E-1189; D-713 to E-1189; R-714 to E-1189; F-715 to E-1189; T-716 to E-1189; N-717 to E-1189; R-718 to E-1189; A-719 to E-1189; V-720 to E-1189; L-721 to E-1189; L-722 to E-1189; S-723 to E-1189; S-724 to E-1189; G-725 to E-1189; Q-726 to E-1189; E-727 to E-1189; L-728 to E-1189; C-729 to E-1189; E-730 to E-1189; R-731 to E-1189; I-732 to E-1189; N-733 to E-1189; F-734 to E-1189; H-735 to E-1189; V-736 to E-1189; L-737 to E-1189; D-738 to E-1189; T-739 to E-1189; A-740 to E-1189; D-741 to E-1189; Y-742 to E-1189; V-743 to E-1189; K-744 to E-1189; P-745 to E-1189; V-746 to E-1189; T-747 to E-1189; F-748 to E-1189; S-749 to E-1189; V-750 to E-1189; E-751 to E-1189; Y-752 to E-1189; S-753 to E-1189; L-754 to E-1189; E-755 to E-1189; D-756 to E-1189; P-757 to E-1189; D-758 to E-1189; H-759 to E-1189; G-760 to E-1189; P-761 to E-1189; M-762 to E-1189; L-763 to E-1189; D-764 to E-1189; D-765 to E-1189; G-766 to E-1189; W-767 to E-1189; P-768 to E-1189; T-769 to E-1189; T-770 to E-1189; L-771 to E-1189; R-772 to E-1189; V-773 to E-1189; S-774 to E-1189; V-775 to E-1189; P-776 to E-1189; F-777 to E-1189; W-778 to E-1189; N-779 to E-1189; G-780 to E-1189; C-781 to E-1189; N-782 to E-1189; E-783 to E-1189; D-784 to E-1189; E-785 to E-1189; H-786 to E-1189; C-787 to E-1189; V-788 to E-1189; P-789 to E-1189; D-790 to E-1189; L-791 to E-1189; V-792 to E-1189; L-793 to E-1189; D-794 to E-1189; A-795 to E-1189; R-796 to E-1189; S-797 to E-1189; D-798 to E-1189; L-799 to E-1189; P-800 to E-1189; T-801 to E-1189; A-802 to E-1189; M-803 to E-1189; E-804 to E-1189; Y-805 to E-1189; C-806 to E-1189; Q-807 to E-1189; R-808 to E-1189; V-809 to E-1189; L-810 to E-1189; R-811 to E-1189; K-812 to E-1189; P-813 to E-1189; A-814 to E-1189; Q-815 to E-1189; D-816 to E-1189; C-817 to E-1189; S-818 to E-1189; A-819 to E-1189; Y-820 to E-1189; T-821 to E-1189; L-822 to E-1189; S-823 to E-1189; F-824 to E-1189; D-825 to E-1189; T-826 to E-1189; T-827 to E-1189; V-828 to E-1189; F-829 to E-1189; I-830 to E-1189; I-831 to E-1189; E-832 to E-1189; S-833 to E-1189; T-834 to E-1189; R-835 to E-1189; Q-836 to E-1189; R-837 to E-1189; V-838 to E-1189; A-839 to E-1189; V-840 to E-1189; E-841 to E-1189; A-842 to E-1189; T-843 to E-1189; L-844 to E-1189; E-845 to E-1189; N-846 to E-1189; R-847 to E-1189; G-848 to E-1189; E-849 to E-1189; N-850 to E-1189; A-851 to E-1189; Y-852 to E-1189; S-853 to E-1189; T-854 to E-1189; V-855 to E-1189; L-856 to E-1189; N-857 to E-1189; I-858 to E-1189; S-859 to E-1189; Q-860 to E-1189; S-861 to E-1189; A-862 to E-1189; N-863 to E-1189; L-864 to E-1189; Q-865 to E-1189; F-866 to E-1189; A-867 to E-1189; S-868 to E-1189; L-869 to E-1189; I-870 to E-1189; Q-871 to E-1189; K-872 to E-1189; E-873 to E-1189; D-874 to E-1189; S-875 to E-1189; D-876 to E-1189; G-877 to E-1189; S-878 to E-1189; I-879 to E-1189; E-880 to E-1189; C-881 to E-1189; V-882 to E-1189; N-883 to E-1189; E-884 to E-1189; E-885 to E-1189; R-886 to E-1189; R-887 to E-1189; L-888 to E-1189; Q-889 to E-1189; K-890 to E-1189; Q-891 to E-1189; V-892 to E-1189; C-893 to E-1189; N-894 to E-1189; V-895 to E-1189; S-896 to E-1189; Y-897 to E-1189; P-898 to E-1189; F-899 to E-1189; F-900 to E-1189; R-901 to E-1189; A-902 to E-1189; K-903 to E-1189; A-904 to E-1189; K-905 to E-1189; V-906 to E-1189; A-907 to E-1189; F-908 to E-1189; R-909 to E-1189; L-910 to E-1189; D-911 to E-1189; F-912 to E-1189; E-913 to E-1189; F-914 to E-1189; S-915 to E-1189; K-916 to E-1189; S-917 to E-1189; I-918 to E-1189; F-919 to E-1189; L-920 to E-1189; H-921 to E-1189; H-922 to E-1189; L-923 to E-1189; E-924 to E-1189; I-925 to E-1189; E-926 to E-1189; L-927 to E-1189; A-928 to E-1189; A-929 to E-1189; G-930 to E-1189; S-931 to E-1189; D-932 to E-1189; S-933 to E-1189; N-934 to E-1189; E-935 to E-1189; R-936 to E-1189; D-937 to E-1189; S-938 to E-1189; T-939 to E-1189; K-940 to E-1189; E-941 to E-1189; D-942 to E-1189; N-943 to E-1189; V-944 to E-1189; A-945 to E-1189; P-946 to E-1189; L-947 to E-1189; R-948 to E-1189; F-949 to E-1189; H-950 to E-1189; L-951 to E-1189; K-952 to E-1189; Y-953 to E-1189; E-954 to E-1189; A-955 to E-1189; D-956 to E-1189; V-957 to E-1189; L-958 to E-1189; F-959 to E-1189; T-960 to E-1189; R-961 to E-1189; S-962 to E-1189; S-963 to E-1189; S-964 to E-1189; L-965 to E-1189; S-966 to E-1189; H-967 to E-1189; Y-968 to E-1189; E-969 to E-1189; V-970 to E-1189; K-971 to E-1189; L-972 to E-1189; N-973 to E-1189; S-974 to E-1189; S-975 to E-1189; L-976 to E-1189; E-977 to E-1189; R-978 to E-1189; Y-979 to E-1189; D-980 to E-1189; G-981 to E-1189; I-982 to E-1189; G-983 to E-1189; P-984 to E-1189; P-985 to E-1189; F-986 to E-1189; S-987 to E-1189; C-988 to E-1189; I-989 to E-1189; F-990 to E-1189; R-991 to E-1189; I-992 to E-1189; Q-993 to E-1189; N-994 to E-1189; L-995 to E-1189; G-996 to E-1189; L-997 to E-1189; F-998 to E-1189; P-999 to E-1189; I-1000 to E-1189; H-1001 to E-1189; G-1002 to E-1189; I-1003 to E-1189; M-1004 to E-1189; M-1005 to E-1189; K-1006 to E-1189; I-1007 to E-1189; T-1008 to E-1189; I-1009 to E-1189; P-1010 to E-1189; I-1011 to E-1189; A-1012 to E-1189; T-1013 to E-1189; R-1014 to E-1189; S-1015 to E-1189; G-1016 to E-1189; N-1017 to E-1189; R-1018 to E-1189; L-1019 to E-1189; L-1020 to E-1189; K-1021 to E-1189; L-1022 to E-1189; R-1023 to E-1189; D-1024 to E-1189; F-1025 to E-1189; L-1026 to E-1189; T-1027 to E-1189; D-1028 to E-1189; E-1029 to E-1189; V-1030 to E-1189; A-1031 to E-1189; N-1032 to E-1189; T-1033 to E-1189; S-1034 to E-1189; C-1035 to E-1189; N-1036 to E-1189; I-1037 to E-1189; W-1038 to E-1189; G-1039 to E-1189; N-1040 to E-1189; S-1041 to E-1189; T-1042 to E-1189; E-1043 to E-1189; Y-1044 to E-1189; R-1045 to E-1189; P-1046 to E-1189; T-1047 to E-1189; P-1048 to E-1189; V-1049 to E-1189; E-1050 to E-1189; E-1051 to E-1189; D-1052 to E-1189; L-1053 to E-1189; R-1054 to E-1189; R-1055 to E-1189; A-1056 to E-1189; P-1057 to E-1189; Q-1058 to E-1189; L-1059 to E-1189; N-1060 to E-1189; H-1061 to E-1189; S-1062 to E-1189; N-1063 to E-1189; S-1064 to E-1189; D-1065 to E-1189; V-1066 to E-1189; V-1067 to E-1189; S-1068 to E-1189; I-1069 to E-1189; N-1070 to E-1189; C-1071 to E-1189; N-1072 to E-1189; I-1073 to E-1189; R-1074 to E-1189; L-1075 to E-1189; V-1076 to E-1189; P-1077 to E-1189; N-1078 to E-1189; Q-1079 to E-1189; E-1080 to E-1189; I-1081 to E-1189; N-1082 to E-1189; F-1083 to E-1189; H-1084 to E-1189; L-1085 to E-1189; L-1086 to E-1189; G-1087 to E-1189; N-1088 to E-1189; L-1089 to E-1189; W-1090 to E-1189; L-1091 to E-1189; R-1092 to E-1189; S-1093 to E-1189; L-1094 to E-1189; K-1095 to E-1189; A-1096 to E-1189; L-1097 to E-1189; K-1098 to E-1189; Y-1099 to E-1189; K-1100 to E-1189; S-1101 to E-1189; M-1102 to E-1189; K-1103 to E-1189; I-1104 to E-1189; M-1105 to E-1189; V-1106 to E-1189; N-1107 to E-1189; A-1108 to E-1189; A-1109 to E-1189; L-1110 to E-1189; Q-1111 to E-1189; R-1112 to E-1189; Q-1113 to E-1189; F-1114 to E-1189; H-1115 to E-1189; S-1116 to E-1189; P-1117 to E-1189; F-1118 to E-1189; I-1119 to E-1189; F-1120 to E-1189; R-1121 to E-1189; E-1122 to E-1189; E-1123 to E-1189; D-1124 to E-1189; P-1125 to E-1189; S-1126 to E-1189; R-1127 to E-1189; Q-1128 to E-1189; I-1129 to E-1189; V-1130 to E-1189; F-1131 to E-1189; E-1132 to E-1189; I-1133 to E-1189; S-1134 to E-1189; K-1135 to E-1189; Q-1136 to E-1189; E-1137 to E-1189; D-1138 to E-1189; W-1139 to E-1189; Q-1140 to E-1189; V-1141 to E-1189; P-1142 to E-1189; I-1143 to E-1189; W-1144 to E-1189; I-1145 to E-1189; I-1146 to E-1189; V-1147 to E-1189; G-1148 to E-1189; S-1149 to E-1189; T-1150 to E-1189; L-1151 to E-1189; G-1152 to E-1189; G-1153 to E-1189; L-1154 to E-1189; L-1155 to E-1189; L-1156 to E-1189; L-1157 to E-1189; A-1158 to E-1189; L-1159 to E-1189; L-1160 to E-1189; V-1161 to E-1189; L-1162 to E-1189; A-1163 to E-1189; L-1164 to E-1189; W-1165 to E-1189; K-1166 to E-1189; L-1167 to E-1189; G-1168 to E-1189; F-1169 to E-1189; F-1170 to E-1189; R-1171 to E-1189; S-1172 to E-1189; A-1173 to E-1189; R-1174 to E-1189; R-1175 to E-1189; R-1176 to E-1189; R-1177 to E-1189; E-1178 to E-1189; P-1179 to E-1189; G-1180 to E-1189; L-1181 to E-1189; D-1182 to E-1189; P-1183 to E-1189; T-1184 to E-1189; of SEQ ID NO:187. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.) may still be retained. For example the ability of the shortened all mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an all mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six all amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the a11 polypeptide shown in FIGS. 11A-F, up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1−m1 of FIGS. 11A-F, where m1 is an integer from 6 to 1189 corresponding to the position of the amino acid residue in FIGS. 11A-F. Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the a11 polypeptide of the invention shown as SEQ ID NO:187 include polypeptides comprising the amino acid sequence of residues: M-1 to L-1188; M-1 to V-1187; M-1 to K-1186; M-1 to P-1185; M-1 to T-1184; M-1 to P-1183; M-1 to D-1182; M-1 to L-1181; M-1 to G-1180; M-1 to P-1179; M-1 to E-1178; M-1 to R-1177; M-1 to R-1176; M-1 to R-1175; M-1 to R-1174; M-1 to A-1173; M-1 to S-1172; M-1 to R-1171; M-1 to F-1170; M-1 to F-1169; M-1 to G-1168; M-1 to L-1167; M-1 to K-1166; M-1 to W-1165; M-1 to L-1164; M-1 to A-1163; M-1 to L-1162; M-1 to V-1161; M-1 to L-1160; M-1 to L-1159; M-1 to A-1158; M-1 to L-1157; M-1 to L-1156; M-1 to L-1155; M-1 to L-1154; M-1 to G-1153; M-1 to G-1152; M-1 to L-1151; M-1 to T-1150; M-1 to S-1149; M-1 to G-1148; M-1 to V-1147; M-1 to I-1146; M-1 to I-1145; M-1 to W-1144; M-1 to I-1143; M-1 to P-1142; M-1 to V-1141; M-1 to Q-1140; M-1 to W-1139; M-1 to D-1138; M-1 to E-1137; M-1 to Q-1136; M-1 to K-1135; M-1 to S-1134; M-1 to I-1133; M-1 to E-1132; M-1 to F-1131; M-1 to V-1130; M-1 to I-1129; M-1 to Q-1128; M-1 to R-1127; M-1 to S-1126; M-1 to P-1125; M-1 to D-1124; M-1 to E-1123; M-1 to E-1122; M-1 to R-1121; M-1 to F-1120; M-1 to I-1119; M-1 to F-1118; M-1 to P-1117; M-1 to S-1116; M-1 to H-1115; M-1 to F-1114; M-1 to Q-1113; M-1 to R-1112; M-1 to Q-1111; M-1 to L-1110; M-1 to A-1109; M-1 to A-1108; M-1 to N-1107; M-1 to V-1106; M-1 to M-1105; M-1 to I-1104; M-1 to K-1103; M-1 to M-1102; M-1 to S-1101; M-1 to K-1100; M-1 to Y-1099; M-1 to K-1098; M-1 to L-1097; M-1 to A-1096; M-1 to K-1095; M-1 to L-1094; M-1 to S-1093; M-1 to R-1092; M-1 to L-1091; M-1 to W-1090; M-1 to L-1089; M-1 to N-1088; M-1 to G-1087; M-1 to L-1086; M-1 to L-1085; M-1 to H-1084; M-1 to F-1083; M-1 to N-1082; M-1 to I-1081; M-1 to E-1080; M-1 to Q-1079; M-1 to N-1078; M-1 to P-1077; M-1 to V-1076; M-1 to L-1075; M-1 to R-1074; M-1 to I-1073; M-1 to N-1072; M-1 to C-1071; M-1 to N-1070; M-1 to I-1069; M-1 to S-1068; M-1 to V-1067; M-1 to V-1066; M-1 to D-1065; M-1 to S-1064; M-1 to N-1063; M-1 to S-1062; M-1 to H-1061; M-1 to N-1060; M-1 to L-1059; M-1 to Q-1058; M-1 to P-1057; M-1 to A-1056; M-1 to R-1055; M-1 to R-1054; M-1 to L-1053; M-1 to D-1052; M-1 to E-1051; M-1 to E-1050; M-1 to V-1049; M-1 to P-1048; M-1 to T-1047; M-1 to P-1046; M-1 to R-1045; M-1 to Y-1044; M-1 to E-1043; M-1 to T-1042; M-1 to S-1041; M-1 to N-1040; M-1 to G-1039; M-1 to W-1038; M-1 to I-1037; M-1 to N-1036; M-1 to C-1035; M-1 to S-1034; M-1 to T-1033; M-1 to N-1032; M-1 to A-1031; M-1 to V-1030; M-1 to E-1029; M-1 to D-1028; M-1 to T-1027; M-1 to L-1026; M-1 to F-1025; M-1 to D-1024; M-1 to R-1023; M-1 to L-1022; M-1 to K-1021; M-1 to L-1020; M-1 to L-1019; M-1 to R-1018; M-1 to N-1017; M-1 to G-1016; M-1 to S-1015; M-1 to R-1014; M-1 to T-1013; M-1 to A-1012; M-1 to I-1011; M-1 to P-1010; M-1 to I-1009; M-1 to T-1008; M-1 to I-1007; M-1 to K-1006; M-1 to M-1005; M-1 to M-1004; M-1 to I-1003; M-1 to G-1002; M-1 to H-1001; M-1 to I-1000; M-1 to P-999; M-1 to F-998; M-1 to L-997; M-1 to G-996; M-1 to L-995; M-1 to N-994; M-1 to Q-993; M-1 to I-992; M-1 to R-991; M-1 to F-990; M-1 to I-989; M-1 to C-988; M-1 to S-987; M-1 to F-986; M-1 to P-985; M-1 to P-984; M-1 to G-983; M-1 to I-982; M-1 to G-981; M-1 to D-980; M-1 to Y-979; M-1 to R-978; M-1 to E-977; M-1 to L-976; M-1 to S-975; M-1 to S-974; M-1 to N-973; M-1 to L-972; M-1 to K-971; M-1 to V-970; M-1 to E-969; M-1 to Y-968; M-1 to H-967; M-1 to S-966; M-1 to L-965; M-1 to S-964; M-1 to S-963; M-1 to S-962; M-1 to R-961; M-1 to T-960; M-1 to F-959; M-1 to L-958; M-1 to V-957; M-1 to D-956; M-1 to A-955; M-1 to E-954; M-1 to Y-953; M-1 to K-952; M-1 to L-951; M-1 to H-950; M-1 to F-949; M-1 to R-948; M-1 to L-947; M-1 to P-946; M-1 to A-945; M-1 to V-944; M-1 to N-943; M-1 to D-942; M-1 to E-941; M-1 to K-940; M-1 to T-939; M-1 to S-938; M-1 to D-937; M-1 to R-936; M-1 to E-935; M-1 to N-934; M-1 to S-933; M-1 to D-932; M-1 to S-931; M-1 to G-930; M-1 to A-929; M-1 to A-928; M-1 to L-927; M-1 to E-926; M-1 to I-925; M-1 to E-924; M-1 to L-923; M-1 to H-922; M-1 to H-921; M-1 to L-920; M-1 to F-919; M-1 to I-918; M-1 to S-917; M-1 to K-916; M-1 to S-915; M-1 to F-914; M-1 to E-913; M-1 to F-912; M-1 to D-911; M-1 to L-910; M-1 to R-909; M-1 to F-908; M-1 to A-907; M-1 to V-906; M-1 to K-905; M-1 to A-904; M-1 to K-903; M-1 to A-902; M-1 to R-901; M-1 to F-900; M-1 to F-899; M-1 to P-898; M-1 to Y-897; M-1 to S-896; M-1 to V-895; M-1 to N-894; M-1 to C-893; M-1 to V-892; M-1 to Q-891; M-1 to K-890; M-1 to Q-889; M-1 to L-888; M-1 to R-887; M-1 to R-886; M-1 to E-885; M-1 to E-884; M-1 to N-883; M-1 to V-882; M-1 to C-881; M-1 to E-880; M-1 to I-879; M-1 to S-878; M-1 to G-877; M-1 to D-876; M-1 to S-875; M-1 to D-874; M-1 to E-873; M-1 to K-872; M-1 to Q-871; M-1 to I-870; M-1 to L-869; M-1 to S-868; M-1 to A-867; M-1 to F-866; M-1 to Q-865; M-1 to L-864; M-1 to N-863; M-1 to A-862; M-1 to S-861; M-1 to Q-860; M-1 to S-859; M-1 to I-858; M-1 to N-857; M-1 to L-856; M-1 to V-855; M-1 to T-854; M-1 to S-853; M-1 to Y-852; M-1 to A-851; M-1 to N-850; M-1 to E-849; M-1 to G-848; M-1 to R-847; M-1 to N-846; M-1 to E-845; M-1 to L-844; M-1 to T-843; M-1 to A-842; M-1 to E-841; M-1 to V-840; M-1 to A-839; M-1 to V-838; M-1 to R-837; M-1 to Q-836; M-1 to R-835; M-1 to T-834; M-1 to S-833; M-1 to E-832; M-1 to I-831; M-1 to I-830; M-1 to F-829; M-1 to V-828; M-1 to T-827; M-1 to T-826; M-1 to D-825; M-1 to F-824; M-1 to S-823; M-1 to L-822; M-1 to T-821; M-1 to Y-820; M-1 to A-819; M-1 to S-818; M-1 to C-817; M-1 to D-816; M-1 to Q-815; M-1 to A-814; M-1 to P-813; M-1 to K-812; M-1 to R-811; M-1 to L-810; M-1 to V-809; M-1 to R-808; M-1 to Q-807; M-1 to C-806; M-1 to Y-805; M-1 to E-804; M-1 to M-803; M-1 to A-802; M-1 to T-801; M-1 to P-800; M-1 to L-799; M-1 to D-798; M-1 to S-797; M-1 to R-796; M-1 to A-795; M-1 to D-794; M-1 to L-793; M-1 to V-792; M-1 to L-791; M-1 to D-790; M-1 to P-789; M-1 to V-788; M-1 to C-787; M-1 to H-786; M-1 to E-785; M-1 to D-784; M-1 to E-783; M-1 to N-782; M-1 to C-781; M-1 to G-780; M-1 to N-779; M-1 to W-778; M-1 to F-777; M-1 to P-776; M-1 to V-775; M-1 to S-774; M-1 to V-773; M-1 to R-772; M-1 to L-771; M-1 to T-770; M-1 to T-769; M-1 to P-768; M-1 to W-767; M-1 to G-766; M-1 to D-765; M-1 to D-764; M-1 to L-763; M-1 to M-762; M-1 to P-761; M-1 to G-760; M-1 to H-759; M-1 to D-758; M-1 to P-757; M-1 to D-756; M-1 to E-755; M-1 to L-754; M-1 to S-753; M-1 to Y-752; M-1 to E-751; M-1 to V-750; M-1 to S-749; M-1 to F-748; M-1 to T-747; M-1 to V-746; M-1 to P-745; M-1 to K-744; M-1 to V-743; M-1 to Y-742; M-1 to D-741; M-1 to A-740; M-1 to T-739; M-1 to D-738; M-1 to L-737; M-1 to V-736; M-1 to H-735; M-1 to F-734; M-1 to N-733; M-1 to I-732; M-1 to R-731; M-1 to E-730; M-1 to C-729; M-1 to L-728; M-1 to E-727; M-1 to Q-726; M-1 to G-725; M-1 to S-724; M-1 to S-723; M-1 to L-722; M-1 to L-721; M-1 to V-720; M-1 to A-719; M-1 to R-718; M-1 to N-717; M-1 to T-716; M-1 to F-715; M-1 to R-714; M-1 to D-713; M-1 to G-712; M-1 to G-711; M-1 to E-710; M-1 to D-709; M-1 to L-708; M-1 to H-707; M-1 to A-706; M-1 to R-705; M-1 to P-704; M-1 to T-703; M-1 to Y-702; M-1 to R-701; M-1 to R-700; M-1 to E-699; M-1 to D-698; M-1 to M-697; M-1 to T-696; M-1 to A-695; M-1 to N-694; M-1 to Y-693; M-1 to R-692; M-1 to I-691; M-1 to G-690; M-1 to V-689; M-1 to T-688; M-1 to T-687; M-1 to T-686; M-1 to Q-685; M-1 to F-684; M-1 to H-683; M-1 to P-682; M-1 to A-681; M-1 to L-680; M-1 to F-679; M-1 to I-678; M-1 to P-677; M-1 to T-676; M-1 to F-675; M-1 to C-674; M-1 to L-673; M-1 to F-672; M-1 to A-671; M-1 to A-670; M-1 to L-669; M-1 to C-668; M-1 to T-667; M-1 to A-666; M-1 to D-665; M-1 to R-664; M-1 to G-663; M-1 to S-662; M-1 to R-661; M-1 to K-660; M-1 to C-659; M-1 to D-658; M-1 to R-657; M-1 to H-656; M-1 to F-655; M-1 to I-654; M-1 to N-653; M-1 to I-652; M-1 to K-651; M-1 to S-650; M-1 to P-649; M-1 to E-648; M-1 to F-647; M-1 to H-646; M-1 to L-645; M-1 to S-644; M-1 to A-643; M-1 to N-642; M-1 to I-641; M-1 to Q-640; M-1 to V-639; M-1 to V-638; M-1 to P-637; M-1 to R-636; M-1 to S-635; M-1 to W-634; M-1 to L-633; M-1 to I-632; M-1 to V-631; M-1 to A-630; M-1 to N-629; M-1 to G-628; M-1 to L-627; M-1 to A-626; M-1 to G-625; M-1 to V-624; M-1 to A-623; M-1 to L-622; M-1 to D-621; M-1 to I-620; M-1 to L-619; M-1 to G-618; M-1 to D-617; M-1 to E-616; M-1 to N-615; M-1 to L-614; M-1 to D-613; M-1 to L-612; M-1 to Q-611; M-1 to G-610; M-1 to H-609; M-1 to I-608; M-1 to S-607; M-1 to C-606; M-1 to G-605; M-1 to F-604; M-1 to Y-603; M-1 to Q-602; M-1 to L-601; M-1 to G-600; M-1 to T-599; M-1 to A-598; M-1 to L-597; M-1 to E-596; M-1 to S-595; M-1 to A-594; M-1 to T-593; M-1 to I-592; M-1 to R-591; M-1 to Q-590; M-1 to K-589; M-1 to P-588; M-1 to T-587; M-1 to K-586; M-1 to L-585; M-1 to I-584; M-1 to S-583; M-1 to G-582; M-1 to R-581; M-1 to F-580; M-1 to G-579; M-1 to H-578; M-1 to F-577; M-1 to I-576; M-1 to Y-575; M-1 to I-574; M-1 to A-573; M-1 to G-572; M-1 to A-571; M-1 to H-570; M-1 to N-569; M-1 to D-568; M-1 to E-567; M-1 to L-566; M-1 to P-565; M-1 to A-564; M-1 to G-563; M-1 to V-562; M-1 to V-561; M-1 to V-560; M-1 to D-559; M-1 to N-558; M-1 to Y-557; M-1 to S-556; M-1 to D-555; M-1 to Q-554; M-1 to N-553; M-1 to L-552; M-1 to D-551; M-1 to R-550; M-1 to V-549; M-1 to S-548; M-1 to A-547; M-1 to I-546; M-1 to S-545; M-1 to S-544; M-1 to G-543; M-1 to F-542; M-1 to R-541; M-1 to A-540; M-1 to N-539; M-1 to Q-538; M-1 to Y-537; M-1 to S-536; M-1 to H-535; M-1 to S-534; M-1 to D-533; M-1 to K-532; M-1 to L-531; M-1 to T-530; M-1 to G-529; M-1 to N-528; M-1 to Y-527; M-1 to V-526; M-1 to F-525; M-1 to R-524; M-1 to N-523; M-1 to Q-522; M-1 to R-521; M-1 to L-520; M-1 to E-519; M-1 to Y-518; M-1 to V-517; M-1 to Y-516; M-1 to V-515; M-1 to K-514; M-1 to G-513; M-1 to R-512; M-1 to E-511; M-1 to R-510; M-1 to G-509; M-1 to E-508; M-1 to N-507; M-1 to F-506; M-1 to Y-505; M-1 to M-504; M-1 to P-503; M-1 to A-502; M-1 to G-501; M-1 to V-500; M-1 to L-499; M-1 to L-498; M-1 to V-497; M-1 to D-496; M-1 to T-495; M-1 to V-494; M-1 to G-493; M-1 to D-492; M-1 to G-491; M-1 to D-490; M-1 to I-489; M-1 to D-488; M-1 to V-487; M-1 to S-486; M-1 to T-485; M-1 to I-484; M-1 to E-483; M-1 to S-482; M-1 to G-481; M-1 to F-480; M-1 to Y-479; M-1 to S-478; M-1 to G-477; M-1 to I-476; M-1 to Q-475; M-1 to Q-474; M-1 to G-473; M-1 to R-472; M-1 to M-471; M-1 to A-470; M-1 to Q-469; M-1 to H-468; M-1 to I-467; M-1 to T-466; M-1 to L-465; M-1 to S-464; M-1 to R-463; M-1 to N-462; M-1 to N-461; M-1 to H-460; M-1 to M-459; M-1 to T-458; M-1 to F-457; M-1 to L-456; M-1 to I-455; M-1 to V-454; M-1 to K-453; M-1 to G-452; M-1 to T-451; M-1 to H-450; M-1 to N-449; M-1 to F-448; M-1 to R-447; M-1 to P-446; M-1 to A-445; M-1 to G-444; M-1 to A-443; M-1 to V-442; M-1 to Y-441; M-1 to V-440; M-1 to R-439; M-1 to G-438; M-1 to Q-437; M-1 to R-436; M-1 to S-435; M-1 to S-434; M-1 to V-433; M-1 to V-432; M-1 to S-431; M-1 to T-430; M-1 to V-429; M-1 to T-428; M-1 to Y-427; M-1 to G-426; M-1 to L-425; M-1 to Y-424; M-1 to A-423; M-1 to G-422; M-1 to H-421; M-1 to N-420; M-1 to K-419; M-1 to L-418; M-1 to E-417; M-1 to E-416; M-1 to P-415; M-1 to F-414; M-1 to E-413; M-1 to K-412; M-1 to L-411; M-1 to Y-410; M-1 to S-409; M-1 to E-408; M-1 to R-407; M-1 to L-406; M-1 to P-405; M-1 to I-404; M-1 to V-403; M-1 to K-402; M-1 to G-401; M-1 to A-400; M-1 to S-399; M-1 to T-398; M-1 to E-397; M-1 to K-396; M-1 to L-395; M-1 to V-394; M-1 to A-393; M-1 to G-392; M-1 to N-391; M-1 to W-390; M-1 to D-389; M-1 to Y-388; M-1 to A-387; M-1 to G-386; M-1 to V-385; M-1 to A-384; M-1 to G-383; M-1 to L-382; M-1 to L-381; M-1 to V-380; M-1 to G-379; M-1 to D-378; M-1 to E-377; M-1 to V-376; M-1 to V-375; M-1 to H-374; M-1 to S-373; M-1 to S-372; M-1 to F-371; M-1 to G-370; M-1 to T-369; M-1 to Q-368; M-1 to S-367; M-1 to M-366; M-1 to E-365; M-1 to L-364; M-1 to G-363; M-1 to F-362; M-1 to S-361; M-1 to T-360; M-1 to E-359; M-1 to N-358; M-1 to K-357; M-1 to N-356; M-1 to T-355; M-1 to G-354; M-1 to E-353; M-1 to L-352; M-1 to S-351; M-1 to F-350; M-1 to I-349; M-1 to R-348; M-1 to D-347; M-1 to G-346; M-1 to L-345; M-1 to A-344; M-1 to D-343; M-1 to V-342; M-1 to I-341; M-1 to D-340; M-1 to K-339; M-1 to L-338; M-1 to A-337; M-1 to A-336; M-1 to E-335; M-1 to D-334; M-1 to T-333; M-1 to V-332; M-1 to N-331; M-1 to F-330; M-1 to F-329; M-1 to H-328; M-1 to K-327; M-1 to D-326; M-1 to D-325; M-1 to P-324; M-1 to D-323; M-1 to S-322; M-1 to A-321; M-1 to I-320; M-1 to Y-319; M-1 to K-318; M-1 to I-317; M-1 to E-316; M-1 to N-315; M-1 to L-314; M-1 to F-313; M-1 to T-312; M-1 to E-311; M-1 to P-310; M-1 to N-309; M-1 to I-308; M-1 to G-307; M-1 to R-306; M-1 to R-305; M-1 to N-304; M-1 to Y-303; M-1 to Y-302; M-1 to G-301; M-1 to L-300; M-1 to V-299; M-1 to A-298; M-1 to V-297; M-1 to A-296; M-1 to Y-295; M-1 to R-294; M-1 to T-293; M-1 to V-292; M-1 to N-291; M-1 to D-290; M-1 to R-289; M-1 to E-288; M-1 to S-287; M-1 to Q-286; M-1 to Q-285; M-1 to I-284; M-1 to V-283; M-1 to K-282; M-1 to E-281; M-1 to L-280; M-1 to D-279; M-1 to P-278; M-1 to S-277; M-1 to D-276; M-1 to H-275; M-1 to S-274; M-1 to E-273; M-1 to G-272; M-1 to D-271; M-1 to T-270; M-1 to I-269; M-1 to V-268; M-1 to I-267; M-1 to M-266; M-1 to V-265; M-1 to K-264; M-1 to K-263; M-1 to A-262; M-1 to G-261; M-1 to K-260; M-1 to R-259; M-1 to G-258; M-1 to G-257; M-1 to K-256; M-1 to Q-255; M-1 to F-254; M-1 to A-253; M-1 to E-252; M-1 to S-251; M-1 to R-250; M-1 to A-249; M-1 to F-248; M-1 to E-247; M-1 to I-246; M-1 to G-245; M-1 to F-244; M-1 to A-243; M-1 to T-242; M-1 to R-241; M-1 to T-240; M-1 to E-239; M-1 to T-238; M-1 to G-237; M-1 to G-236; M-1 to R-235; M-1 to Q-234; M-1 to E-233; M-1 to I-232; M-1 to H-231; M-1 to S-230; M-1 to A-229; M-1 to A-228; M-1 to E-227; M-1 to V-226; M-1 to V-225; M-1 to D-224; M-1 to K-223; M-1 to V-222; M-1 to S-221; M-1 to R-220; M-1 to Y-219; M-1 to D-218; M-1 to N-217; M-1 to L-216; M-1 to H-215; M-1 to F-214; M-1 to E-213; M-1 to H-212; M-1 to V-211; M-1 to V-210; M-1 to D-209; M-1 to E-208; M-1 to G-207; M-1 to Y-206; M-1 to Q-205; M-1 to V-204; M-1 to V-203; M-1 to G-202; M-1 to V-201; M-1 to Q-200; M-1 to I-199; M-1 to Q-198; M-1 to G-197; M-1 to P-196; M-1 to G-195; M-1 to I-194; M-1 to Y-193; M-1 to F-192; M-1 to K-191; M-1 to K-190; M-1 to L-189; M-1 to I-188; M-1 to N-187; M-1 to I-186; M-1 to L-185; M-1 to F-184; M-1 to H-183; M-1 to Q-182; M-1 to V-181; M-1 to E-180; M-1 to V-179; M-1 to W-178; M-1 to P-177; M-1 to Y-176; M-1 to I-175; M-1 to S-174; M-1 to N-173; M-1 to S-172; M-1 to G-171; M-1 to D-170; M-1 to L-169; M-1 to V-168; M-1 to I-167; M-1 to V-166; M-1 to I-165; M-1 to D-164; M-1 to M-163; M-1 to Y-162; M-1 to T-161; M-1 to Q-160; M-1 to C-159; M-1 to R-158; M-1 to Q-157; M-1 to L-156; M-1 to A-155; M-1 to P-154; M-1 to A-153; M-1 to V-152; M-1 to T-151; M-1 to K-150; M-1 to S-149; M-1 to F-148; M-1 to R-147; M-1 to F-146; M-1 to N-145; M-1 to S-144; M-1 to N-143; M-1 to V-142; M-1 to R-141; M-1 to S-140; M-1 to C-139; M-1 to M-138; M-1 to G-137; M-1 to T-136; M-1 to T-135; M-1 to Y-134; M-1 to Y-133; M-1 to S-132; M-1 to S-131; M-1 to G-130; M-1 to C-129; M-1 to E-128; M-1 to H-127; M-1 to S-126; M-1 to W-125; M-1 to L-124; M-1 to P-123; M-1 to S-122; M-1 to C-121; M-1 to A-120; M-1 to L-119; M-1 to F-118; M-1 to S-117; M-1 to N-116; M-1 to D-115; M-1 to K-114; M-1 to P-113; M-1 to N-112; M-1 to T-111; M-1 to A-110; M-1 to L-109; M-1 to S-108; M-1 to L-107; M-1 to G-106; M-1 to L-105; M-1 to R-104; M-1 to M-103; M-1 to N-102; M-1 to D-101; M-1 to K-100; M-1 to R-99; M-1 to E-98; M-1 to S-97; M-1 to V-96; M-1 to N-95; M-1 to S-94; M-1 to L-93; M-1 to T-92; M-1 to V-91; M-1 to R-90; M-1 to G-89; M-1 to L-88; M-1 to N-87; M-1 to L-86; M-1 to K-85; M-1 to T-84; M-1 to C-83; M-1 to N-82; M-1 to G-81; M-1 to H-80; M-1 to I-79; M-1 to V-78; M-1 to P-77; M-1 to C-76; M-1 to K-75; M-1 to Y-74; M-1 to V-73; M-1 to D-72; M-1 to G-71; M-1 to T-70; M-1 to K-69; M-1 to Q-68; M-1 to Y-67; M-1 to G-66; M-1 to N-65; M-1 to T-64; M-1 to E-63; M-1 to L-62; M-1 to P-61; M-1 to A-60; M-1 to G-59; M-1 to V-58; M-1 to V-57; M-1 to L-56; M-1 to W-55; M-1 to K-54; M-1 to N-53; M-1 to G-52; M-1 to S-51; M-1 to I-50; M-1 to D-49; M-1 to H-48; M-1 to Q-47; M-1 to Q-46; M-1 to V-45; M-1 to T-44; M-1 to Y-43; M-1 to G-42; M-1 to F-41; M-1 to F-40; M-1 to A-39; M-1 to T-38; M-1 to R-37; M-1 to S-36; M-1 to G-35; M-1 to P-34; M-1 to I-33; M-1 to V-32; M-1 to R-31; M-1 to P-30; M-1 to K-29; M-1 to R-28; M-1 to T-27; M-1 to D-26; M-1 to M-25; M-1 to N-24; M-1 to F-23; M-1 to T-22; M-1 to D-21; M-1 to T-20; M-1 to F-19; M-1 to G-18; M-1 to P-17; M-1 to W-16; M-1 to L-15; M-1 to S-14; M-1 to L-13; M-1 to A-12; M-1 to W-11; M-1 to A-10; M-1 to V-9; M-1 to V-8; M-1 to L-7; M-1 to G-6; of SEQ ID NO:187. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:186 which have been determined from the following related cDNA genes:









(SEQ ID NO: 192)



HEEAB54R,


 



(SEQ ID NO: 193)



HRDAF83R,


 



(SEQ ID NO: 194)



HOUBC62R,


 



(SEQ ID NO: 195)



HCDBI19R,


 



(SEQ ID NO: 196)



HOHCU94R,


 



(SEQ ID NO: 197)



HOACC13R,


 



(SEQ ID NO: 198)



HCDAP21R



and


 



(SEQ ID NO: 199)



HNHHA34R,


 



(SEQ ID NO: 200)



HOHEA75R,



and


 



(SEQ ID NO: 201)



HNGEL59R.






A polynucleotide encoding a polypeptide of the present invention is obtained from human ovary, small intestine, fetal heart, fetal brain, large intestine, osteoblasts, human trabecular bone cells, mesangial cells, adipocytes, osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow tissues and cells. The polynucleotide of this invention was discovered in a human osteoblast II cDNA library.
Based on the sequence similarity to the human integrin alpha 1 subunit, translation product of this gene is expected to share at least some biological activities with integrin proteins, and specifically the integrin alpha 1 protein. Such activities are known in the art, some of which are described elsewhere herein.
Specifically, polynucleotides and polypeptides of the invention, including antibodies, are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response. Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders. The full-length protein should be a secreted protein, based upon homology to the integrin family. Therefore, it is secreted into serum, urine, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection. In addition, expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancer.
Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers.
Alternatively, the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.
Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA).
Alternatively, this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis. Thus, aberrant expression of this gene product in tissues—particularly adult tissues—may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus, this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
This gene is expressed almost exclusively in osteoblasts, human trabecular bone cells, mesangial cells, adipocytes, and to a lesser extent in osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow.
Therefore, polynucleotides and polypeptides of the invention, including antibodies, are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, disorders of the skeletal system, connective tissues, and immune and hematopoietic diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the connective tissue and skeletal system, expression of this gene at significantly higher or lower levels is detected in certain tissues or cell types (e.g. immune, hematopoietic, skeletal, bone, cartilage, developmental, reproductive, secretory, and cancerous and wounded tissues) or bodily fluids or cell types (e.g., lymph, serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
The tissue distribution in osteoblasts and homology to integrin alpha subunit 10 indicates that the protein products of this gene are useful for the treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis and treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (i.e., spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. Such a use is consistent with the observed homology to integrin family members, in conjunction with the tissue distribution in bone marrow cells.
Integrins play pivotal roles in cell migration, inflammation, proliferation, and cellular infiltration. Thus, the present invention is expected to share at least some of these activities. Representative uses are described in the “Immune Activity” and “infectious disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Based upon the tissue distribution of this protein, antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.
Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly described elsewhere herein.
Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:186 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 4981 of SEQ ID NO:186, b is an integer of 15 to 4995, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:186, and where b is greater than or equal to a+14.
Tables
Description of Table 1A
Table 1A summarizes information concerning certain polynucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “cDNA Clone ID”, for a cDNA clone related to each contig sequence disclosed in Table 1A. The cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCCTM Deposit No: Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.
SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein.
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCCTM, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods
The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.
Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPTTM (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENETM Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from STRATAGENETM.
Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIESTM, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIESTM. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIESTM. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCCTM. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCCTM Deposit No:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCCTM Deposit No:Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCCTM Deposit No:Z.
Description of Tables 1B.1 and 1B.2
Tables 1B.1 and 1B.2 summarize some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifiers (SEQ ID NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column of Tables 1B.1 and 1B.2 provide the gene numbers in the application for each clone identifier. The second column of Tables 1B.1 and 1B.2 provide unique clone identifiers, “cDNA Clone ID”, for cDNA clones related to each contig sequence disclosed in Table 1A and/or Tables 1B.1 and 1B.2. The third column of Tables 1B.1 and 1B.2 provide unique contig identifiers, “Contig ID:” for each of the contig sequences disclosed in these tables. The fourth column of Tables 1B.1 and 1B.2 provide the sequence identifiers, “SEQ ID NO:X”, for each of the contig sequences disclosed in Table 1A and/or Tables 1B.1 and 1B.2. Table 1B.1
The fifth column of Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineates the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1B.1 as SEQ ID NO:Y (column 6). Column 7 of Table 1B.1 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y). Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wis.). This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 1B.1 as “Predicted Epitopes”. In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1B.1. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8 of Table 1B.1 (“Cytologic Band”) provides the chromosomal location of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from OMIMTM (“Online Mendelian Inheritance in Man,” McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000 (world wide web at ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM identification number is disclosed in Table 1B.1, column 9 labeled “OMIM Disease Reference(s)”. Table 5 is a key to the OMIM reference identification numbers (Table 5, column 1), and provides a description of the associated disease in Table 5, column 2.
Table 1B.2
Column 5 of Table 1B.2, “Tissue Distribution” shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention. The first code number shown in Table 1B.2 column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested. The second number in column 5 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO:X) was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
Description of Table 1C
Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID NO:A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof). Table 1C in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 228 to 235 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.
Description of Tables 1D, 1E, 1E.1 and 1E.2
Table 1D: In preferred embodiments, the present invention encompasses a method of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder listed as listed in the “Preferred Indications” column of Table 1D, column 3; comprising administering to a patient (in which such detection, prevention, treatment, and/or amelioration is desired) a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by, for example, Table 1A or 1D (in the same row as the disease or disorder to be treated is listed in the “Preferred Indications” column of Tables 1D) in an amount effective to detect, prevent, diagnose, prognosticate, treat, or ameliorate the disease or disorder. In preferred embodiments, the present invention encompasses a method of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder (such as an immune, cardiovascular, cancer, or other proliferative disease or disorder), comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Tables 1A, 1B.1, 1B.2, and 1C, in an amount effective to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate the disease or disorder.
As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to prevent, treat, or ameliorate the associated disease.
The present invention encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of detecting, diagnosing, treating, preventing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, diagnose, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.
In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.
The “Preferred Indication” column describes diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
The recitation of “Cancer” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).
In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.
The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.
The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.
The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.
The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.
The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.
The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).
The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.
The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.
The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.
The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.
The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.
Table 1E provides information related to biological activities and preferred indications for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention and indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating.
Tables 1E.1 and 1E.2 provide information related to biological activities for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Tables 1E.2 also provide information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column of Table 1E.1 (“Gene No.”) provides the gene number in the application for each clone identifier. The second column of Table 1E.1 (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, and 1C. The third column of Table 1E.1 (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column of Table 1E.1 (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides).
In Table 1E.2, each of the biological activities of Table 1E.1 are listed by “Biological Activity Number” and the corresponding “Biological Activity” and are followed by an “Exemplary Activity Assay” column and a “Preferred Indication” column; however, for some biological activities no “Exemplary Activity Assay” or “Preferred Indication” is given. The “Exemplary Activity Assay” column describes the biological activity listed in the column that precedes it and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The “Preferred Indication” column also refers to the biological activity listed in the preceding column and describes disease(s) or disorder(s) that may be detected, diagnosed, prevented, treated, or ameliorated by the nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof).
Tables 1E, 1E.1, and 1E.2 describe the use of FMAT technology, inter alia, for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system that provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound fluorophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for high throughput screening using fluorometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).
Tables 1E, 1E.1, and 1E.2 also describe the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).
Description of Table 1F
Polynucleotides encoding polypeptides of the present invention can be used in assays to test for one or more biological activities. One such biological activity which may be tested includes the ability of polynucleotides and polypeptides of the invention to stimulate up-regulation or down-regulation of expression of particular genes and proteins. Hence, if polynucleotides and polypeptides of the present invention exhibit activity in altering particular gene and protein expression patterns, it is likely that these polynucleotides and polypeptides of the present invention may be involved in, or capable of effecting changes in, diseases associated with the altered gene and protein expression profiles. Hence, polynucleotides, polypeptides, or antibodies of the present invention could be used to treat said associated diseases.
TaqMan® assays may be performed to assess the ability of polynucleotides (and polypeptides they encode) to alter the expression pattern of particular “target” genes. TaqMan® reactions are performed to evaluate the ability of a test agent to induce or repress expression of specific genes in different cell types. TaqMan® gene expression quantification assays (“TaqMan® assays”) are well known to, and routinely performed by, those of ordinary skill in the art. TaqMan® assays are performed in a two step reverse transcription/polymerase chain reaction (RT-PCR). In the first (RT) step, cDNA is reverse transcribed from total RNA samples using random hexamer primers. In the second (PCR) step, PCR products are synthesized from the cDNA using gene specific primers.
To quantify gene expression the TaqMan® PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold® DNA Polymerase to cleave a TaqMan® probe (distinct from the primers) during PCR. The TaqMan® probe contains a reporter dye at the 5′-end of the probe and a quencher dye at the 3′ end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. AmpliTaq Gold® DNA Polymerase then cleaves the probe between the reporter and quencher when the probe hybridizes to the target, resulting in increased fluorescence of the reporter (see FIG. 2). Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.
After the probe fragments are displaced from the target, polymerization of the strand continues. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected.
For test sample preparation, vector controls or constructs containing the coding sequence for the gene of interest are transfected into cells, such as for example 293T cells, and supernatants collected after 48 hours. For cell treatment and RNA isolation, multiple primary human cells or human cell lines are used; such cells may include but are not limited to, Normal Human Dermal Fibroblasts, Aortic Smooth Muscle, Human Umbilical Vein Endothelial Cells, HepG2, Daudi, Jurkat, U937, Caco, and THP-1 cell lines. Cells are plated in growth media and growth is arrested by culturing without media change for 3 days, or by switching cells to low serum media and incubating overnight. Cells are treated for 1, 6, or 24 hours with either vector control supernatant or sample supernatant (or purified/partially purified protein preparations in buffer). Total RNA is isolated; for example, by using TRIZOLTM extraction or by using the Ambion RNAqueousTM-4PCR RNA isolation system. Expression levels of multiple genes are analyzed using TaqMan®, and expression in the test sample is compared to control vector samples to identify genes induced or repressed. Each of the above described techniques are well known to, and routinely performed by, those of ordinary skill in the art.
Table 1F indicates particular disease classes and preferred indications for which polynucleotides, polypeptides, or antibodies of the present invention may be used in detecting, diagnosing, preventing, treating and/or ameliorating said diseases and disorders based on “target” gene expression patterns which may be up- or down-regulated by polynucleotides (and the encoded polypeptides) corresponding to each indicated cDNA Clone ID (shown in Table 1F, Column 2).
Thus, in preferred embodiments, the present invention encompasses a method of detecting, diagnosing, preventing, treating, and/or ameliorating a disease or disorder listed in the “Disease Class” and/or “Preferred Indication” columns of Table 1F; comprising administering to a patient in which such detection, diagnosis, prevention, or treatment is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, diagnose, prevent, treat, or ameliorate the disease or disorder. The first and second columns of Table 1F show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, diagnosing, preventing, treating, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
In another embodiment, the present invention also encompasses methods of detecting, diagnosing, preventing, treating, or ameliorating a disease or disorder listed in the “Disease Class” or “Preferred Indication” Columns of Table 1F; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
The “Disease Class” Column of Table 1F provides a categorized descriptive heading for diseases, disorders, and/or conditions (more fully described below) that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
The “Preferred Indication” Column of Table 1F describes diseases, disorders, and/or conditions that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
The “Cell Line” and “Exemplary Targets” Columns of Table 1F indicate particular cell lines and target genes, respectively, which may show altered gene expression patterns (i.e., up- or down-regulation of the indicated target gene) in TaqMan® assays, performed as described above, utilizing polynucleotides of the cDNA Clone ID shown in the corresponding row. Alteration of expression patterns of the indicated “Exemplary Target” genes is correlated with a particular “Disease Class” and/or “Preferred Indication” as shown in the corresponding row under the respective column headings.
The “Exemplary Accessions” Column indicates GenBank Accessions (available online through the National Center for Biotechnology Information (NCBI) at the world wide web at ncbi.nlm.nih.gov/) which correspond to the “Exemplary Targets” shown in the adjacent row.
The recitation of “Cancer” in the “Disease Class” Column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate neoplastic diseases and/or disorders (e.g., leukemias, cancers, etc., as described below under “Hyperproliferative Disorders”).
The recitation of “Immune” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
The recitation of “Angiogenesis” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), diseases and/or disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”), diseases and/or disorders involving cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), diseases and/or disorders involving angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), to promote or inhibit cell or tissue regeneration (e.g., as described below under “Regeneration”), or to promote wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”). Highly preferred indications include diagnosis, prevention, treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti-Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.”
The recitation of “Diabetes” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diabetes (including diabetes mellitus types I and II), as well as diseases and/or disorders associated with, or consequential to, diabetes (e.g. as described below under “Endocrine Disorders,” “Renal Disorders,” and “Gastrointestinal Disorders”).
Description of Table 2
Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, “Clone ID:”, corresponding to a cDNA clone disclosed in Tables 1A, 1B.1, and/or 1B.2. The second column provides the unique contig identifier, “Contig ID:” corresponding to contigs in Tables 1B.1 and 1B.2 and allowing for correlation with the information in Tables 1B.1 and 1B.2. The third column provides the sequence identifier, “SEQ ID NO:X”, for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”) as further described below. The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, “Score/Percent Identity”, provides a quality score or the percent identity, of the hit disclosed in columns five and six. Columns 8 and 9, “NT From” and “NT To” respectively, delineate the polynucleotides in “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
Description of Table 3
Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, “cDNA Clone ID”, for a cDNA clone related to contig sequences disclosed in Tables 1B.1 and 1B.2. The second column provides the sequence identifier, “SEQ ID NO:X”, for contig sequences disclosed in Tables 1A, 1B.1, and/or 1B.2. The third column provides the unique contig identifier, “Contig ID:”, for contigs disclosed in Tables 1B.1 and 1B.2. In specific embodiments of the invention, for each “Contig ID” listed in the third column of Table 3, preferably excluded are one or more polynucleotides comprising, or alternatively consisting of, SEQ ID NO:X referenced in the second column of Table 3 and described by the general formula of a−b, whereas a and b are uniquely defined integers determined for the corresponding SEQ ID NO:X referred to in column 2 of Table 3. The fourth column provides a range of values for the unique integer ‘a’ where ‘a’ is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth column provides a range of values for the unique integer ‘b’ where ‘b’ is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a+14. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are at least five, ten, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, or more of the available material having the accession numbers identified in the sixth column of Table 3 (including for example, the actual sequence contained in an identified BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least five, ten, or more of the available material having the accession numbers identified in the sixth column of Table 3 (including for example, the actual sequence contained in an identified BAC clone). In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.
Description of Table 4
Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 provides the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a_” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.
Description of Table 5
Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B.1, column 9. OMIM reference identification numbers (column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000 (world wide web at ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B.1, column 8 as determined using the Morbid Map database.
Description of Table 6
Table 6 summarizes some of the ATCCTM Deposits, Deposit dates, and ATCCTM designation numbers of deposits made with the ATCCTM in connection with the present application. These deposits were made in addition to those described in Table 1A.
Description of Table 7
Table 7 shows the cDNA libraries sequenced, and ATCCTM designation numbers and vector information relating to these cDNA libraries.
The first column shows the first four letters indicating the Library from which each library clone was derived. The second column indicates the catalogued tissue description for the corresponding libraries. The third column indicates the vector containing the corresponding clones. The fourth column shows the ATCCTM deposit designation for each library clone as indicated by the deposit information in Table 6.
Definitions
The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
In the present invention, a “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof (e.g., the polypeptide delineated in columns fourteen and fifteen of Table 1A); a nucleic acid sequence contained in SEQ ID NO:X (as described in column 5 of Table 1A and/or column 4 of Tables 1B.1 and 1B.2) or the complement thereof a cDNA sequence contained in Clone ID: (as described in column 2 of Table 1A and/or Tables 1B.1 and 1B.2 and contained within a library deposited with the ATCCTM); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 (EXON From-To) of Table 1C or a fragment or variant thereof or a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region (with or without a natural or artificial signal sequence), the protein coding region as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
In the present invention, “SEQ ID NO:X” was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X is deposited at Human Genome Sciences, Inc. (HGS) in a catalogued and archived library. As shown, for example, in column 2 of Tables 1B.1 and 1B.2, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID:). Each Clone ID is unique to an individual clone and the Clone ID is all the information needed to retrieve a given clone from the HGS library. Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID: to determine the library source by reference to Tables 6 and 7. Table 7 lists the deposited cDNA libraries by name and links each library to an ATCCTM Deposit. Library names contain four characters, for example, “HTWE.” The name of a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example “HTWEP07”. As mentioned below, Tables 1A, 1B.1 and 1B.2 correlate the Clone ID names with SEQ ID NO:X. Thus, starting with an SEQ ID NO:X, one can use Tables 1A, 1B.1, 1B.2, 6, and 7 to determine the corresponding Clone ID, which library it came from and which ATCCTM deposit the library is contained in. Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCCTM is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCCTM deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein), the polynucleotide sequence delineated in columns 7 and 8 of Table 1A or the complement thereof, the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID: (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones deposited with the ATCCTM, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1C or the complement thereof. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.
Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).
Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
“SEQ ID NO:X” refers to a polynucleotide sequence described, for example, in Tables 1A, 1B.1, 1B.2, 1C, 2, and/or 3, while “SEQ ID NO:Y” refers to a polypeptide sequence described in, for example, column 11 of Table 1A; column 6 of Table 1B.1; and/or column 3 of Table 1E or 1E.1. SEQ ID NO:X is identified by an integer specified in, for example, column 5 of Table 1A; column 4 of Table 1B.1 or 1B.2; column 2 of Table 1C; column 3 of Table 2; and/or column 2 of Table 3. The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. “Clone ID:” refers to a cDNA clone described in, for example, column 2 of Tables 1A, 1B.1, 1B.2, 1D, 1E, 1E.1, and/or 1F; and/or column 1 of Table 1C, 2, and/or 3.
“A polypeptide having functional activity” refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity (e.g. activity useful in treating, preventing and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders), antigenicity (ability to bind [or compete with a polypeptide for binding] to an anti-polypeptide antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
The polypeptides of the invention can be assayed for functional activity (e.g. biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay secreted polypeptides (including fragments, variants, derivatives, and analogs) of the invention for activity using assays as described in the examples section below.
“A polypeptide having biological activity” refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
Tables
Table 1A
Table 1A summarizes information concerning certain polynucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence disclosed in Table 1A. Third column, the cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCCTM Deposit No:Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.
SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein.
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCCTM, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods
The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.
Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPTTM (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENETM Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from STRATAGENETM.
Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIESTM, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIESTM. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIESTM. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCCTM. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCCTM Deposit No:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCCTM Deposit No:Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCCTM Deposit No:Z.





















TABLE 1A














5′ NT
















of

First
Last






ATCC TM



5′ NT
3′ NT
5′ NT
First
AA
AA
AA
First
Last




Deposit

NT SEQ
Total
of
of
of
AA of
SEQ
of
of
AA of
AA


Gene
cDNA
No: Z and

ID
NT
Clone
Clone
Start
Signal
ID
Sig
Sig
Secreted
of


No.
Clone ID
Date
Vector
NO: X
Seq.
Seq.
Seq.
Codon
Pep
NO: Y
Pep
Pep
Portion
ORF





























1
HNFIP24
97903
pBluescript TM
1
902
46
816
19
19
2
1
26
27
234




Feb. 26, 1997
















209049
















May 15, 1997














2
HETBY74
97904
Uni-ZAP XR
7
1923
30
1923
45
45
8
1
33
34
193




Feb. 26, 1997
















209050
















May 15, 1997














3
HTEEB42
97922
Uni-ZAP XR
9
1022
20
1022
59
59
10
1
22
23
298




Mar. 7, 1997
















209070
















May 22, 1997














4
HEMCM42
97978
Uni-ZAP XR
11
1041
48
1007
58
58
12
1
29
30
113




Mar. 27, 1997
















209075
















May 22, 1997














4
HEQCC55
209965
pCMVSport
13
1052
30
1052
62
62
14
1
27
28
112




Jun. 11, 1998
3.0













4
HEQCC55
209965
pCMVSport
15
1000
1
1000
25
25
16
1
27
28
129




Jun. 11, 1998
3.0













4
HEMCM42
97978
Uni-ZAP XR
15
1000
1
1000
25
25
16
1
27
28
129




Mar. 27, 1997
















209075
















May 22, 1997














4
HEQCC55
209965
pCMVSport
17
1037
1
1037
57
57
18
1
27
28
155




Jun. 11, 1998
3.0













5
HEMAE80
97975
Uni-ZAP XR
20
996
1
945
12
12
21
1
24
25
136




Apr. 4, 1997
















209081
















May 29, 1997














5
HEMAE80
97975
Uni-ZAP XR
22
1092
1
1092
61
61
23
1
24
25
136




Apr. 4, 1997
















209081
















May 29, 1997














6
HRDFB85
97977
Uni-ZAP XR
28
1705
23
1697
233
233
29
1
21
22
201




Apr. 4, 1997
















209082
















May 29, 1997














7
HDTAW95
209007
pCMVSport
38
1288
412
1288
571
571
39
1


16




Apr. 28, 1997
2.0















209083
















May 29, 1997














8
HEMCV19
209010
Uni-ZAP XR
46
941
33
931
79
79
47
1
23
24
178




Apr. 28, 1997
















209085
















May 29, 1997














8
HEMCV19
209010
Uni-ZAP XR
48
736
1
736
84
84
49
1
22
23
181




Apr. 28, 1997
















209085
















May 29, 1997














8
HEMCV19
209010
Uni-ZAP XR
50
864
1
864
125
125
51
1
21
22
185




Apr. 28, 1997
















209085
















May 29, 1997














9
HETBX14
209010
Uni-ZAP XR
61
1292
303
1292
207
207
62
1
18
19
250




Apr. 28, 1997
















209085
















May 29, 1997














9
HETBX14
209010
Uni-ZAP XR
63
1146
157
1146

74
64
1
13
14
53




Apr. 28, 1997
















209085
















May 29, 1997














9
HETBX14
209010
Uni-ZAP XR
65
1146
157
1146

74
66
1
14
15
53




Apr. 28, 1997
















209085
















May 29, 1997














10
HLHSK94
209011
pBluescript TM
70
1974
1
1794
112
112
71
1
26
27
379




Apr. 28, 1997














10
HLHSK94
209011
pBluescript
72
1789
1
1789
112
112
73
1
25
26
379




Apr. 28, 1997














10
HLHSK94
209011
pBluescript
74
1974
1
1794
112
112
75
1
25
26
379




Apr. 28, 1997














11
HLHFP03
209126
Uni-ZAP XR
76
613
1
613
224
224
77
1
20
21
116




Jun. 19, 1997














11
HLHFP03
209126
Uni-ZAP XR
78
613
1
613
224
224
79
1
19
20
116




Jun. 19, 1997














12
HHTLF25
209125
ZAP Express
83
697
1
661
142
142
84
1
26
27
111




Jun. 19, 1997














13
HTADX17
209124
Uni-ZAP XR
90
1140
22
1140
84
84
91
1
24
25
142




Jun. 19, 1997














13
HTADX17
209124
Uni-ZAP XR
92
1147
0
1148
92
92
93
1
23
24
142




Jun. 19, 1997














13
HTADX17
209124
Uni-ZAP XR
94
1140
22
1140
84
84
95
1
19
20
142




Jun. 19, 1997














14
HJACG02
209215
pBluescript
102
553
1
553
47
47
103
1
23
24
108




Aug. 21, 1997
SK−













14
HJACG02
209215
pBluescript
104
575
1
575
66
66
105
1
22
23
108




Aug. 21, 1997
SK−













15
HKGAJ54
209224
pSport1
107
1346
1
1346
31
31
108
1
27
28
303




Aug. 28, 1997














15
HKGAJ54
209224
pSport1
109
1332
1
1332
24
24
110
1
27
28
340




Aug. 28, 1997














16
HSVAK93
209563
Uni-ZAP XR
116
571
1
571
21
21
117
1
24
25
183




Dec. 18, 1997














16
HE8CH92
209580
Uni-ZAP XR
118
1282
1
1282
31
31
119
1
24
25
378




Jan. 14, 1998














16
HSVAK93
209563
Uni-ZAP XR
120
1240
1
1240
24
24
121
1
24
25
378




Dec. 18, 1997














17
HSDEK49
209603
Uni-ZAP XR
123
1590
96
1590
126
126
124
1
21
22
305




Jan. 29, 1998














17
HSDEK49
209603
Uni-ZAP XR
125
1782
1
1782
60
60
126
1
19
20
399




Jan. 29, 1998














18
HWBAO62
209603
pCMVSport
127
1903
1
1903
52
52
128
1
30
31
212




Jan. 29, 1998
3.0













18
HWBAO62
209603
pCMVSport
129
1940
1
1940
81
81
130
1
30
31
101




Jan. 29, 1998
3.0













19
HWHGU54
209782
pCMVSport
131
1445
1
1445
145
145
132
1
19
20
414




Apr. 20, 1998
3.0













20
HCEJQ69
209782
Uni-ZAP XR
134
1777
1
1777
39
39
135
1
26
27
473




Apr. 20, 1998














20
HCEJQ69
209782
Uni-ZAP XR
136
1774
1
1774
39
39
137
1
26
27
550




Apr. 20, 1998














20
HCEJQ69
209782
Uni-ZAP XR
138
1777
1
1777
39
39
139
1
26
27
380




Apr. 20, 1998














21
HT5GJ57
209889,
UNI-ZAP TM
159
1797
92
1797
122
122
160
1
25
26
190




May 22, 1998
XR













21
HT5GJ57
209889
Uni-ZAP XR
161
1773
1
1773
105
105
162
1
25
26
243




May 22, 1998














22
HPIBX03
209965
Uni-ZAP XR
169
2209
1
2178
81
81
170
1
29
30
709




Jun. 11, 1998














23
HDPBO81
203070
pCMVSport
174
3798
1
3798
265
265
175
1
26
27
348




Jul. 27, 1998
3.0













23
HDPBO81
203070
pCMVSport
176
3793
1
3793
255
255
177
1
26
27
348




Jul. 27, 1998
3.0













24
HWBFY57
203648
pCMVSport
179
1796
1
1796
113
113
180
1
19
20
290




Feb. 9, 1999
3.0













25
HYABV21
PTA-3105
pCMVSport
182
2738
1
2738
55
55
183
1
16
17
301




Feb. 23, 2001
3.0













25
HYABV21
PTA-3105
pCMVSport
184
729
28
729
63
63
185
1
16
17
222




Feb. 23, 2001
3.0













26
HOHBY69
203331
pCMVSport
186
4995
1
4995
82
82
187
1
22
23
1189




Oct. 8, 1998
2.0













26
HOHBY69
203331
pCMVSport
188
4631
1
4631
84
84
189
1
22
23
1034




Oct. 8, 1998
2.0













27
HDHMA45
203331
pCMVSport
202
2184
1
2184
199
199
203
1
33
34
413




Oct. 8, 1998
2.0













27
HDHMA45
203331
pCMVSport
204
2190
1
2190
204
204
205
1
33
34
413




Oct. 8, 1998
2.0













28
HMADJ14
PTA-622
Uni-ZAP XR
206
1364
15
1364
278
278
207
1
68
69
352




Sep. 2, 1999














28
HMADJ14
PTA-622
Uni-ZAP XR
208
1583
1
1583
264
264
209
1
25
26
257




Sep. 2, 1999














28
HMADJ14
PTA-622
Uni-ZAP XR
210
1444
91
1444
125
125
211
1
25
26
257




Sep. 2, 1999














28
HMADJ14
203979
Uni-ZAP XR
212
1444
91
1444
125
125
213
1
25
26
257




Apr. 29, 1999














28
HMADJ14
PTA-622
Uni-ZAP XR
214
1892
1
1891
264
264
215
1
25
26
291




Sep. 2, 1999














28
HMADJ14
PTA-622
Uni-ZAP XR
216
1439
1
1439
47
47
217
1
1
2
242




Sep. 2, 1999














29
HEAAL31
97903
Uni-ZAP XR
218
991
374
970
60
60
219
1
24
25
177




Feb. 26, 1997
















209049
















May 15, 1997














29
HEAAL31
97903
Uni-ZAP XR
220
2416
1387
2413
1473
1473
221
1
18
19
25




Feb. 26, 1997
















209049
















May 15, 1997














30
HEMFA84
203499
Uni-ZAP XR
222
985
1
985
42
42
223
1
17
18
257




Dec. 1, 1998














31
HDPPA04
PTA-867
pCMVSport
224
2406
1
2406
271
271
225
1
19
20
283




Oct. 26, 1999
3.0













31
HDPPA04
PTA-867
pCMVSport
226
1675
1
1613

1003
227
1
11
12
23




Oct. 26, 1999
3.0













31
HDPPA04
PTA-867
pCMVSport
228
786
1
786
261
261
229
1
19
20
93




Oct. 26, 1999
3.0













32
HE2OA95
203960
Uni-ZAP XR
230
1671
1
1671

1224
231
1
9
10
35




Apr. 26, 1999














33
HKABZ65
209683
pCMVSport
232
1189
1
1189
77
77
233
1
17
18
243




Mar. 20, 1998
2.0













33
HKABZ65
209683
pCMVSport
234
1191
1
1191
69
69
235
1
17
18
243




Mar. 20, 1998
2.0









Tables 1B.1 and 1B.2

The first column in Tables 1B.1 and 1B.2 provides the gene number in the application corresponding to the clone identifier. The second column in Tables 1B.1 and 1B.2 provides a unique “Clone ID:” for the cDNA clone related to each contig sequence disclosed in Tables 1B.1 and 1B.2. This clone ID references the cDNA clone which contains at least the 5′ most sequence of the assembled contig and at least a portion of SEQ ID NO:X was determined by directly sequencing the referenced clone. The referenced clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein. The third column in Tables 1B.1 and 1B.2 provides a unique “Contig ID” identification for each contig sequence. The fourth column in Tables 1B.1 and 1B.2 provides the “SEQ ID NO:” identifier for each of the contig polynucleotide sequences disclosed in Tables 1B.1 and 1B.2.
Table 1B.1
The fifth column in Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence “SEQ ID NO:X” that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1B.1, column 6, as SEQ ID NO:Y. Where the nucleotide position number “To” is lower than the nucleotide position number “From”, the preferred ORF is the reverse complement of the referenced polynucleotide sequence. The sixth column in Table 1B.1 provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated by “ORF (From-To)”. Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. Column 7 in Table 1B.1 lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN (Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South Park Street Madison, Wis.). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted epitopes as described in Table 1B.1. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly.
Column 8 in Table 1B.1 (“Cytologic Band”) provides a chromosomal map location for certain polynucleotides of the invention (e.g., polynucleotides corresponding to SEQ ID NO:X). Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Each sequence in the UniGene database is assigned to a “cluster”; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often available for one or more sequence(s) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster.
A modified version of the computer program BLASTN (Altshul, et al., J. Mol. Biol. 215:403-410 (1990), and Gish, and States, Nat. Genet. 3:266-272) (1993) was used to search the UniGene database for EST or cDNA sequences that contain exact or near-exact matches to a polynucleotide sequence of the invention (the ‘Query’). A sequence from the UniGene database (the ‘Subject’) was said to be an exact match if it contained a segment of 50 nucleotides in length such that 48 of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same UniGene cluster, and mapping data was available for this cluster, it is indicated in Table 1B.1 under the heading “Cytologic Band”. Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.
Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIMTM (“Online Mendelian Inheritance in Man”; McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000; world wide web at ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of a polynucleotide of the invention (Query sequence) was associated with a disease in the Morbid Map database, an OMIM reference identification number was noted in column 9, Table 1B.1, labeled “OMIM Disease Reference(s). Table 5 is a key to the OMIM reference identification numbers (Table 5, column 1), and provides a description of the associated disease in Table 5, column 2.
Table 1B.2
Column 5, in Table 1B.2, provides an expression profile and library code:count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1B.2, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention. The first number in Table 1B.2, column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4. The second number in column 5 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33P dCTP, using oligo (dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.















TABLE 1B.1









ORF
AA





Gene
cDNA
Contig
SEQ ID
(From-
SEQ ID
Predicted
Cytologic



No:
Clone ID
ID:
NO: X
To)
NO: Y
Epitopes
Band
OMIM Disease Reference(s)























3
HTEEB42
206980
9
59-952
10
Met-1 to His-7.
21q21.2



4
HEMCM42
407085
11
58-399
12
Pro-35 to Trp-










42, Ala-53 to










Asp-62, Arg-










103 to Pro-113.




4
HEMCM42
1352150
15
25-411
16
Pro-35 to Trp-










42, Ala-53 to










Asp-62, Arg-










103 to Phe-110,










Ile-114 to Glu-










120.




4
HEQCC55
884824
13
62-397
14
Pro-35 to Trp-










42, Pro-65 to










Asp-72, Thr-86










to Phe-93, Ile-










97 to Glu-103.




4
HEQCC55
1352368
15
25-411
16
Pro-35 to Trp-
16p13.3
141750, 141800, 141800,








42, Ala-53 to

141800, 141800, 141850,








Asp-62, Arg-

141850, 141850, 141850,








103 to Phe-110,

141850, 156850, 186580,








Ile-114 to Glu-

191092, 600140, 600273,








120.

601313, 601785


4
HEQCC55
748227
17
57-524
18
Pro-35 to Trp-










42, Pro-65 to










Asp-72, Thr-86










to Glu-92, Pro-










96 to Gly-104,










Ser-138 to Gly-










154.




5
HEMAE80
409495
20
12-422
21
Ser-91 to Lys-










98.




5
HEMAE80
1310948
22
61-471
23
Ser-91 to Lys-
4p16-p15
225500, 600593, 602363








98.




6
HRDFB85
411020
28
233-838 
29
Gly-147 to Met-
7q11.23
116860, 129900, 233700,








152, Cys-177 to

600079








Lys-188.




7
HDTAW95
412472
38
571-621 
39





8
HEMCV19
423219
46
79-615
47
Thr-19 to Ala-










33, Leu-54 to










Asp-82, Pro-89










to Ala-97, Pro-










100 to Lys-125,










Ser-127 to Phe-










135, Gly-164 to










Leu-169, Cys-










173 to Arg-178.




8
HEMCV19
1352162
48
84-626
49
Thr-19 to Ser-
19q12-q13.1
164731, 172400, 172400,








31, Leu-54 to

180901, 180901, 221770,








Asp-82, Pro-89

248600, 600918, 602716








to Ala-97, Pro-










100 to Lys-125,










Ser-127 to Phe-










135.




8
HEMCV19
1352161
50
125-679 
51
Thr-19 to Ala-










33, Leu-54 to










Asp-82, Pro-89










to Ala-97, Pro-










100 to Lys-125,










Ser-127 to Phe-










135, Ser-180 to










Ser-185.




9
HETBX14
806447
61
207-956 
62
Glu-27 to Trp-
19q13.3-q13.4
113900, 126340, 126391,








35, Leu-77 to

130410, 134790, 138570,








Ala-89, Pro-96

160900, 173850, 191044,








to Asn-109,

258501, 600040, 600138,








Ser-149 to Arg-

602225, 602225








156, Gln-172 to










Ile-182, Glu-










193 to Gly-204,










Glu-245 to Asn-










250.




9
HETBX14
422659
63
74-235
64
Gly-12 to Ser-










22, Pro-34 to










Ser-53.




10
HLHSK94
1307727
72
112-1251
73
Gly-29 to Glu-
12q14.3
181430, 600808, 602116








34, Arg-71 to










Arg-76, Thr-










176 to Cys-182,










Gly-184 to Glu-










199, Lys-277 to










Lys-287, Ser-










292 to Cys-305,










Gly-318 to Tyr-










341, Gln-358 to










Tyr-377.




10
HLHSK94
422828
74
112-1251
75
Gly-29 to Glu-










34, Arg-71 to










Arg-76, Thr-










176 to Cys-182,










Gly-184 to Glu-










199.




11
HLHFP03
460467
76
224-574 
77
Tyr-28 to Phe-










34, Thr-54 to










Val-60, Tyr-73










to Thr-82.




11
HLHFP03
460467
78
224-574 
79
Tyr-28 to Phe-










34, Thr-54 to










Val-60, Tyr-73










to Thr-82.




12
HHTLF25
461438
83
142-474 
84
Ala-28 to Ser-
19q13.1
164731, 172400, 172400,








33, Ala-76 to

180901, 180901, 221770,








Lys-111.

248600, 600918, 602716


13
HTADX17
457172
90
84-512
91
Glu-15 to Arg-










23, Asn-79 to










Gly-84.




13
HTADX17
753289
92
92-520
93
Glu-15 to Arg-
1q23.1
107300, 131210, 136132,








23, Asn-79 to

145001, 173610, 601652








Gly-84, Ser-101










to Gly-106, Ser-










111 to Asn-116.




13
HTADX17
457172
94
84-512
95
Glu-15 to Arg-










23, Asn-79 to










Gly-84.




14
HJACG02
509948
102
47-373
103
Val-54 to Asp-










59.




14
HJACG02
1307789
104
66-392
105
Val-54 to Asp-
19p13.3
108725, 120700, 133171,








59.

136836, 145981, 147141,










164953, 188070, 600957,










601238, 601846, 602216,










602477


15
HKGAJ54
498303
107
31-942
108
Ala-55 to Thr-










62, His-164 to










Gly-175, Ala-










197 to Glu-202.




15
HKGAJ54
1300770
109
 24-1046
110
Ala-55 to Thr-










62, His-164 to










Gly-175, Ala-










197 to Thr-204,










Ser-212 to Ala-










220, Gln-226 to










Glu-233, Pro-










252 to Gly-263,










Arg-318 to Glu-










326, Ser-331 to










Pro-340.




16
HE8CH92
609866
118
 31-1167
119
Ser-44 to Ser-51,










Cys-53 to Cys-










64, Val-76 to










Lys-83, Pro-102










to Gly-108,










Arg-133 to Thr-










162, Thr-204 to










Ala-209, Asp-










235 to Glu-241,










Lys-270 to Ala-










282, Ala-286 to










Gly-297, Ser-










346 to Arg-351,










Gly-368 to Gly-










374.




16
HSVAK93
597462
116
21-569
117
Ser-44 to Ser-51,










Cys-53 to Cys-










64, Val-76 to










Lys-83, Pro-102










to Gly-108,










Arg-133 to Thr-










162, Thr-169 to










Lys-183.




16
HSVAK93
1352228
120
 24-1157
121
Ser-44 to Ser-51,










Cys-53 to Cys-










64, Val-76 to










Lys-83, Pro-102










to Gly-108,










Arg-133 to Thr-










162, Thr-204 to










Ala-209, Asp-










235 to Glu-241,










Lys-270 to Ala-










282, Ala-286 to










Gly-297, Ser-










346 to Arg-351,










Gly-368 to Gly-










374.




17
HSDEK49
625998
123
126-1043
124
Val-29 to Val-










37, Asp-71 to










His-76, Gln-78










to Gly-84, Met-










105 to His-110,










Trp-117 to Gly-










122, Gln-136 to










Lys-141, Leu-










143 to Ala-149,










Thr-162 to Asp-










174, Ser-181 to










Lys-186, Arg-










214 to Glu-220,










Glu-232 to Glu-










238, Cys-249 to










Asp-265.




17
HSDEK49
1352253
125
 60-1256
126
Val-29 to Val-
Xq12-q13.3
300011, 300011, 300011,








37, Asp-71 to

300127, 305450, 309605,








His-76, Gln-78

313700, 313700, 313700,








to Gly-84, Met-

313700, 313700, 314580








105 to His-110,










Trp-117 to Asn-










123, Lys-179 to










Pro-187, Gly-










218 to Asp-224,










Leu-237 to Ala-










243, Thr-256 to










Asp-268, Ser-










275 to Lys-280,










Arg-308 to Glu-










314, Glu-326 to










Glu-332, Cys-










343 to Asp-359.




18
HWBAO62
838164
127
52-687
128
Ile-40 to Glu-45,










Cys-63 to Val-










69, Glu-83 to










Asn-94, Pro-










107 to Cys-115,










Phe-137 to Ser-










143, Ser-159 to










Thr-167, Glu-










200 to Tyr-210.




18
HWBAO62
625914
129
81-386
130
Ile-40 to Glu-45,










Cys-63 to Val-










69, Glu-83 to










Phe-95.




19
HWHGU54
695695
131
145-1389
132
Phe-25 to Tyr-










30, Gln-37 to










Arg-42, Lys-










106 to Leu-112,










Leu-123 to Leu-










130, Gln-142 to










Phe-150, Gln-










183 to Lys-188,










Asp-219 to Glu-










226, Lys-359 to










Glu-366.




20
HCEJQ69
1243825
134
 39-1457
135
Thr-41 to Gly-

104170, 104170, 104170,








47, Pro-170 to

115470, 142360, 188400,








Asp-176, Arg-

188400, 217095, 600850,








257 to Trp-262,

601607








Gln-276 to Ser-










283, Phe-278 to










Val-285, Arg-










323 to Gly-331,










Glu-348 to Ser-










354, Arg-362 to










Gly-385, Pro-










364 to Arg-377,










Arg-407 to Leu-










431, Pro-409 to










Ser-427, Gly-










438 to Gly-448,










Gly-440 to Ala-










449.




20
HCEJQ69
1243825
134
 39-1457
135
Thr-41 to Gly-
22q11
104170, 104170, 104170,








47, Pro-170 to

115470, 142360, 188400,








Asp-176, Leu-

188400, 217095, 600850,








257 to Trp-262,

601607








Gln-276 to Ser-










283, Arg-323 to










Leu-330, Pro-










364 to Arg-377,










Arg-407 to Leu-










431, Gly-438 to










Gly-448.




20
HCEJQ69
872582
136
 39-1688
137
Thr-41 to Gly-










47, Pro-170 to










Asp-176, Arg-










257 to Trp-262,










Gln-276 to Ser-










283, Phe-278 to










Val-285, Arg-










323 to Gly-331,










Glu-348 to Ser-










354, Arg-362 to










Gly-385, Pro-










364 to Arg-377,










Arg-407 to Leu-










431, Pro-409 to










Ser-427, Gly-










438 to Gly-448,










Gly-440 to Ala-










449, Pro-500 to










Ser-514, Pro-










521 to His-528.




20
HCEJQ69
872582
136
 39-1688
137
Thr-41 to Gly-










47, Pro-170 to










Asp-176, Leu-










257 to Trp-262,










Gln-276 to Ser-










283, Arg-323 to










Leu-330, Pro-










364 to Phe-374,










Pro-401 to Ser-










409, Arg-415 to










Arg-427, Ala-










474 to Gln-484,










Pro-500 to Ser-










514, Pro-521 to










His-528.




20
HCEJQ69
609999
138
 39-1178
139
Thr-41 to Gly-










47, Pro-170 to










Asp-176, Arg-










257 to Trp-262,










Gln-276 to Ser-










283, Phe-278 to










Val-285, Arg-










323 to Gly-331,










Glu-348 to Ser-










354.




20
HCEJQ69
609999
138
 39-1178
139
Thr-41 to Gly-










47, Pro-170 to










Asp-176, Leu-










257 to Trp-262,










Gln-276 to Ser-










283, Arg-323 to










Leu-330, Pro-










362 to Val-374.




21
HT5GJ57
740767
159
122-694 
160
Ser-29 to Thr-
7q11.23
116860, 129900, 233700,








57, Pro-74 to

600079








Lys-79, Pro-85










to Glu-107, Tyr-










118 to Tyr-136,










Gln-144 to Gln-










152, Ala-182 to










Glu-188.




21
HT5GJ57
1299921
161
105-836 
162
Ser-29 to Thr-
7q11.23
116860, 129900, 233700,








57, Pro-74 to

600079








Lys-79, Pro-85










to Glu-107,










Tyr-118 to Tyr-










136, Gln-144 to










Gln-152, Ala-










182 to Asn-195,










Arg-203 to Val-










208, Leu-212 to










Ser-217, Gly-










222 to Val-234.




22
HPIBX03
743314
169
 81-2207
170

3p24.3-p22.1
154705, 182280, 190160,










227646, 261510, 600163,










601154


23
HDPBO81
892018
174
265-1308
175
Asp-53 to Tyr-










61, Pro-105 to










Ile-128, Arg-










133 to Leu-140,










Gln-182 to Ala-










188, Pro-205 to










Asn-218, Gly-










259 to Ala-264,










Asn-290 to Ser-










302, Glu-307 to










Tyr-314, Tyr-










317 to Lys-332.




23
HDPBO81
790188
176
255-1301
177
Asp-53 to Tyr-










61, Pro-105 to










Ile-128, Arg-










133 to Leu-140,










Gln-182 to Ala-










188, Pro-205 to










Asn-218, Gly-










259 to Ser-264.




24
HWBFY57
837478
179
113-985 
180
Ser-69 to Arg-










79, Ile-82 to










Arg-89, Pro-129










to Ser-137, Leu-










146 to Lys-151.




25
HYABV21
1281466
182
55-960
183





25
HYABV21
1213593
184
63-728
185





26
HOHBY69
827480
186
 82-3648
187
Phe-23 to Arg-
15q22.3-q23
118485, 151670, 231680,








31, Leu-62 to

272800, 272800, 272800,








Asp-72, Val-96

276700, 600374, 601780








to Asp-101,










Thr-111 to Asn-










116, Glu-128 to










Thr-135, Val-










142 to Ser-149,










Asn-217 to Val-










222, Glu-233 to










Arg-241, Gly-










272 to Leu-280,










Gln-286 to Thr-










293, Tyr-303 to










Ile-308, Gly-










354 to Thr-360,










Glu-408 to Lys-










419, Glu-508 to










Lys-514, Arg-










521 to Val-526,










Gly-529 to Phe-










542, Asp-551 to










Tyr-557, Thr-










587 to Thr-593,










His-656 to Asp-










665, Met-697 to










Arg-705, Asp-










709 to Thr-716,










Glu-755 to Gly-










760, Asn-779 to










His-786, Leu-










810 to Asp-816,










Leu-844 to Ala-










851, Gln-871 to










Gly-877, Glu-










884 to Gln-889,










Ser-931 to Asn-










943, Ser-974 to










Ile-982, Gly-










1039 to Gln-










1058, Arg-1121










to Arg-1127,










Ser-1134 to Trp-










1139, Ser-1172










to Pro-1183.




26
HOHBY69
815681
188
 84-3185
189
Phe-23 to Arg-










31, Leu-62 to










Asp-72, Val-96










to Asp-101,










Thr-111 to Asn-










116, Glu-128 to










Thr-135, Val-










142 to Ser-149,










Asn-217 to Val-










222, Glu-233 to










Arg-241, Gly-










272 to Leu-280,










Gln-286 to Thr-










293, Tyr-303 to










Ile-308, Gly-










354 to Thr-360,










Glu-408 to Lys-










419, Glu-508 to










Lys-514, Arg-










521 to Val-526,










Gly-529 to Phe-










542, Asp-551 to










Tyr-557, Thr-










587 to Thr-593,










His-656 to Asp-










665, Met-697 to










Arg-705, Asp-










709 to Thr-716,










Glu-755 to Gly-










760, Asn-779 to










His-786, Leu-










810 to Asp-816,










Leu-844 to Ala-










851, Gln-871 to










Gly-877, Glu-










884 to Gln-889,










Ser-931 to Asn-










943, Ser-974 to










Ile-982.




27
HDHMA45
902513
202
199-1440
203
Ala-145 to Ser-
11q









154, Ala-258 to










Tyr-263, Ala-










287 to Arg-297,










Thr-306 to Met-










316.




27
HDHMA45
812764
204
204-1445
205
Ala-145 to Ser-










154, Ala-258 to










Tyr-263, Ala-










287 to Arg-297,










Thr-306 to Met-










316.




28
HMADJ14
1099342
206
278-1333
207
Asp-229 to Gln-
8q22
216550, 259730








236, Asn-244 to










Lys-250, Trp-










258 to Asn-266.




28
HMADJ14
889659
208
264-1037
209
Asp-229 to Gln-










236, Asn-244 to










Phe-251.




28
HMADJ14
843725
212
125-898 
213
Asp-229 to Gln-
8q22
216550, 259730








236, Asn-244 to










Phe-251.




28
HMADJ14
795479
214
264-1139
215
Asp-229 to Cys-










239, Asn-244 to










Glu-253, Thr-










271 to Ser-278.




28
HMADJ14
426068
216
47-772
217





30
HEMFA84
608198
222
42-815
223
Leu-23 to Asp-
17p13.3
113721, 247200, 600059,








34, Cys-97 to

601545








Pro-106, Ser-










202 to Gly-208,










Pro-251 to Gly-










257.




31
HDPPA04
904765
224
271-1122
225
Lys-61 to Arg-










72, Arg-95 to










Tyr-100, Ala-










121 to Ile-126,










Asn-163 to Gly-










172, Lys-183 to










Asn-189, Ser-










211 to His-218,










Leu-251 to Val-










269.




31
HDPPA04
905419
226
1003-1074 
227
Ser-16 to Lys-










23.




31
HDPPA04
905418
228
261-542 
229
Lys-61 to Arg-










72.




32
HE2OA95
637595
230
1224-1331 
231

2q32.3
600258, 602087


33
HKABZ65
862030
232
77-808
233
Ser-25 to Ala-










31, Gln-146 to










Ser-151, His-










231 to Asn-236.




33
HKABZ65
665424
234
69-800
235
Ser-25 to Ala-










31, Gln-146 to










Ser-151, His-










231 to Asn-236.




















TABLE 1B.2






cDNA Clone


Tissue Distribution Library Code: Count (see Table 4


Gene No:
ID
Contig ID:
SEQ ID NO: X
for Library Codes)



















3
HTEEB42
206980
9
AR174: 12, AR191: 12, AR190: 11, AR244: 11,






AR181: 11, AR291: 10, AR186: 10, AR180: 10,






AR175: 10, AR192: 10, AR1899, AR1769, AR2409,






AR2699, AR2419, AR1789, AR2709, AR1778, AR2668,






AR2688, AR1838, AR2738, AR2748, AR1658, AR2477,






AR1647, AR1987, AR1847, AR1667, AR1627, AR2027,






AR1617, AR1637, AR2467, AR2456, AR1976, AR2896,






AR1736, AR2016, AR2676, AR1826, AR2716, AR0526,






AR2066, AR3096, AR1856, AR1886, AR2756, AR2636,






AR2515, AR2365, AR2845, AR1945, AR2955, AR2555,






AR2355, AR2775, AR2995, AR1795, AR0555, AR2905,






AR1045, AR0335, AR1935, AR2284, AR2304, AR2044,






AR1964, AR1704, AR2854, AR2564, AR1724, AR2724,






AR2574, AR2624, AR2054, AR2334, AR3084, AR2614,






AR1954, AR2524, AR3004, AR2234, AR0894, AR2874,






AR2384, AR2434, AR2144, AR2964, AR2374, AR2654,






AR2504, AR2394, AR2884, AR2984, AR2243, AR2943,






AR2293, AR2483, AR3163, AR2073, AR2863, AR3123,






AR2973, AR2643, AR1993, AR0613, AR2933, AR0533,






AR2273, AR0603, AR3113, AR2113, AR2493, AR2253,






AR2923, AR2193, AR2583, AR0393, AR2153, AR3133,






AR2823, AR2263, AR2603, AR2312, AR2422, AR2032,






AR1712, AR1682, AR2102, AR2002, AR2592, AR2342,






AR0962, AR2322, AR1692, AR2222, AR2542, AR2182,






AR2212, AR2532, AR2832, AR2131, AR2161, AR2171,






AR3101 L0794: 4, H0624: 2, H0038: 2, L0375: 2,






S0330: 2, L0750: 2, L0779: 2, H0031: 1, H0644: 1,






H0124: 1, H0591: 1, H0616: 1, H0264: 1, H0623: 1,






L0770: 1, L0637: 1, L0805: 1, L0663: 1, L0749: 1,






L0777: 1, L0780: 1 and L0599: 1.


4
HEMCM42
407085
11


4
HEQCC55
884824
13


4
HEQCC55
1352368
15
AR216: 11, AR217: 10, AR2149, AR2079, AR2638,






AR1958, AR1658, AR2538, AR2248, AR2428, AR0538,






AR1648, AR1638, AR2468, AR1618, AR2228, AR2458,






AR1668, AR1708, AR1628, AR3087, AR1977, AR2127,






AR3097, AR2237, AR3127, AR1987, AR3116, AR2506,






AR2546, AR2056, AR2436, AR2136, AR2746, AR1686,






AR2646, AR1935, AR2965, AR2015, AR2725, AR2385,






AR0335, AR2755, AR1755, AR2824, AR3134, AR2214,






AR2914, AR2834, AR2254, AR1744, AR2354, AR1044,






AR2614, AR1714, AR2774, AR2974, AR2884, AR1774,






AR3004, AR1834, AR2954, AR1694, AR3164, AR1924,






AR2704, AR1814, AR0894, AR2664, AR2893, AR2693,






AR1783, AR2263, AR1733, AR1723, AR2393, AR2683,






AR2993, AR2903, AR2933, AR1893, AR1963, AR1853,






AR2313, AR2573, AR2403, AR2853, AR2473, AR1763,






AR0393, AR2103, AR2713, AR2553, AR1913, AR2673,






AR2043, AR1823, AR0963, AR2623, AR2003, AR1793,






AR2373, AR2273, AR1993, AR2863, AR0603, AR2343,






AR2333, AR2323, AR0612, AR1902, AR2942, AR2872,






AR2582, AR1882, AR2292, AR0552, AR2302, AR2152,






AR2282, AR2032, AR2362, AR2112, AR2192, AR2181,






AR2561 L0803: 5, L0755: 5, L0666: 4, S0418: 3,






H0059: 3, H0494: 3, S0420: 2, H0086: 2, H0551: 2,






H0413: 2, L0763: 2, L3904: 2, L0646: 2, L0800: 2,






L0775: 2, L0659: 2, L0809: 2, H0144: 2, H0435: 2,






H0670: 2, L0731: 2, S0342: 1, H0294: 1, S0180: 1,






H0734: 1, S0046: 1, S0278: 1, H0437: 1, H0392: 1,






H0544: 1, H0545: 1, L0471: 1, H0012: 1, H0375: 1,






H0286: 1, S0250: 1, H0039: 1, H0553: 1, H0628: 1,






H0646: 1, L0769: 1, L5565: 1, L0761: 1, L0764: 1,






L0773: 1, L0662: 1, L0649: 1, L0804: 1, L0774: 1,






L0806: 1, L0653: 1, L0657: 1, L0512: 1, L0789: 1,






L0663: 1, S0406: 1, L0743: 1, L0754: 1, L0750: 1,






L0780: 1, L0581: 1, L0603: 1 and H0665: 1.


4
HEMCM42
1352150
15
AR205: 54, AR202: 41, AR206: 35, AR204: 29,






AR244: 19, AR192: 15, AR194: 11, AR296: 10,






AR198: 10, AR274: 10, AR2319, AR2419, AR2468,






AR1867, AR0606, AR1046, AR1826, AR2346, AR2896,






AR2515, AR2435, AR0525, AR2824, AR0614, AR2194,






AR2664, AR0534, AR0554, AR2704, AR2923, AR2683,






AR0393, AR2983, AR2933, AR2903, AR2693, AR3163,






AR2833, AR1833, AR0333, AR2372, AR0892, AR2482,






AR1772, AR2292, AR2182, AR2402, AR3102, AR3122,






AR3002, AR1752, AR2772, AR2672, AR3131, AR1851,






AR2561, AR2991, AR0961 H0494: 9, L0803: 5,






L0755: 5, S0418: 4, H0551: 4, L3904: 4, L0666: 4,






L2260: 4, H0435: 4, H0734: 3, H0510: 3, H0059: 3,






S0436: 3, S0420: 2, H0545: 2, H0086: 2, H0628: 2,






H0413: 2, L0763: 2, L0646: 2, L0800: 2, L0775: 2,






L0659: 2, L0809: 2, L0663: 2, H0144: 2, H0670: 2,






S0380: 2, S0406: 2, L0731: 2, L0581: 2, S0342: 1,






H0294: 1, S0180: 1, S0212: 1, H0484: 1, S0408: 1,






S0046: 1, H0393: 1, S0278: 1, H0437: 1, H0392: 1,






H0587: 1, H0599: 1, H0748: 1, H0544: 1, L0471: 1,






H0012: 1, H0375: 1, H0286: 1, S0250: 1, H0039: 1,






H0553: 1, H0646: 1, H0652: 1, S0144: 1, S0422: 1,






L0769: 1, L5565: 1, L0761: 1, L0764: 1, L0773: 1,






L0662: 1, L0649: 1, L0804: 1, L0774: 1, L0806: 1,






L0653: 1, L0657: 1, L0512: 1, L0789: 1, H0519: 1,






H0593: 1, S0126: 1, H0684: 1, H0672: 1, H0555: 1,






L0743: 1, L0754: 1, L0750: 1, L0780: 1, L0603: 1 and






H0665: 1.


4
HEQCC55
748227
17


5
HEMAE80
409495
20


5
HEMAE80
1310948
22
AR055: 113, AR060: 64, AR185: 56, AR283: 50,






AR299: 49, AR300: 47, AR240: 43, AR277: 42,






AR282: 42, AR104: 41, AR039: 41, AR089: 36,






AR316: 33, AR218: 31, AR219: 31, AR096: 28,






AR313: 16 S0126: 7, L0748: 6, S0338: 3, L0759: 3,






S0318: 2, S0046: 1, H0251: 1, S0334: 1, S0336: 1,






S0316: 1, S0312: 1, S0314: 1, H0056: 1, L0769: 1,






L0659: 1, L0747: 1, L0749: 1, L0750: 1, L0777: 1,






L0780: 1, S0194: 1 and S0276: 1.


6
HRDFB85
411020
28
AR205: 15, AR1949, AR2448, AR2467, AR2737,






AR2637, AR2046, AR2705, AR2435, AR2065, AR3105,






AR2745, AR1984, AR2414, AR1864, AR1824, AR0394,






AR3094, AR3123, AR2823, AR2473, AR2533, AR2403,






AR0893, AR2713, AR2893, AR0523, AR2693, AR2773,






AR0603, AR2513, AR2653, AR1833, AR3163, AR1923,






AR0532, AR2982, AR2672, AR2842, AR2992, AR0612,






AR1842, AR1752, AR2132, AR2342, AR2682, AR0552,






AR2952, AR2752, AR2662, AR2272, AR2492, AR2932,






AR3002, AR0332, AR2912, AR2942, AR2902, AR1852,






AR2312, AR2852, AR3132, AR0962, AR2962, AR2382,






AR2261, AR2371, AR2331, AR2181, AR2291, AR2811,






AR2861, AR1041, AR2831, AR2921, AR2591, AR2561






S0436: 29, S0406: 23, S0440: 20, S0442: 16, S0358: 15,






H0622: 15, S0354: 14, S0408: 12, S0444: 11, H0435: 11,






S0360: 10, S0356: 9, H0039: 9, L0666: 9, L0751: 9,






H0553: 8, H0506: 8, H0689: 7, L0748: 7, H0661: 6,






L0659: 6, L0809: 6, L0665: 6, S0376: 5, L3818: 5,






L0743: 5, H0484: 4, H0483: 4, H0059: 4, L0764: 4,






L0771: 4, L0662: 4, S0330: 4, H0696: 4, H0615: 3,






H0688: 3, H0040: 3, L0763: 3, L0364: 3, L0774: 3,






L0663: 3, H0683: 3, H0670: 3, H0672: 3, L0750: 3,






L0731: 3, S0434: 3, L0601: 3, H0663: 2, H0549: 2,






H0046: 2, H0014: 2, H0124: 2, H0494: 2, S0450: 2,






L0374: 2, L0765: 2, L0804: 2, L0378: 2, L0805: 2,






L0653: 2, L0529: 2, H0593: 2, H0658: 2, S0152: 2,






L0439: 2, L0779: 2, H0294: 1, S0430: 1, H0675: 1,






H0676: 1, H0749: 1, H0370: 1, H0587: 1, H0642: 1,






T0109: 1, H0204: 1, L0040: 1, L0738: 1, H0620: 1,






H0024: 1, H0356: 1, H0375: 1, L0194: 1, T0023: 1,






L0483: 1, H0033: 1, H0424: 1, H0644: 1, H0264: 1,






H0488: 1, H0413: 1, H0334: 1, S0352: 1, S0306: 1,






H0647: 1, H0646: 1, H0652: 1, L0373: 1, L0646: 1,






L0648: 1, L0794: 1, L0803: 1, L0375: 1, L0376: 1,






L0806: 1, L0658: 1, L0382: 1, L0528: 1, L5623: 1,






L0664: 1, S0374: 1, H0690: 1, H0682: 1, H0648: 1,






S0328: 1, H0478: 1, S0432: 1, L0744: 1, L0747: 1,






L0749: 1, L0755: 1, L0759: 1, H0707: 1, L0596: 1,






H0543: 1, S0462: 1 and H0352: 1.


7
HDTAW95
412472
38
AR219: 109, AR218: 78, AR282: 11, AR3164,






AR2993, AR3132, AR3002, AR2402, AR0391, AR0551






S0360: 9, L0794: 8, L0659: 6, S0436: 6, H0622: 5,






S0126: 4, H0170: 3, H0587: 3, S0422: 3, L0800: 3,






L0649: 3, S0390: 3, S0028: 3, L0777: 3, L0757: 3,






H0662: 2, H0586: 2, H0013: 2, H0150: 2, H0551: 2,






L0770: 2, L0761: 2, L0773: 2, H0682: 2, H0648: 2,






S0406: 2, L0743: 2, L0751: 2, L0779: 2, S0026: 2,






S0194: 2, H0506: 2, H0624: 1, S0212: 1, S0356: 1,






S0354: 1, S0376: 1, H0643: 1, H0486: 1, H0427: 1,






H0156: 1, L0021: 1, L0105: 1, H0031: 1, H0628: 1,






H0316: 1, H0090: 1, H0412: 1, S0450: 1, S0440: 1,






H0647: 1, L0763: 1, L0372: 1, L0646: 1, L0771: 1,






L0648: 1, L0767: 1, L0768: 1, L0805: 1, L0653: 1,






L0606: 1, L0789: 1, L4501: 1, H0144: 1, H0762: 1,






H0547: 1, H0690: 1, H0684: 1, H0435: 1, S0152: 1,






S0332: 1, H0626: 1, S0037: 1, S3014: 1, L0747: 1,






L0480: 1, S0192: 1 and S0196: 1.


8
HEMCV19
423219
46


8
HEMCV19
1352162
48
AR277: 50, AR283: 34, AR219: 33, AR096: 32,






AR240: 32, AR218: 31, AR316: 30, AR089: 23,






AR282: 23, AR039: 23, AR104: 23, AR313: 23,






AR299: 21, AR300: 20, AR185: 20, AR055: 17,






AR060: 17 H0617: 7, S0114: 5, H0486: 5, H0069: 5,






S0344: 5, L0794: 5, H0436: 5, L0748: 5, H0341: 4,






H0255: 4, L0744: 4, S0436: 4, H0295: 3, H0294: 3,






T0049: 3, H0661: 3, S0356: 3, S0476: 3, H0581: 3,






H0271: 3, H0038: 3, H0087: 3, H0529: 3, L0364: 3,






L0766: 3, S0126: 3, L0747: 3, L0749: 3, H0423: 3,






H0740: 2, H0657: 2, S0376: 2, H0497: 2, H0635: 2,






H0616: 2, S0440: 2, S0144: 2, S0142: 2, L0771: 2,






L0378: 2, L0806: 2, L0518: 2, H0658: 2, S0044: 2,






S3014: 2, S0027: 2, L0754: 2, L0777: 2, L0731: 2,






H0445: 2, L0588: 2, L0599: 2, S0424: 2, L2852: 2,






H0556: 1, T0002: 1, S0040: 1, H0717: 1, L2865: 1,






H0656: 1, L2919: 1, H0381: 1, H0402: 1, H0305: 1,






S0360: 1, H0580: 1, H0722: 1, H0728: 1, S0046: 1,






H0747: 1, S0278: 1, H0549: 1, H0586: 1, H0333: 1,






T0060: 1, L0021: 1, H0575: 1, T0048: 1, H0318: 1,






H0421: 1, H0263: 1, H0597: 1, H0530: 1, H0545: 1,






H0041: 1, S0388: 1, H0266: 1, S0334: 1, H0688: 1,






L0483: 1, H0424: 1, H0644: 1, H0628: 1, H0182: 1,






H0316: 1, H0090: 1, H0591: 1, H0063: 1, H0551: 1,






H0264: 1, H0412: 1, H0100: 1, H0280: 1, S0426: 1,






H0743: 1, L0369: 1, L0520: 1, L4497: 1, L0770: 1,






L0769: 1, L3904: 1, L0761: 1, L0772: 1, L0800: 1,






L0662: 1, L5569: 1, L0381: 1, L0775: 1, L0805: 1,






L0654: 1, L0776: 1, L0655: 1, L0657: 1, L0656: 1,






L0382: 1, L0809: 1, L0543: 1, L0791: 1, L0666: 1,






L0664: 1, H0697: 1, H0699: 1, H0689: 1, H0690: 1,






H0660: 1, H0518: 1, H0521: 1, H0696: 1, S0406: 1,






H0576: 1, S3012: 1, L0743: 1, L0780: 1, L0757: 1,






L0759: 1, S0031: 1, L0593: 1, L0595: 1, H0542: 1,






H0543: 1, H0422: 1 and H0506: 1.


8
HEMCV19
1352161
50


9
HETBX14
806447
61
AR277: 75, AR269: 27, AR163: 22, AR165: 22,






AR162: 21, AR161: 21, AR164: 21, AR089: 21,






AR282: 21, AR166: 20, AR173: 17, AR243: 16,






AR183: 16, AR188: 16, AR182: 15, AR268: 14,






AR060: 14, AR196: 14, AR290: 14, AR238: 13,






AR270: 12, AR240: 12, AR246: 12, AR291: 12,






AR193: 11, AR300: 11, AR176: 11, AR245: 11,






AR175: 11, AR180: 11, AR267: 10, AR191: 10,






AR205: 10, AR316: 10, AR254: 10, AR250: 10,






AR242: 10, AR255: 10, AR2339, AR2479, AR0969,






AR2979, AR2379, AR1819, AR0398, AR1988, AR2318,






AR2968, AR2108, AR1978, AR1898, AR2858, AR2758,






AR2138, AR2347, AR0617, AR2397, AR1747, AR2667,






AR2837, AR2007, AR3137, AR2017, AR1797, AR1777,






AR1787, AR2327, AR2727, AR2717, AR2957, AR3087,






AR2197, AR2877, AR2537, AR3127, AR2267, AR2276,






AR2366, AR2886, AR2036, AR2996, AR2286, AR2616,






AR1926, AR1856, AR2746, AR2296, AR1996, AR0536,






AR1906, AR1956, AR2936, AR2606, AR2116, AR2576,






AR0335, AR3115, AR2125, AR2165, AR2645, AR2255,






AR3095, AR2155, AR1695, AR2895, AR2865, AR2185,






AR1685, AR2235, AR2945, AR2175, AR2585, AR2044,






AR2354, AR2244, AR2524, AR1714, AR2624, AR2304,






AR0554, AR1704, AR2143, AR1043, AR1723, AR2633,






AR2563, AR2213, AR2072, AR2222 L0751: 6, L0809:






5, S0328: 5, H0046: 4, L0758: 4, L0764: 3, L0665: 2,






L0747: 2, H0592: 1, H0486: 1, H0263: 1, H0598: 1,






H0063: 1, H0646: 1, L0662: 1, L0657: 1, L0543: 1,






L5622: 1, L0793: 1, L0666: 1, L2654: 1, H0658: 1,






S0330: 1 and S0044: 1.


9
HETBX14
422659
63


10
HLHSK94
1307727
72
AR283: 613, AR104: 327, AR219: 244, AR218: 219,






AR055: 199, AR316: 176, AR313: 160, AR089: 107,






AR060: 106, AR039: 106, AR096: 99, AR299: 86,






AR185: 65, AR240: 56, AR282: 51, AR300: 46,






AR277: 26, AR2524, AR2463, AR2233, AR2353,






AR2533, AR2663, AR1703, AR1693, AR2302, AR2432,






AR2622, AR3122, AR2142, AR3092, AR2612, AR1972,






AR2742, AR1932, AR2552, AR0532, AR2972, AR2252,






AR1682, AR2722, AR1811, AR2171, AR2871, AR2571,






AR2051, AR2911, AR0331, AR1911, AR1901, AR2361,






AR1751 H0046: 44, H0135: 10, H0539: 10, L0455: 7,






S0010: 3, L0456: 3, L0750: 3, L0663: 2, L0746: 2,






L0747: 2, L0779: 2, L0777: 2, H0624: 1, S0116: 1,






H0208: 1, L0717: 1, H0549: 1, H0333: 1, H0013: 1,






L0157: 1, T0006: 1, H0652: 1, L0666: 1, H0144: 1,






L3811: 1, S0328: 1, H0696: 1 and L0439: 1.


10
HLHSK94
422828
74


11
HLHFP03
460467
76
AR194: 6, AR186: 6, AR169: 6, AR170: 5, AR202:






5, AR060: 5, AR206: 5, AR184: 5, AR176: 5,






AR273: 4, AR249: 4, AR248: 4, AR223: 4, AR161:






4, AR162: 4, AR251: 4, AR163: 4, AR061: 4,






AR244: 4, AR052: 4, AR055: 4, AR310: 4, AR282:






4, AR053: 4, AR267: 4, AR253: 3, AR235: 3,






AR183: 3, AR269: 3, AR182: 3, AR312: 3, AR204:






3, AR266: 3, AR192: 3, AR246: 3, AR275: 3,






AR185: 3, AR270: 3, AR283: 3, AR089: 3, AR298:






3, AR295: 3, AR039: 3, AR241: 3, AR271: 3,






AR309: 3, AR181: 3, AR166: 3, AR291: 3, AR263:






3, AR257: 3, AR217: 3, AR316: 3, AR289: 3,






AR299: 3, AR296: 3, AR033: 3, AR238: 3, AR292:






3, AR205: 2, AR247: 2, AR313: 2, AR104: 2,






AR193: 2, AR231: 2, AR213: 2, AR268: 2, AR168:






2, AR284: 2, AR262: 2, AR277: 2, AR237: 2,






AR212: 2, AR243: 2, AR274: 2, AR297: 2, AR286:






2, AR228: 2, AR240: 2, AR233: 2, AR272: 2,






AR285: 2, AR300: 2, AR165: 2, AR229: 2, AR226:






2, AR096: 2, AR293: 2, AR255: 2, AR294: 2,






AR191: 2, AR290: 2, AR164: 2, AR172: 2, AR264:






2, AR227: 2, AR174: 2, AR287: 2, AR198: 2,






AR265: 2, AR232: 2, AR171: 2, AR216: 2, AR177:






2, AR311: 1, AR234: 1, AR175: 1, AR239: 1,






AR203: 1, AR236: 1, AR230: 1, AR196: 1, AR261:






1, AR260: 1, AR259: 1, AR201: 1, AR189: 1,






AR179: 1, L0742: 4 and H0024: 1.


11
HLHFP03
460467
78
AR1946, AR1866, AR1696, AR1705, AR2025, AR0605,






AR2065, AR1845, AR1765, AR2734, AR2494, AR2484,






AR2234, AR1614, AR0554, AR1624, AR2514, AR1634,






AR0614, AR2824, AR2444, AR0524, AR3104, AR0534,






AR2674, AR2533, AR2353, AR1833, AR2693, AR1823,






AR3123, AR2043, AR2663, AR1923, AR2463, AR2753,






AR2703, AR1043, AR1853, AR2983, AR0893, AR2953,






AR2413, AR2713, AR3093, AR1813, AR1663, AR2913,






AR2633, AR2573, AR2173, AR2893, AR2963, AR0333,






AR2383, AR2833, AR2773, AR2923, AR2052, AR2472,






AR2992, AR1932, AR2312, AR2132, AR2682, AR1682,






AR2842, AR2622, AR2372, AR2122, AR2432, AR2742,






AR2972, AR3002, AR2862, AR2282, AR2402, AR2332,






AR2722, AR2852, AR3162, AR1652, AR2292, AR0962,






AR2262, AR2932, AR3132, AR2552, AR2942, AR1912,






AR2902, AR1642, AR1722, AR2642, AR2272, AR1742,






AR0392, AR2872, AR1982, AR2652, AR2322, AR1712,






AR2162, AR1772, AR3111, AR2341, AR1751, AR2391,






AR2031, AR2361, AR2301, AR2181, AR1961, AR2611,






AR2601, AR2591, AR2011, AR1891, AR1791 L0742:






4 and H0024: 1.


12
HHTLF25
461438
83
AR251: 168, AR248: 141, AR249: 139, AR265: 60,






AR253: 50, AR263: 41, AR096: 37, AR244: 32,






AR268: 26, AR264: 24, AR290: 20, AR246: 18,






AR240: 17, AR177: 16, AR267: 14, AR183: 14,






AR270: 13, AR229: 13, AR184: 12, AR055: 11,






AR269: 10, AR274: 9, AR194: 8, AR175: 8, AR247:






7, AR202: 7, AR316: 7, AR234: 7, AR033: 6,






AR180: 6, AR198: 6, AR271: 6, AR182: 6, AR238:






6, AR299: 5, AR206: 5, AR190: 5, AR205: 5,






AR313: 5, AR188: 5, AR275: 5, AR272: 5, AR061:






5, AR196: 5, AR284: 5, AR241: 5, AR273: 5,






AR173: 5, AR189: 5, AR203: 5, AR199: 5, AR237:






5, AR179: 4, AR200: 4, AR039: 4, AR172: 4,






AR191: 4, AR298: 4, AR192: 4, AR181: 4, AR291:






4, AR282: 4, AR289: 4, AR176: 4, AR224: 4,






AR292: 4, AR186: 3, AR226: 3, AR174: 3, AR300:






3, AR165: 3, AR161: 3, AR162: 3, AR266: 3,






AR285: 3, AR163: 3, AR164: 3, AR231: 3, AR052:






3, AR215: 3, AR295: 3, AR212: 3, AR243: 3,






AR309: 3, AR221: 3, AR166: 3, AR169: 3, AR296:






3, AR232: 2, AR053: 2, AR185: 2, AR223: 2,






AR225: 2, AR089: 2, AR104: 2, AR277: 2, AR233:






2, AR213: 2, AR308: 2, AR286: 2, AR256: 2,






AR257: 2, AR310: 2, AR283: 2, AR235: 2, AR217:






2, AR227: 2, AR204: 2, AR288: 2, AR195: 2,






AR281: 2, AR293: 1, AR312: 1, AR214: 1, AR261:






1, AR294: 1, AR236: 1, AR216: 1, AR193: 1,






AR259: 1, AR230: 1, S0144: 10, L0775: 10, S0278:






6, H0638: 5, H0641: 5, L0438: 5, H0580: 4, S0142: 4,






H0521: 4, H0522: 4, L0747: 4, H0441: 3, S0388: 3,






S0428: 3, H0402: 2, S0358: 2, S0408: 2, S0140: 2,






H0392: 2, H0438: 2, H0086: 2, L0520: 2, L0763: 2,






L0770: 2, L0772: 2, L0771: 2, L0774: 2, L0776: 2,






L0526: 2, H0658: 2, L0743: 2, L0439: 2, L0751: 2,






L0754: 2, L0756: 2, L0605: 2, S0134: 1, S0116: 1,






H0662: 1, S0444: 1, S0360: 1, H0637: 1, S0045: 1,






S0222: 1, S6014: 1, H0455: 1, H0592: 1, H0250: 1,






H0069: 1, H0575: 1, T0082: 1, H0036: 1, H0581: 1,






H0457: 1, S0050: 1, S0051: 1, H0399: 1, H0354: 1,






H0594: 1, H0247: 1, H0271: 1, L0055: 1, S0036: 1,






S0038: 1, S0438: 1, H0646: 1, L0769: 1, L0764: 1,






L0375: 1, L0787: 1, S0053: 1, S0374: 1, H0682: 1,






H0648: 1, H0710: 1, L0744: 1, L0755: 1, L0731: 1,






L0758: 1, L0599: 1, L0603: 1, H0423: 1 and H0352: 1.


12
HHTLF25
461438
83
AR251: 168, AR248: 141, AR249: 139, AR265: 60,






AR253: 50, AR263: 41, AR244: 32, AR096: 32,






AR268: 26, AR264: 24, AR290: 20, AR246: 18,






AR240: 17, AR177: 16, AR267: 14, AR183: 14,






AR270: 13, AR229: 13, AR184: 12, AR269: 10,






AR2749, AR1948, AR1758, AR3167, AR2477, AR2027,






AR3137, AR2347, AR0556, AR2996, AR0336, AR1806,






AR1986, AR2716, AR1826, AR2386, AR2065, AR1905,






AR2055, AR1885, AR2755, AR2725, AR0615, AR1965,






AR2845, AR2415, AR2735, AR1735, AR1895, AR2035,






AR1995, AR2375, AR1794, AR0394, AR2004, AR1724,






AR1914, AR2984, AR1924, AR1814, AR2914, AR1044,






AR2894, AR1764, AR2244, AR2924, AR1863, AR2823,






AR2263, AR1743, AR3003, AR1653, AR1613, AR1623,






AR2663, AR2853, AR1633, AR1643, AR2313, AR1853,






AR0523, AR2153, AR2953, AR2123, AR2433, AR3093,






AR2213, AR1663, AR1693, AR2963, AR2322, AR0532,






AR2232, AR2252, AR0892, AR2772, AR2332, AR2132,






AR3082, AR2862, AR2562, AR2572, AR2832, AR3102,






AR2352, AR2172, AR2272, AR2042, AR2882, AR1952,






AR2812, AR2931, AR3121, AR2141, AR2611, AR2941,






AR2361, AR2161, AR1931, AR2591, AR2301 S0144:






10, L0775: 10, S0278: 6, H0638: 5, H0580: 5, H0641: 5,






L0438: 5, H0521: 5, H0740: 4, H0392: 4, H0522: 4,






L0747: 4, S0408: 3, H0749: 3, H0441: 3, H0438: 3,






S0388: 3, S0428: 3, H0658: 3, H0402: 2, S0358: 2,






S0444: 2, S0140: 2, H0747: 2, H0086: 2, S0142: 2,






L0520: 2, L0763: 2, L0770: 2, L0772: 2, L0771: 2,






L0774: 2, L0776: 2, L0526: 2, L0743: 2, L0439: 2,






L0751: 2, L0754: 2, L0756: 2, L0605: 2, S0116: 1,






H0662: 1, S0360: 1, L3646: 1, H0637: 1, S0045: 1,






S0222: 1, S6014: 1, H0455: 1, H0592: 1, H0250: 1,






H0069: 1, H0575: 1, T0082: 1, H0036: 1, H0581: 1,






H0457: 1, S0050: 1, S0051: 1, H0399: 1, H0354: 1,






H0594: 1, H0247: 1, H0271: 1, L0055: 1, S0036: 1,






S0038: 1, S0438: 1, H0646: 1, L0769: 1, L0764: 1,






L0375: 1, L0787: 1, S0053: 1, S0374: 1, H0682: 1,






H0648: 1, H0710: 1, S0152: 1, H0727: 1, L0744: 1,






L0755: 1, L0731: 1, L0758: 1, L0599: 1, L0603: 1,






H0423: 1 and H0352: 1.


13
HTADX17
457172
90
AR227: 8, AR293: 7, AR176: 7, AR233: 7, AR229:






7, AR232: 6, AR179: 6, AR182: 6, AR237: 6,






AR296: 6, AR266: 6, AR060: 5, AR294: 5, AR287:






5, AR252: 4, AR185: 4, AR286: 4, AR239: 4,






AR255: 4, AR200: 4, AR230: 4, AR271: 4, AR221:






3, AR289: 3, AR282: 3, AR297: 3, AR162: 3,






AR089: 3, AR291: 3, AR290: 3, AR275: 3, AR257:






3, AR096: 3, AR316: 3, AR253: 2, AR270: 2,






AR175: 2, AR242: 2, AR228: 2, AR061: 2, AR262:






2, AR269: 2, AR172: 2, AR161: 2, AR283: 2,






AR168: 2, AR183: 2, AR181: 2, AR205: 2, AR300:






2, AR231: 2, AR177: 2, AR267: 2, AR104: 2,






AR261: 2, AR033: 2, AR264: 2, AR225: 2, AR277:






2, AR195: 2, AR215: 2, AR214: 2, AR238: 2,






AR173: 2, AR197: 2, AR201: 2, AR268: 2, AR246:






2, AR299: 2, AR188: 2, AR313: 2, AR288: 2,






AR216: 2, AR285: 2, AR234: 1, AR309: 1, AR169:






1, AR240: 1, AR224: 1, AR174: 1, AR190: 1,






AR165: 1, AR274: 1, AR039: 1, AR178: 1, AR217:






1, AR260: 1, AR196: 1, AR191: 1, AR204: 1,






AR311: 1, AR055: 1, AR166: 1, AR222: 1, AR210:






1, AR171: 1, AR199: 1, AR258: 1, H0069: 5,






H0634: 1 and L0772: 1.


13
HTADX17
753289
92
AR2278, AR2937, AR1767, AR2337, AR2297, AR2326,






AR1796, AR1826, AR2376, AR2966, AR2666, AR0605,






AR2945, AR0555, AR2875, AR2524, AR2864, AR1854,






AR2394, AR2554, AR2004, AR2304, AR2714, AR2213,






AR2893, AR2823, AR2973, AR1623, AR0893, AR2913,






AR2903, AR2753, AR2573, AR0963, AR2532, AR2702,






AR1752, AR2422, AR2282, AR3162, AR0612, AR2622,






AR2692, AR1722, AR1612, AR3002, AR1682, AR2832,






AR1832, AR1812, AR2052, AR2312, AR1772, AR2672,






AR2612, AR0332, AR2642, AR2252, AR1952, AR2772,






AR2152, AR3132, AR2142, AR2382, AR1732, AR1972,






AR0392, AR2012, AR2682, AR1042, AR2462, AR2182,






AR1882, AR2882, AR2162, AR2992, AR2852, AR2341,






AR3091, AR1691, AR2401, AR2241, AR1741, AR1901,






AR1651, AR2741, AR1781, AR2171, AR2601, AR1961,






AR1911, AR2041, AR3111, AR1661, AR2221, AR2101,






AR1711, AR1991, AR2581 H0069: 5, H0634: 1 and






L0772: 1.


13
HTADX17
457172
94


14
HJACG02
509948
102
AR207: 37, AR195: 33, AR283: 32, AR263: 32,






AR264: 29, AR223: 28, AR214: 28, AR089: 28,






AR277: 27, AR222: 27, AR309: 27, AR311: 27,






AR212: 26, AR316: 26, AR169: 26, AR224: 24,






AR096: 24, AR055: 24, AR197: 23, AR213: 23,






AR282: 22, AR104: 22, AR245: 22, AR171: 22,






AR218: 22, AR162: 22, AR192: 21, AR217: 21,






AR161: 21, AR193: 21, AR163: 20, AR308: 20,






AR165: 20, AR168: 20, AR216: 20, AR170: 20,






AR164: 20, AR235: 19, AR172: 19, AR053: 19,






AR166: 19, AR060: 19, AR219: 19, AR242: 19,






AR271: 19, AR299: 19, AR210: 19, AR039: 19,






AR033: 19, AR240: 18, AR225: 18, AR313: 18,






AR312: 18, AR201: 18, AR221: 17, AR261: 17,






AR198: 17, AR246: 17, AR288: 17, AR252: 17,






AR295: 17, AR176: 16, AR177: 16, AR215: 15,






AR297: 15, AR253: 15, AR205: 15, AR270: 15,






AR196: 15, AR185: 15, AR275: 15, AR286: 15,






AR285: 14, AR260: 14, AR287: 14, AR233: 14,






AR236: 14, AR183: 14, AR227: 13, AR175: 13,






AR211: 13, AR300: 13, AR250: 13, AR294: 13,






AR181: 13, AR274: 13, AR272: 13, AR229: 13,






AR174: 12, AR256: 12, AR182: 12, AR234: 12,






AR204: 12, AR269: 12, AR228: 12, AR293: 12,






AR178: 12, AR226: 12, AR268: 11, AR266: 11,






AR173: 11, AR262: 11, AR200: 11, AR243: 11,






AR199: 11, AR258: 11, AR231: 11, AR291: 11,






AR180: 11, AR289: 11, AR247: 11, AR239: 10,






AR257: 10, AR267: 10, AR255: 10, AR188: 10,






AR254: 10, AR203: 10, AR232: 10, AR238: 9,






AR191: 9, AR189: 9, AR190: 9, AR061: 9, AR230:






9, AR296: 9, AR179: 9, AR290: 8, AR237: 7,






S0442: 4, L0764: 4, S0408: 3, H0306: 2, H0263: 2,






H0596: 2, L0800: 2, L0755: 2, S0116: 1, S0358: 1,






H0489: 1, H0597: 1, T0041: 1 and L0772: 1.


14
HJACG02
1307789
104
AR207: 37, AR195: 33, AR283: 32, AR263: 32,






AR264: 29, AR223: 28, AR214: 28, AR089: 28,






AR277: 27, AR222: 27, AR309: 27, AR311: 27,






AR212: 26, AR169: 26, AR316: 25, AR224: 24,






AR096: 24, AR055: 24, AR197: 23, AR213: 23,






AR282: 22, AR104: 22, AR245: 22, AR171: 22,






AR218: 22, AR162: 22, AR192: 21, AR217: 21,






AR161: 21, AR193: 21, AR163: 20, AR308: 20,






AR165: 20, AR168: 20, AR216: 20, AR170: 20,






AR164: 20, AR235: 19, AR172: 19, AR053: 19,






AR166: 19, AR060: 19, AR219: 19, AR242: 19,






AR271: 19, AR299: 19, AR210: 19, AR039: 19,






AR033: 19, AR240: 18, AR225: 18, AR313: 18,






AR312: 18, AR201: 18, AR221: 17, AR261: 17,






AR198: 17, AR246: 17, AR288: 17, AR252: 17,






AR295: 17, AR176: 16, AR177: 16, AR215: 15,






AR297: 15, AR253: 15, AR205: 15, AR270: 15,






AR196: 15, AR185: 15, AR275: 15, AR286: 15,






AR285: 14, AR260: 14, AR287: 14, AR233: 14,






AR236: 14, AR183: 14, AR227: 13, AR175: 13,






AR211: 13, AR300: 13, AR250: 13, AR294: 13,






AR181: 13, AR274: 13, AR272: 13, AR229: 13,






AR174: 12, AR256: 12, AR182: 12, AR234: 12,






AR204: 12, AR269: 12, AR228: 12, AR293: 12,






AR178: 12, AR226: 12, AR268: 11, AR266: 11,






AR173: 11, AR262: 11, AR200: 11, AR243: 11,






AR199: 11, AR258: 11, AR231: 11, AR291: 11,






AR180: 11, AR289: 11, AR247: 11, AR239: 10,






AR257: 10, AR267: 10, AR255: 10, AR188: 10,






AR254: 10, AR203: 10, AR232: 10, AR2389, AR1919,






AR1899, AR1909, AR0619, AR2309, AR2969, AR1799,






AR2908, AR2377 S0442: 4, L0764: 4, S0408: 3,






H0306: 2, H0263: 2, H0596: 2, L0800: 2, L0755: 2,






S0116: 1, S0358: 1, H0489: 1, H0597: 1, T0041: 1 and






L0772: 1.


15
HKGAJ54
498303
107
AR218: 7, AR219: 6, AR242: 6, AR315: 6, AR225:






5, AR248: 4, AR244: 4, AR273: 4, AR263: 4,






AR310: 4, AR280: 4, AR214: 4, AR282: 4, AR314:






3, AR221: 3, AR170: 3, AR311: 3, AR052: 3,






AR213: 3, AR162: 3, AR217: 3, AR281: 3, AR243:






3, AR253: 2, AR270: 2, AR265: 2, AR186: 2,






AR277: 2, AR055: 2, AR207: 2, AR104: 2, AR257:






2, AR316: 2, AR212: 2, AR194: 2, AR312: 2,






AR206: 2, AR089: 2, AR195: 2, AR283: 2, AR171:






2, AR193: 1, AR240: 1, AR284: 1, AR199: 1,






AR039: 1, AR163: 1, AR053: 1, AR183: 1, AR288:






1, AR033: 1, AR309: 1, AR060: 1, AR096: 1,






AR236: 1, AR188: 1, AR266: 1, AR216: 1, AR161:






1, AR201: 1, AR205: 1, S0474: 2, S0400: 1, H0661: 1,






S0045: 1, T0039: 1, S0003: 1, H0412: 1, H0056: 1,






H0494: 1, H0646: 1, H0538: 1, S0422: 1, L0533: 1,






H0539: 1, L0740: 1, L0777: 1, S0436: 1 and L0588: 1.


15
HKGAJ54
1300770
109
AR2187, AR2196, AR2426, AR3156, AR2255, AR2484,






AR2444, AR2734, AR2634, AR3104, AR2804, AR2144,






AR2824, AR3143, AR2213, AR1703, AR3113, AR0523,






AR2133, AR1623, AR2173, AR2813, AR2433, AR2532,






AR2702, AR2652, AR1862, AR2772, AR0552, AR3162,






AR2072, AR1042, AR2572, AR2122, AR1942, AR3122,






AR2062, AR0892, AR1952, AR2832, AR1712, AR1931,






AR2401, AR2841, AR1991, AR0391, AR1631, AR0531,






AR1831, AR2881, AR0331, AR3091, AR0601, AR0961,






AR2361, AR1881, AR2661, AR2161, AR1611, AR2011,






AR2051 S0474: 2, S0400: 1, H0661: 1, S0045: 1,






T0039: 1, S0003: 1, H0412: 1, H0056: 1, H0494: 1,






H0646: 1, H0773: 1, H0538: 1, S0422: 1, L0533: 1,






H0539: 1, L0740: 1, L0777: 1, S0436: 1 and L0588: 1.


16
HSVAK93
597462
116


16
HE8CH92
609866
118
AR290: 19, AR268: 11, AR2679, AR3129, AR3098,






AR2518, AR2707, AR3107, AR0537, AR2826, AR2666,






AR3136, AR0526, AR2656, AR2635, AR0615, AR2695,






AR2935, AR0605, AR0335, AR1825, AR2925, AR2955,






AR2864, AR2484, AR0554, AR2384, AR2414, AR2534,






AR2984, AR2134, AR0394, AR2474, AR2854, AR2894,






AR1754, AR2914, AR2294, AR2944, AR2964, AR0893,






AR2993, AR1773, AR2493, AR0963, AR2843, AR2563,






AR2373, AR2333, AR3163, AR2343, AR2263, AR3003,






AR1843, AR2593, AR1853, AR2313, AR2833, AR1832,






AR2402, AR2322, AR2772, AR1042, AR1862, AR2192,






AR2272, AR2182, AR2582, AR1792 L0769: 7, L0754:






7, H0657: 4, L0764: 4, L0751: 4, L0752: 4, S0408: 3,






S0222: 3, H0327: 3, H0046: 3, H0056: 3, L0756: 3,






S0282: 2, S0356: 2, S0444: 2, H0580: 2, L3653: 2,






H0250: 2, H0318: 2, S0474: 2, H0622: 2, H0625: 2,






L0803: 2, L0809: 2, L0666: 2, L2654: 2, H0648: 2,






H0672: 2, S0380: 2, S0146: 2, L0748: 2, L0745: 2,






L0747: 2, L0777: 2, L0731: 2, L0600: 2, H0556: 1,






H0740: 1, S0116: 1, H0305: 1, H0125: 1, S0410: 1,






H0637: 1, S0476: 1, H0393: 1, H0586: 1, H0643: 1,






H0486: 1, H0013: 1, H0635: 1, H0599: 1, H0575: 1,






S0346: 1, H0052: 1, H0309: 1, H0050: 1, H0024: 1,






H0266: 1, H0271: 1, H0188: 1, H0286: 1, H0615: 1,






H0124: 1, H0135: 1, H0634: 1, H0264: 1, H0413: 1,






T0042: 1, H0396: 1, H0132: 1, H0641: 1, H0652: 1,






S0422: 1, L0639: 1, L0646: 1, L0771: 1, L0773: 1,






L0662: 1, L0766: 1, L0774: 1, L0776: 1, L0655: 1,






L0807: 1, L0659: 1, L0367: 1, L2261: 1, L2440: 1,






L2464: 1, H0726: 1, H0547: 1, H0670: 1, H0521: 1,






H0696: 1, S0404: 1, S0406: 1, L0740: 1, L0749: 1,






L0779: 1, L0755: 1, L0759: 1, H0445: 1, S0434: 1,






S0026: 1, H0653: 1, H0667: 1, S0242: 1, S0412: 1,






H0506: 1 and: 1.


16
HSVAK93
1352228
120
AR205: 32, AR269: 30, AR275: 23, AR234: 22,






AR281: 21, AR299: 20, AR315: 17, AR194: 17,






AR231: 17, AR271: 17, AR274: 16, AR282: 16,






AR177: 16, AR202: 15, AR283: 15, AR266: 15,






AR246: 15, AR310: 14, AR280: 14, AR273: 14,






AR265: 14, AR033: 14, AR244: 13, AR268: 13,






AR206: 13, AR204: 13, AR053: 13, AR175: 13,






AR289: 12, AR052: 12, AR192: 12, AR309: 12,






AR314: 12, AR237: 12, AR039: 12, AR247: 12,






AR198: 12, AR184: 11, AR238: 11, AR295: 11,






AR284: 11, AR243: 11, AR270: 11, AR213: 11,






AR241: 11, AR277: 11, AR291: 10, AR055: 10,






AR312: 10, AR183: 10, AR285: 10, AR256: 10,






AR290: 10, AR267: 10, AR263: 10, AR292: 10,






AR2869, AR2969, AR2519, AR2989, AR1859, AR2408,






AR1868, AR0618, AR3168, AR3008, AR2938, AR2278,






AR2328, AR0898, AR2947, AR1827, AR3137, AR2297,






AR2267, AR1047, AR2187, AR0966, AR2336, AR2196,






AR2486, AR2535, AR2595, AR1795, AR2495, AR2585,






AR0605 L0438: 4, L0439: 4, S0116: 3, L0749: 3,






H0663: 2, H0309: 2, H0032: 2, H0616: 2, L0646: 2,






L0774: 2, S0428: 2, L0748: 2, L0777: 2, H0445: 2,






H0556: 1, S0134: 1, H0586: 1, H0632: 1, H0013: 1,






H0427: 1, H0575: 1, H0318: 1, H0421: 1, H0050: 1,






S0050: 1, H0051: 1, H0598: 1, H0413: 1, H0494: 1,






H0396: 1, S0344: 1, L0769: 1, L0772: 1, L0666: 1,






L0664: 1, L0665: 1, S0216: 1, H0726: 1, H0435: 1,






S0378: 1, H0521: 1, S0146: 1, L0779: 1, L0755: 1 and






L0731: 1.


17
HSDEK49
625998
123


17
HSDEK49
1352253
125
AR290: 45, AR268: 37, AR240: 23, AR267: 22,






AR269: 16, AR270: 14, AR234: 10, AR055: 10,






AR238: 10, AR1849, AR2928, AR2918, AR1798,






AR1838, AR2847, AR1777, AR1826, AR0606, AR2995,






AR2955, AR2855, AR2445, AR2935, AR1755, AR0964,






AR1853, AR2293, AR2493, AR2963, AR3163, AR2313,






AR2983, AR2893, AR1043, AR2373, AR2862, AR0892,






AR2262, AR2042, AR2662, AR2822, AR2942, AR2272,






AR3132, AR2472, AR3002, AR2332, AR2482, AR2592,






AR2752, AR2562, AR0391, AR0331, AR2771, AR2631,






AR0611, AR2581, AR2321, AR2711, AR2831, AR3101






H0031: 7, L0439: 7, L0754: 7, L3388: 6, L0731: 6,






S0002: 5, H0580: 4, H0575: 3, H0309: 3, L0438: 3,






H0555: 3, L0758: 3, S0360: 2, L3649: 2, H0553: 2,






S0344: 2, S0426: 2, L0775: 2, S0330: 2, L0747: 2,






L0779: 2, S0260: 2, L0599: 2, L0603: 2, H0739: 1,






H0170: 1, S0116: 1, S0354: 1, S0444: 1, L3645: 1,






H0270: 1, S0280: 1, H0590: 1, H0581: 1, H0251: 1,






H0014: 1, H0355: 1, H0030: 1, H0644: 1, H0674: 1,






H0090: 1, H0063: 1, S0142: 1, L0770: 1, L0769: 1,






L0651: 1, L0776: 1, L0659: 1, L0519: 1, L0664: 1,






H0682: 1, L0749: 1, L0752: 1, S0031: 1 and H0506: 1.


18
HWBAO62
838164
127
AR252: 43, AR264: 25, AR311: 20, AR308: 19,






AR245: 16, AR254: 15, AR250: 15, AR246: 14,






AR309: 13, AR195: 13, AR197: 13, AR201: 13,






AR263: 13, AR212: 12, AR272: 11, AR193: 10,






AR1749, AR2059, AR0539, AR2079, AR2438, AR2008,






AR2258, AR1988, AR2538, AR2238, AR1888, AR2228,






AR2247, AR3127, AR1897, AR1707, AR1637, AR2137,






AR1927, AR2216, AR1616, AR1966, AR1626, AR1916,






AR1656, AR2426, AR1646, AR1736, AR1805, AR1785,






AR1695, AR2115, AR2405, AR2105, AR2745, AR1905,






AR1725, AR2885, AR1665, AR2035, AR1815, AR1995,






AR2165, AR2905, AR2575, AR2185, AR2615, AR1845,






AR2695, AR2044, AR2974, AR1684, AR1764, AR2144,






AR2304, AR1834, AR2874, AR2624, AR2354, AR2554,






AR2684, AR2704, AR2674, AR2664, AR1793, AR2173,






AR2193, AR1853, AR1713, AR1773, AR2963, AR1753,






AR2823, AR3133, AR0393, AR0963, AR2153, AR2753,






AR2363, AR1823, AR0893, AR2343, AR2473, AR3163,






AR2953, AR0333, AR2313, AR2913, AR2383, AR2653,






AR2392, AR2892, AR3002, AR0522, AR2772, AR2852,






AR2712, AR2332, AR2292, AR2282, AR2582, AR2372,






AR2322, AR0602, AR2932, AR2942, AR2602, AR2862,






AR2992, AR2262, AR2062, AR3102, AR0612, AR2732,






AR1862, AR2921, AR2561, AR1041, AR2811, AR2271,






AR2831 H0580: 1 and H0427: 1.


18
HWBAO62
625914
129


19
HWHGU54
695695
131
AR223: 5, AR169: 4, AR171: 4, AR221: 4, AR224:






4, AR264: 4, AR214: 4, AR261: 3, AR235: 3,






AR263: 3, AR195: 3, AR225: 3, AR311: 3, AR168:






3, AR216: 3, AR222: 3, AR238: 3, AR183: 3,






AR172: 3, AR297: 2, AR212: 2, AR162: 2, AR161:






2, AR251: 2, AR170: 2, AR217: 2, AR269: 2,






AR207: 2, AR272: 2, AR228: 2, AR288: 2, AR308:






2, AR237: 2, AR231: 2, AR163: 2, AR266: 2,






AR312: 2, AR176: 2, AR282: 2, AR165: 2, AR257:






2, AR262: 2, AR277: 2, AR200: 2, AR196: 2,






AR198: 2, AR173: 2, AR254: 2, AR213: 2, AR166:






2, AR180: 2, AR226: 2, AR181: 2, AR234: 2,






AR298: 2, AR189: 2, AR096: 2, AR236: 2, AR089:






2, AR271: 2, AR197: 2, AR246: 2, AR295: 2,






AR193: 2, AR239: 2, AR274: 1, AR178: 1, AR061:






1, AR227: 1, AR300: 1, AR177: 1, AR039: 1,






AR164: 1, AR188: 1, AR267: 1, AR247: 1, AR287:






1, AR229: 1, AR211: 1, AR243: 1, AR201: 1,






AR191: 1, AR204: 1, AR190: 1, AR179: 1, AR270:






1, AR182: 1, AR055: 1, AR230: 1, AR294: 1,






AR199: 1, AR285: 1, AR291: 1, AR290: 1, AR316:






1, AR286: 1, AR296: 1, AR060: 1, AR309: 1,






AR210: 1, H0586: 3 and L0777: 2.


19
HWHGU54
695695
131
AR2235, AR1694, AR1714, AR2214, AR2244, AR2644,






AR2144, AR2613, AR2353, AR2633, AR1953, AR2253,






AR3113, AR1683, AR2163, AR2223, AR2383, AR1833,






AR1723, AR2972, AR2122, AR1622, AR1612, AR2512,






AR1702, AR2172, AR2692, AR2072, AR2722, AR2282,






AR2882, AR3082, AR2372, AR2312, AR1632, AR2662,






AR3122, AR1762, AR2822, AR1652, AR2572, AR2622,






AR2772, AR2002, AR1962, AR1982, AR1732, AR2542,






AR2132, AR1662, AR1802, AR2262, AR1812, AR2342,






AR2982, AR1892, AR2362, AR2712, AR1972, AR0892,






AR2462, AR2952, AR1932, AR2392, AR2741, AR1781,






AR0611, AR2271, AR3001, AR1771, AR1641, AR1881,






AR2671, AR2471, AR0961, AR2871, AR2291, AR2111,






AR2431, AR2011, AR1911, AR2041, AR1901, AR1791,






AR2701, AR1821, AR2301, AR2941, AR1991, AR2851,






AR2911, AR2901, AR3161, AR2861, AR2961, AR0601,






AR3091, AR2101 H0586: 3 and L0777: 2.


20
HCEJQ69
1243825
134
AR273: 54, AR251: 54, AR186: 47, AR249: 31,






AR052: 30, AR292: 30, AR241: 24, AR184: 24,






AR061: 23, AR259: 23, AR310: 23, AR206: 22,






AR194: 22, AR298: 21, AR248: 20, AR265: 18,






AR274: 18, AR244: 16, AR198: 15, AR237: 15,






AR243: 15, AR202: 14, AR309: 14, AR229: 14,






AR033: 14, AR227: 13, AR053: 13, AR271: 13,






AR204: 13, AR096: 12, AR104: 12, AR275: 12,






AR192: 12, AR185: 12, AR313: 12, AR284: 11,






AR296: 11, AR293: 10, AR233: 10, AR312: 10,






AR282: 10, AR294: 10, AR232: 10, AR055: 10,






AR283: 9, AR263: 9, AR300: 9, AR215: 9, AR266:






9, AR183: 9, AR247: 9, AR219: 9, AR256: 9,






AR231: 8, AR175: 8, AR213: 8, AR267: 8, AR177:






8, AR238: 8, AR089: 8, AR218: 8, AR289: 8,






AR039: 8, AR246: 7, AR277: 7, AR253: 7, AR295:






7, AR285: 7, AR299: 7, AR205: 7, AR182: 7,






AR240: 7, AR286: 7, AR221: 7, AR226: 7, AR316:






6, AR234: 6, AR291: 6, AR269: 6, AR270: 6,






AR060: 6, AR170: 6, AR290: 5, AR223: 5, AR179:






5, AR268: 5, AR214: 5, AR172: 5, AR224: 5,






AR258: 5, AR245: 4, AR171: 4, AR216: 4, AR168:






4, AR222: 4, AR165: 4, AR254: 4, AR164: 4,






AR217: 3, AR161: 3, AR225: 3, AR197: 3, AR162:






3, AR166: 3, AR163: 3, AR308: 3, AR272: 3,






AR264: 3, AR257: 3, AR169: 3, AR311: 2, AR195:






2, AR212: 2, AR199: 2, AR210: 2, AR235: 2,






AR180: 2, AR242: 2, AR188: 2, AR181: 2, AR288:






2, AR262: 2, AR173: 2, AR201: 2, AR178: 2,






AR207: 2, AR189: 2, AR261: 2, AR196: 2, AR203:






2, AR255: 2, AR297: 2, AR236: 2, AR176: 1,






AR211: 1, AR193: 1, AR287: 1, AR230: 1, AR200:






1, AR191: 1, AR174: 1, AR239: 1, AR190: 1,






AR228: 1, H0052: 3, L0439: 3, H0194: 1, H0546: 1,






H0615: 1, H0135: 1, H0087: 1, L0759: 1, S0436: 1 and






S0456: 1.


20
HCEJQ69
1243825
134
AR273: 54, AR251: 54, AR186: 47, AR249: 31,






AR052: 30, AR292: 30, AR241: 24, AR184: 24,






AR061: 23, AR259: 23, AR310: 23, AR206: 22,






AR194: 22, AR298: 21, AR248: 20, AR265: 18,






AR274: 18, AR244: 16, AR198: 15, AR314: 15,






AR237: 15, AR243: 15, AR202: 14, AR309: 14,






AR229: 14, AR280: 14, AR033: 14, AR227: 13,






AR053: 13, AR271: 13, AR204: 13, AR315: 13,






AR104: 12, AR275: 12, AR192: 12, AR185: 12,






AR313: 12, AR284: 11, AR296: 11, AR293: 10,






AR233: 10, AR096: 10, AR281: 10, AR312: 10,






AR282: 10, AR294: 10, AR219: 10, AR232: 10,






AR0399, AR2839, AR2639, AR3009, AR2159, AR0559,






AR2669, AR1839, AR2479, AR2569, AR2318, AR1758,






AR2138, AR2678, AR1778, AR2388, AR2898, AR2467,






AR2777, AR2537, AR0897, AR2957, AR2857, AR2997,






AR2187, AR2057, AR1827, AR2407, AR2867, AR2217,






AR2267, AR3166, AR2346, AR2916, AR2696, AR2706,






AR0606, AR1706, AR2905, AR2235, AR1795, AR2685,






AR2145, AR1725, AR2245, AR2585, AR2454, AR1714,






AR2164, AR1684, AR2224, AR1654, AR2544, AR1644,






AR2173, AR1613, AR2253, AR1973, AR1623, AR1663,






AR1633, AR3083, AR2723, AR2643, AR2573, AR1693,






AR3112, AR1952, AR2122, AR1992, AR2102, AR2352,






AR1802, AR2422, AR1882, AR1812, AR2882, AR2622,






AR1732, AR2012, AR1782, AR2072, AR1892, AR2612,






AR1962, AR2032, AR2552, AR2972, AR2362, AR1761,






AR2111, AR1931, AR2871, AR2301, AR2001, AR1911,






AR1741, AR2391, AR1901, AR2281 H0052: 3, L0439:






3, H0194: 1, H0546: 1, H0615: 1, H0135: 1, H0087: 1,






L3841: 1, L0759: 1, S0436: 1 and S0456: 1.


20
HCEJQ69
872582
136


20
HCEJQ69
609999
138


21
HT5GJ57
740767
159
AR213: 5, AR264: 4, AR224: 4, AR254: 4, AR253:






4, AR207: 4, AR053: 3, AR197: 3, AR161: 3,






AR162: 3, AR163: 3, AR250: 3, AR221: 3, AR171:






3, AR311: 3, AR212: 3, AR165: 3, AR215: 3,






AR282: 3, AR217: 2, AR309: 2, AR263: 2, AR193:






2, AR195: 2, AR089: 2, AR168: 2, AR166: 2,






AR164: 2, AR312: 2, AR308: 2, AR223: 2, AR183:






2, AR267: 2, AR169: 2, AR198: 2, AR266: 2,






AR033: 2, AR216: 2, AR271: 2, AR096: 1, AR210:






1, AR192: 1, AR277: 1, AR272: 1, AR240: 1,






AR286: 1, AR313: 1, AR060: 1, AR180: 1, AR204:






1, AR299: 1, AR039: 1, AR246: 1, AR104: 1,






AR201: 1, AR270: 1, AR205: 1, AR055: 1, AR225:






1, AR247: 1, AR258: 1, AR182: 1, AR274: 1,






AR214: 1, AR287: 1, AR243: 1, H0584: 3, H0341:






3, H0271: 3, H0318: 2, H0090: 2, H0264: 2, S0052: 2,






H0656: 1, H0580: 1, S0278: 1, H0431: 1, H0013: 1,






H0457: 1, H0284: 1, S0003: 1, S0144: 1, S0142: 1,






S0002: 1, H0702: 1, H0522: 1 and H0445: 1.


21
HT5GJ57
1299921
161
AR2135, AR2644, AR2244, AR2544, AR2534, AR2074,






AR0533, AR1973, AR1613, AR1623, AR1633, AR2503,






AR2213, AR1713, AR3113, AR2123, AR1653, AR2153,






AR2172, AR3092, AR2632, AR1932, AR2822, AR1952,






AR1682, AR1662, AR0892, AR1642, AR3122, AR3082,






AR2232, AR1832, AR2672, AR1692, AR1982, AR2662,






AR0332, AR2162, AR2712, AR2101, AR1921, AR2721,






AR2401, AR2861, AR2771, AR0601, AR1801, AR2041,






AR3131, AR2461, AR1041, AR2011, AR0961, AR2701,






AR2051, AR2251, AR2471, AR2991, AR2581, AR1821,






AR2741, AR2141, AR2871, AR2431 H0584: 3, H0341:






3, H0318: 2, H0271: 2, H0090: 2, H0264: 2, S0052: 2,






H0521: 2, H0556: 1, H0740: 1, H0656: 1, H0580: 1,






S0278: 1, H0431: 1, H0013: 1, H0284: 1, S0003: 1,






S0144: 1, S0142: 1, S0002: 1, H0702: 1, L3832: 1,






H0522: 1 and H0445: 1.


22
HPIBX03
743314
169
AR202: 71, AR194: 67, AR244: 58, AR281: 55,






AR206: 54, AR241: 40, AR310: 38, AR315: 37,






AR265: 36, AR283: 35, AR280: 35, AR246: 35,






AR314: 34, AR104: 29, AR284: 29, AR186: 28,






AR273: 26, AR292: 26, AR052: 25, AR198: 25,






AR205: 25, AR243: 25, AR033: 23, AR039: 23,






AR263: 22, AR271: 20, AR096: 20, AR282: 20,






AR298: 20, AR251: 20, AR192: 19, AR204: 19,






AR289: 18, AR259: 17, AR275: 17, AR274: 17,






AR277: 17, AR055: 17, AR313: 16, AR291: 16,






AR266: 16, AR286: 16, AR312: 15, AR285: 15,






AR299: 15, AR218: 15, AR295: 15, AR240: 15,






AR316: 14, AR309: 14, AR300: 14, AR247: 14,






AR219: 14, AR294: 13, AR053: 13, AR213: 13,






AR089: 12, AR061: 12, AR248: 12, AR249: 12,






AR232: 12, AR177: 12, AR184: 12, AR296: 11,






AR293: 11, AR268: 11, AR185: 11, AR270: 11,






AR182: 10, AR227: 10, AR256: 10, AR290: 10,






AR183: 10, AR269: 10, AR1759, AR2269, AR2589,






AR2299, AR2389, AR2678, AR0608, AR2318, AR2378,






AR1707, AR2337, AR2537, AR2347, AR2236, AR2256,






AR1726, AR1715, AR1685, AR1765, AR2145, AR2175,






AR2165, AR2245, AR2154, AR2214, AR1794, AR1614,






AR1644, AR1664, AR2574, AR2224, AR2004, AR1623,






AR1633, AR1803, AR2363, AR2613, AR1733, AR2553,






AR1693, AR2723, AR2643, AR1653, AR2283, AR2883,






AR2303, AR2873, AR1963, AR1813, AR2623, AR2392,






AR1912, AR1932, AR1882, AR1902, AR1972, AR2102,






AR1782, AR1952, AR1892, AR2122, AR2012, AR1741,






AR2601, AR2971, AR2031 H0031: 4, S0442: 2, S0358:






2, S0150: 2, L0768: 2, S0152: 2, S0444: 1, S0132: 1,






S0476: 1, H0620: 1, H0494: 1, S0002: 1, S0426: 1,






L0776: 1, S0126: 1 and L0596: 1.


23
HDPBO81
892018
174
AR0533, AR2513, AR2623, AR2872, AR1642, AR2022,






AR2522, AR1652, AR2822, AR1952, AR1682, AR1762,






AR2612, AR2252, AR2572, AR2542, AR2972, AR2672,






AR2942, AR2852, AR1722, AR2152, AR1622, AR2702,






AR3092, AR2261, AR2141, AR2121, AR1931, AR2911,






AR2131, AR1631, AR2171, AR2501, AR1751, AR0601,






AR3121, AR2771, AR1611, AR1811, AR2341, AR1821,






AR1961, AR1801, AR3001, AR0891, AR2211, AR2741






H0521: 6, L0804: 2, H0634: 1 and S0440: 1.


23
HDPBO81
790188
176


24
HWBFY57
837478
179
AR207: 12, AR245: 10, AR197: 10, AR250: 10,






AR2019, AR2429, AR1959, AR2548, AR2528, AR2488,






AR1847, AR1937, AR2127, AR0967, AR2646, AR1986,






AR1666, AR2536, AR1656, AR1615, AR1625, AR1645,






AR3085, AR2145, AR1635, AR2245, AR3115, AR2495,






AR2465, AR2434, AR1924, AR2054, AR2714, AR2224,






AR0534, AR2174, AR2234, AR2404, AR2724, AR2773,






AR1743, AR2363, AR2043, AR2823, AR2633, AR2163,






AR2133, AR1813, AR1823, AR1703, AR3123, AR2303,






AR2883, AR1683, AR2703, AR2683, AR3163, AR2973,






AR1833, AR1802, AR3092, AR2692, AR0602, AR3132,






AR2852, AR0892, AR2962, AR2472, AR1782, AR1772,






AR2292, AR2902, AR2952, AR0332, AR3002, AR2752,






AR0522, AR2912, AR2892, AR2842, AR2662, AR0392,






AR2342, AR2312, AR2612, AR2862, AR2982, AR2922,






AR1732, AR1882, AR2872, AR2572, AR2992, AR0552,






AR2062, AR2932, AR2282, AR1792, AR2111, AR2811,






AR2621, AR2331, AR1851, AR2941, AR2261, AR2831,






AR2321, AR2251, AR2351, AR2671, AR2741, AR2271,






AR1861, AR1941, AR1751, AR0611, AR1891, AR1991,






AR1041, AR2381, AR1711, AR2101 L0794: 6, H0580:






4, H0457: 3, L0785: 1, S0360: 1, S0002: 1, L0804: 1,






L0806: 1, L0805: 1 and L0789: 1.


25
HYABV21
1281466
182
L0766: 6, L0748: 3, H0656: 2, H0264: 2, H0583: 1,






H0650: 1, H0657: 1, L0655: 1, L0659: 1, L0790: 1,






H0518: 1 and L0749: 1.


25
HYABV21
1213593
184


26
HOHBY69
827480
186
AR2445, AR2213, AR1683, AR2843, AR2823, AR1842,






AR2242, AR1952, AR3102, AR1622, AR1612, AR1632,






AR2641, AR1811, AR1821, AR1861, AR1711, AR2771,






AR3081, AR2831, AR2631 S0250: 5, H0545: 2, S0210:






2, S0040: 1, S0342: 1, H0661: 1, S0418: 1, S0360: 1,






H0549: 1, H0251: 1, H0544: 1, S0003: 1, H0124: 1,






L0564: 1, L0807: 1, L0565: 1, S0037: 1, L0744: 1,






L0757: 1 and S0276: 1.


26
HOHBY69
815681
188


27
HDHMA45
902513
202
AR2259, AR2778, AR2148, AR2238, AR2158, AR1657,






AR1717, AR1646, AR1706, AR1686, AR1666, AR2246,






AR2226, AR2356, AR1726, AR1625, AR2165, AR1615,






AR2825, AR2175, AR2645, AR1635, AR2975, AR2215,






AR2885, AR1805, AR3115, AR2074, AR2124, AR2614,






AR2634, AR1784, AR2874, AR2574, AR2524, AR1833,






AR1763, AR1923, AR0603, AR2913, AR3093, AR0893,






AR2403, AR3083, AR2893, AR1813, AR1963, AR1733,






AR2853, AR2833, AR2393, AR2623, AR2953, AR3163,






AR2333, AR2963, AR2323, AR2003, AR2863, AR3123,






AR1953, AR2283, AR2993, AR2133, AR2343, AR1913,






AR2933, AR2383, AR2943, AR0963, AR1043, AR2473,






AR2293, AR3003, AR1843, AR2663, AR2423, AR2713,






AR2112, AR1692, AR2552, AR2452, AR2362, AR3132,






AR2312, AR2582, AR2692, AR2012, AR2682, AR1982,






AR2032, AR0392, AR2602, AR1792, AR1742, AR1902,






AR2302, AR0552, AR1752, AR2902, AR2752, AR1852,






AR0332, AR1772, AR1892, AR2702, AR2102, AR2052,






AR2272, AR1882, AR2532, AR2432, AR2672, AR1822,






AR2262, AR3102, AR2742, AR2022, AR2731, AR2721,






AR1991, AR0531, AR2371, AR2541, AR2181, AR0611,






AR2561, AR2651, AR2921, AR1931, AR2841 L0794:






5, L0769: 4, L0749: 3, S0110: 1, H0572: 1, H0050: 1,






L0765: 1, L0756: 1, L0755: 1 and L0758: 1.


27
HDHMA45
812764
204


28
HMADJ14
1099342
206
AR184: 30, AR298: 28, AR259: 25, AR292: 24,






AR249: 18, AR251: 17, AR229: 14, AR314: 13,






AR237: 13, AR294: 13, AR296: 12, AR293: 12,






AR233: 11, AR284: 11, AR227: 11, AR315: 11,






AR182: 11, AR280: 10, AR266: 10, AR0399, AR2319,






AR0619, AR0339, AR2488, AR2328, AR2868, AR2678,






AR2818, AR2698, AR2268, AR2567, AR2897, AR2857,






AR2347, AR2907, AR2387, AR1837, AR1777, AR2957,






AR1756, AR0556, AR2916, AR1866, AR1856, AR1766,






AR0895, AR2705, AR2685, AR2585, AR3005, AR1615,






AR1625, AR1635, AR2285, AR2355, AR2635, AR2825,






AR3135, AR3104, AR1794, AR0964, AR2194, AR1814,






AR2014, AR1044, AR2184, AR3164, AR1724, AR0604,






AR2774, AR2614, AR2994, AR2234, AR2023, AR1683,






AR2833, AR0533, AR2453, AR2363, AR1653, AR2473,






AR1643, AR2173, AR2553, AR3093, AR2713, AR2303,






AR1663, AR2883, AR1913, AR2623, AR2403, AR1783,






AR2533, AR2573, AR2523, AR2153, AR3113, AR2393,






AR1963, AR1903, AR2873, AR2643, AR1713, AR2253,






AR1733, AR2723, AR2002, AR2162, AR2742, AR2422,






AR2972, AR2142, AR1932, AR1882, AR2032, AR2222,






AR2242, AR2432, AR2752, AR2042, AR3122, AR1892,






AR1992, AR2062, AR2072, AR1952, AR2652, AR2461,






AR2111, AR3081, AR1741, AR1971, AR1801, AR2601,






AR0521, AR2211 S0144: 6, S0278: 5, S0344: 3,






S0002: 3, H0521: 3, S0003: 2, H0575: 1, S0214: 1,






H0068: 1, H0591: 1, L0806: 1, L0805: 1, L0776: 1 and






L0791: 1.


28
HMADJ14
889659
208


28
HMADJ14
843725
210


28
HMADJ14
843725
212
AR184: 30, AR298: 28, AR259: 25, AR292: 24, AR249: 18,






AR251: 17, AR229: 14, AR314: 13, AR237: 13, AR294: 13,






AR296: 12, AR293: 12, AR233: 11, AR284: 11, AR227: 11,






AR315: 11, AR182: 11, AR280: 10, AR266: 10, AR039: 9,






AR231: 9, AR061: 9, AR033: 9, AR248: 8, AR232: 8,






AR286: 8, AR267: 8, AR281: 8, AR269: 8, AR226: 8,






AR256: 7, AR289: 7, AR285: 7, AR234: 7, AR290: 7,






AR238: 7, AR183: 7, AR177: 7, AR295: 7, AR175: 6,






AR055: 6, AR291: 6, AR186: 6, AR185: 6, AR176: 6,






AR089: 5, AR270: 5, AR268: 5, AR258: 5, AR300: 5,






AR161: 5, AR162: 5, AR163: 5, AR228: 5, AR235: 5,






AR263: 5, AR282: 5, AR313: 5, AR310: 4, AR179: 4,






AR096: 4, AR219: 4, AR181: 4, AR201: 4, AR104: 4,






AR218: 4, AR316: 4, AR172: 4, AR060: 4, AR277: 4,






AR261: 4, AR299: 4, AR223: 4, AR202: 3, AR168: 3,






AR283: 3, AR053: 3, AR245: 3, AR236: 3, AR165: 3,






AR247: 3, AR164: 3, AR217: 3, AR255: 3, AR309: 3,






AR271: 3, AR230: 3, AR166: 3, AR288: 3, AR191: 3,






AR262: 3, AR240: 3, AR178: 3, AR253: 3, AR257: 3,






AR252: 3, AR215: 3, AR311: 3, AR239: 3, AR196: 3,






AR190: 3, AR287: 3, AR264: 3, AR171: 3, AR225: 3,






AR173: 3, AR272: 3, AR200: 2, AR216: 2, AR274: 2,






AR242: 2, AR297: 2, AR214: 2, AR193: 2, AR188: 2,






AR203: 2, AR222: 2, AR224: 2, AR243: 2, AR275: 2,






AR204: 2, AR312: 2, AR189: 2, AR199: 2, AR206: 2,






AR207: 2, AR195: 2, AR265: 2, AR246: 1, AR211: 1,






AR308: 1, AR174: 1, AR197: 1, AR180: 1, AR260: 1,






AR052: 1, AR221: 1, S0144: 6, S0278: 5, S0344: 3,






S0002: 3, H0521: 3, S0003: 2, H0575: 1, S0214: 1,






H0068: 1, H0591: 1, L0806: 1, L0805: 1, L0776: 1 and






L0791: 1.


28
HMADJ14
795479
214


28
HMADJ14
426068
216


30
HEMFA84
608198
222
AR0606, AR0894, AR0554, AR2194, AR2823, AR3163,






AR0963, AR1043, AR2183, AR2403, AR3132, AR2992,






AR2472, AR2922, AR2832, AR0392, AR2462, AR2942,






AR1852, AR3002, AR2481, AR2681, AR2771, AR3121,






AR0331, AR1831, AR2631, AR2671, AR3101, AR0531,






AR2381 L0747: 5, H0556: 4, L0769: 4, L0806: 3,






L5622: 3, L0790: 3, L0779: 3, H0265: 2, H0013: 2,






L0666: 2, L0439: 2, L0740: 2, L0749: 2, L0752: 2,






L0757: 2, L0596: 2, T0049: 1, S0046: 1, S0132: 1,






S0476: 1, L0717: 1, H0549: 1, H0600: 1, H0559: 1,






H0046: 1, H0083: 1, H0266: 1, S0314: 1, H0622: 1,






H0674: 1, H0135: 1, H0090: 1, L0564: 1, H0494: 1,






S0440: 1, L0772: 1, L0800: 1, L0644: 1, L0794: 1,






L0766: 1, L0776: 1, L0807: 1, L0809: 1, L0791: 1,






L0665: 1, H0144: 1, H0660: 1, S0406: 1, L0780: 1,






S0434: 1 and L0595: 1.


31
HDPPA04
904765
224
AR2517, AR1806, AR2524, AR1944, AR2494, AR1974,






AR1693, AR1783, AR2353, AR2413, AR1903, AR1842,






AR1722, AR2902, AR2712, AR1912, AR1742, AR2252,






AR1662, AR2732, AR1752, AR2682, AR2242, AR2142,






AR2912, AR1642, AR2822, AR1682, AR2532, AR1652,






AR2952, AR2122, AR1832, AR2461, AR2871, AR0961,






AR3111, AR2721, AR1881, AR3081, AR2041, AR2771,






AR3101, AR2971, AR2011, AR2191, AR1861, AR2101,






AR2131, AR2301, AR1891, AR2811, AR3131, AR2571,






AR2021 H0521: 4, L0731: 4, H0591: 2, H0641: 2,






L0794: 2, T0049: 1, S0476: 1, H0004: 1, H0494: 1,






L0791: 1, H0522: 1, L0758: 1 and S0452: 1.


31
HDPPA04
905419
226


31
HDPPA04
905418
228


32
HE2OA95
637595
230
AR223: 26, AR089: 25, AR216: 25, AR224: 23, AR222: 22,






AR214: 22, AR215: 21, AR308: 21, AR210: 21, AR272: 21,






AR221: 19, AR219: 18, AR235: 18, AR211: 17, AR261: 17,






AR225: 17, AR218: 17, AR309: 17, AR217: 16, AR283: 16,






AR311: 16, AR172: 16, AR196: 16, AR168: 16, AR274: 15,






AR169: 15, AR171: 15, AR199: 15, AR316: 15, AR104: 15,






AR188: 14, AR170: 14, AR264: 13, AR096: 13, AR295: 12,






AR313: 12, AR312: 11, AR285: 11, AR165: 11, AR236: 11,






AR189: 11, AR055: 11, AR247: 11, AR164: 11, AR263: 11,






AR299: 10, AR166: 10, AR282: 10, AR161: 10, AR162: 10,






AR200: 10, AR297: 9, AR163: 9, AR293: 9, AR288: 9,






AR262: 9, AR296: 9, AR240: 9, AR060: 8, AR191: 8,






AR039: 8, AR291: 8, AR181: 8, AR277: 8, AR203: 8,






AR177: 8, AR255: 7, AR275: 7, AR231: 7, AR185: 7,






AR190: 7, AR174: 6, AR290: 6, AR287: 6, AR173: 6,






AR175: 6, AR286: 6, AR300: 6, AR183: 5, AR270: 5,






AR269: 5, AR267: 5, AR258: 5, AR239: 5, AR294: 5,






AR179: 5, AR289: 5, AR256: 5, AR268: 5, AR257: 5,






AR061: 4, AR266: 4, AR230: 4, AR178: 4, AR234: 4,






AR176: 4, AR232: 4, AR238: 4, AR229: 4, AR237: 3,






AR180: 3, AR260: 3, AR226: 3, AR233: 3, AR227: 3,






AR182: 3, AR228: 2, AR033: 2, AR197: 2, AR195: 2,






AR205: 1, AR201: 1, L0005: 4, L0439: 4, H0013: 3,






H0674: 3, H0144: 3, H0170: 2, L0157: 2, L0435: 2,






L0540: 2, L0438: 2, L0756: 2, L0759: 2, S0045: 1,






H0599: 1, H0178: 1, H0572: 1, H0050: 1, H0024: 1,






H0051: 1, S6028: 1, L0142: 1, H0673: 1, H0616: 1,






H0551: 1, H0623: 1, L0809: 1, L0666: 1, H0520: 1,






H0547: 1, H0519: 1, S0126: 1, H0539: 1, H0696: 1,






H0694: 1, L0742: 1, L0777: 1, L0758: 1, L0592: 1 and






L0595: 1.


33
HKABZ65
862030
232
AR313: 41, AR242: 32, AR039: 28, AR165: 25,






AR163: 25, AR164: 24, AR161: 24, AR162: 24,






AR166: 24, AR089: 24, AR096: 23, AR173: 22,






AR196: 20, AR193: 20, AR299: 20, AR300: 20,






AR258: 20, AR180: 19, AR175: 19, AR178: 18,






AR240: 18, AR229: 18, AR234: 18, AR185: 17,






AR247: 17, AR218: 17, AR262: 17, AR179: 16,






AR285: 16, AR183: 16, AR269: 16, AR293: 15,






AR174: 15, AR199: 15, AR182: 15, AR181: 15,






AR238: 14, AR191: 14, AR296: 14, AR236: 14,






AR257: 14, AR316: 14, AR270: 14, AR226: 13,






AR219: 13, AR297: 13, AR277: 13, AR264: 12,






AR200: 12, AR312: 12, AR195: 12, AR213: 12,






AR192: 12, AR203: 12, AR268: 12, AR212: 12,






AR294: 12, AR286: 11, AR060: 11, AR230: 11,






AR177: 11, AR189: 11, AR233: 11, AR260: 10,






AR231: 10, AR198: 10, AR290: 10, AR188: 10,






AR204: 10, AR0539, AR2879, AR2889, AR2559,






AR2959, AR0339, AR2619, AR2829, AR1049, AR2459,






AR2439, AR2359, AR2288, AR3088, AR2638, AR2758,






AR2918, AR2018, AR2748, AR2377, AR1977, AR2397,






AR2247, AR3117, AR1767, AR2677, AR1727, AR2567,






AR2237, AR2057, AR1716, AR2276, AR1686, AR2146,






AR2076, AR1696, AR2256, AR2526, AR2506, AR2716,






AR2156, AR1706, AR2116, AR2216, AR3095, AR2835,






AR2665, AR2545, AR2225, AR1905, AR2105, AR2165,






AR2175, AR2325, AR0555, AR2894, AR2534, AR2464,






AR2723, AR0612 H0494: 1


33
HKABZ65
665424
234









Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “cDNA Clone ID”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID: A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof). Table 1C from U.S. patent application Ser. No. 10/472,532, filed Sep. 20, 2003, is herein incorporated by reference. Table 1C in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 228 to 235 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.












TABLE 1C





cDNA Clone
SEQ ID
CONTIG

SEQ ID
EXON


ID
NO: X
ID:
BAC ID: A
NO: B
From-To




















HETBX14
61
806447
AC011473
247
 1-205







 727-1286







1995-2071







2865-3025







3352-3617







4434-4573







4958-5431


HETBX14
61
806447
AC011473
248
 1-77







241-299







745-906







1417-1520







2346-2501







2754-2852







2923-3002







3962-4096







5422-5791


HLHFP03
76
460467
AC011976
251
 1-467







1023-1116







2101-2317







2529-2630







2860-3097


HLHFP03
76
460467
AC011976
252
 1-391


HTADX17
90
457172
AL357565
249
 1-937







 945-1293







2068-2336







2732-3109


HTADX17
90
457172
AL357565
250
 1-104


HJACG02
102
509948
AC008763

 1-47







243-371







736-823







1144-1381


HJACG02
102
509948
AC008763

 1-32







407-497







 818-1357







2275-2380







2384-2560


HT5GJ57
159
740767
AC005081
253
 1-149







1029-1450







4881-5071







5302-5397







6004-6132







6888-6926







7229-7329







9066-9254







9805-9848







 9852-10211







10276-10315







10619-10807







10855-11472







11695-11852







12358-12468







13770-13847







14053-14259







14695-14822







16101-16553







18233-18690







18750-19965


HT5GJ57
159
740767
AC016675
254
 1-149







1029-1450







1623-1790







4908-5098







5329-5424







6031-6159







6915-6953







7256-7356







9095-9306







9834-9877







 9881-10239







10304-10343







10647-10835







10883-11500







11724-11881







13799-13876







14082-14288







14725-14852







16131-16583







18262-18719







18757-19994







20161-20420


HT5GJ57
159
740767
AC005081
255
 1-233


HT5GJ57
159
740767
AC016675
256
 1-720







2099-2265







2776-3550







3946-4146







4976-5104







5781-6171







6476-7039







7384-7738







7837-8009







8434-8593







11644-11746









Tables 1D, 1E, 1E.1, and 1E.2: The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.
The present invention encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder (such as a disease listed in the “Preferred Indications” columns of Tables 1D, 1E, 1E.2, or 1F, and in particular, an immune, cardiovascular, cancer, or other proliferative disease or disorder) comprising administering to a patient in which such detection, treatment, prevention, and/or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in preventing, treating, diagnosing, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.
In another embodiment, the present invention also encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder (such as a disease listed in the “Preferred Indications” columns of Tables 1D, 1E, 1E.2, or 1F, and in particular, an immune, cardiovascular, cancer, or other proliferative disease or disorder); comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D or in the “Preferred Indications” columns of Tables 1E, 1E.2, or 1F.
The “Preferred Indications” columns of Tables 1D, 1E, 1E.2, and 1F describe diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
The recitation of “Cancer” in the “Preferred Indications” columns indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).
In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.
The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.
The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.
The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.
The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.
The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.
The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).
The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.
The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.
The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.
The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.
The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.









TABLE 1D





Gene No.
cDNA Clone ID
Preferred Indications

















3
HTEEB42
Cancer


4
HEMCM42
Cancer


4
HEQCC55
Cancer


5
HEMAE80
Cardiovascular, Musculoskeletal,




Reproductive


6
HRDFB85
Cancer


7
HDTAW95
Cancer


8
HEMCV19
Cancer


9
HETBX14
Cancer


10
HLHSK94
Cancer


11
HLHFP03
Respiratory


11
HLHFP03
Respiratory


12
HHTLF25
Cancer


13
HTADX17
Immune/Hematopoetic, Reproductive


13
HTADX17
Immune/Hematopoetic, Reproductive


14
HJACG02
Digestive, Immune/Hematopoetic


15
HKGAJ54
Cancer


16
HE8CH92
Cancer


16
HSVAK93
Cancer


17
HSDEK49
Cancer


18
HWBAO62
Connective/Epithelial, Immune/




Hematopoetic


19
HWHGU54
Connective/Epithelial


20
HCEJQ69
Cancer


21
HT5GJ57
Cancer


21
HT5GJ57
Cancer


22
HPIBX03
Cancer


23
HDPBO81
Digestive, Immune/Hematopoetic,




Reproductive


24
HWBFY57
Digestive, Immune/Hematopoetic


25
HYABV21
Immune/Hematopoietic


26
HOHBY69
Cancer


27
HDHMA45
Cardiovascular, Neural/Sensory


28
HMADJ14
Connective/Epithelial,




Immune/Hematopoetic, Musculoskeletal


28
HMADJ14
Connective/Epithelial,




Immune/Hematopoietic, Musculoskeletal


30
HEMFA84
Cancer


31
HDPPA04
Cardiovascular, Connective/




Epithelial, Immune/Hematopoetic


32
HE2OA95
Cancer


33
HKABZ65
Connective/Epithelial









Table 1E provides information related to biological activities and preferred indications for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention as well as indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, diagnosing, preventing, and/or treating.












TABLE 1E







AA







SEQ





Gene
cDNA
ID
Biological




No.
Clone ID
NO: Y
Activity
Exemplary Activity Assay
Preferred Indication




















3
HTEEB42
10
Regulation of
Assays for the regulation of
A highly preferred indication is





transcription of
transcription of Malic Enzyme
‘diabetes mellitus. An additional highly





Malic Enzyme
are well-known in the art and
preferred indication is a complication





in hepatocytes
may be used or routinely
associated with diabetes (e.g., diabetic






modified to assess the ability of
retinopathy, diabetic nephropathy, kidney






polypeptides of the invention
disease (e.g., renal failure, nephropathy






(including antibodies and
and/or other diseases and disorders as






agonists or antagonists of the
described in the “Renal Disorders” section






invention) to regulate
below), diabetic neuropathy, nerve disease






transcription of Malic Enzyme,
and nerve damage (e.g., due to diabetic






a key enzyme in lipogenesis.
neuropathy), blood vessel blockage, heart






Malic enzyme is involved in
disease, stroke, impotence (e.g., due to






lipogenesisand its expression is
diabetic neuropathy or blood vessel






stimulted by insulin. ME
blockage), seizures, mental confusion,






promoter contains two direct
drowsiness, nonketotic hyperglycemic-






repeat (DR1)-like elements
hyperosmolar coma, cardiovascular disease






MEp and MEd identified as
(e.g., heart disease, atherosclerosis,






putative PPAR response
microvascular disease, hypertension,






elements. ME promoter may
stroke, and other diseases and disorders as






also responds to AP1 and other
described in the “Cardiovascular Disorders”






transcription factors.
section below), dyslipidemia, endocrine






Exemplary assays that may be
disorders (as described in the “Endocrine






used or routinely modified to
Disorders” section below), neuropathy,






test for regulation of
vision impairment (e.g., diabetic






transcription of Malic Enzyme
retinopathy and blindness), ulcers and






(in hepatocytes) by
impaired wound healing, and infection






polypeptides of the invention
(e.g., infectious diseases and disorders as






(including antibodies and
described in the “Infectious Diseases”






agonists or antagonists of the
section below, especially of the urinary






invention) include assays
tract and skin), carpal tunnel syndrome and






disclosed in: Streeper, R. S., et
Dupuytren's contracture). An






al., Mol Endocrinol,
additional highly preferred indication is






12(11): 1778-91 (1998); Garcia-
obesity and/or complications associated






Jimenez, C., et al., Mol
with obesity. Additional highly preferred






Endocrinol, 8(10): 1361-9
indications include weight loss or






(1994); Barroso, I., et al., J Biol
alternatively, weight gain.






Chem, 274(25): 17997-8004
Aditional highly preferred indications are






(1999); Ijpenberg, A., et al., J
complications associated with insulin






Biol Chem, 272(32): 20108-20117
resistance.






(1997); Berger, et al.,







Gene 66: 1-10 (1988); and,







Cullen, B., et al., Methods in







Enzymol. 216: 362-368 (1992),







the contents of each of which is







herein incorporated by







reference in its entirety.







Hepatocytes that may be used







according to these assays are







publicly available (e.g., through







the ATCC TM) and/or may be







routinely generated. Exemplary







hepatocytes that may be used







according to these assays







includes the mouse 3T3-L1 cell







line. 3T3-L1 is a mouse







preadipocyte cell line







(adherent). It is a continuous







substrain of 3T3 fibroblasts







developed through clonal







isolation. Cells undergo a pre-







adipocyte to adipose-like







conversion under appropriate







differentiation culture







conditions.



4
HEQCC55
16
Production of
MCP-1 FMAT. Assays for
A highly preferred embodiment of the





MCP-1
immunomodulatory proteins
invention includes a method for stimulating






that are produced by a large
(e.g., increasing) MCP-1 production. An






variety of cells and act to
alternative highly preferred embodiment of






induce chemotaxis and
the invention includes a method for






activation of monocytes and T
inhibiting (e.g., reducing) MCP-1






cells are well known in the art
production. A highly preferred indication






and may be used or routinely
is infection (e.g., an infectious disease as






modified to assess the ability of
described below under “Infectious






polypeptides of the invention
Disease”). Additional highly preferred






(including antibodies and
indications include inflammation and






agonists or antagonists of the
inflammatory disorders. Preferred






invention) to mediate
indications include blood disorders (e.g., as






immunomodulation, induce
described below under “Immune Activity”,






chemotaxis, and modulate
“Blood-Related Disorders”, and/or






immune cell activation.
“Cardiovascular Disorders”). Highly






Exemplary assays that test for
preferred indications include autoimmune






immunomodulatory proteins
diseases (e.g., rheumatoid arthritis,






evaluate the production of cell
systemic lupus erythematosis, multiple






surface markers, such as
sclerosis and/or as described below) and






monocyte chemoattractant
immunodeficiencies (e.g., as described






protein (MCP), and the
below). Preferred indications also






activation of monocytes and T
include anemia, pancytopenia, leukopenia,






cells. Such assays that may be
thrombocytopenia, Hodgkin's disease,






used or routinely modified to
acute lymphocytic anemia (ALL),






test immunomodulatory and
plasmacytomas, multiple myeloma,






differentiation activity of
Burkitt's lymphoma, arthritis, AIDS,






polypeptides of the invention
granulomatous disease, inflammatory






(including antibodies and
bowel disease, sepsis, neutropenia,






agonists or antagonists of the
neutrophilia, psoriasis, suppression of






invention) include assays
immune reactions to transplanted organs






disclosed in Miraglia et al., J
and tissues, hemophilia, hypercoagulation,






Biomolecular Screening 4: 193-204
diabetes mellitus, endocarditis, meningitis






(1999); Rowland et al.,
(bacterial and viral), Lyme Disease,






“Lymphocytes: a practical
asthma, and allergy Preferred indications






approach” Chapter 6: 138-160
also include neoplastic diseases (e.g.,






(2000); Satthaporn and Eremin,
leukemia, lymphoma, and/or as described






J R Coll Surg Ednb 45(1): 9-19
below under “Hyperproliferative






(2001); and Verhasselt et al., J
Disorders”). Highly preferred indications






Immunol 158: 2919-2925
include neoplasms and cancers, such as,






(1997), the contents of each of
leukemia, lymphoma, prostate, breast, lung,






which are herein incorporated
colon, pancreatic, esophageal, stomach,






by reference in its entirety.
brain, liver, and urinary cancer. Other






Human dendritic cells that may
preferred indications include benign






be used according to these
dysproliferative disorders and pre-






assays may be isolated using
neoplastic conditions, such as, for example,






techniques disclosed herein or
hyperplasia, metaplasia, and/or dysplasia.






otherwise known in the art.







Human dendritic cells are







antigen presenting cells in







suspension culture, which,







when activated by antigen







and/or cytokines, initiate and







upregulate T cell proliferation







and functional activities.



4
HEQCC55
16
Production of
Assays for production of IL-13
Highly preferred indications include allergy





IL-13 and
and activation of T-cells are
and asthma. Additional highly preferred





activation of T-
well known in the art and may
indications include immune and





cells.
be used or routinely modified to
hematopoietic disorders (e.g., as described






assess the ability of
below under “Immune Activity”, and






polypeptides of the invention
“Blood-Related Disorders”), autoimmune






(including antibodies and
diseases (e.g., rheumatoid arthritis,






agonists or antagonists of the
systemic lupus erythematosis, Crohn''s






invention) to stimulate or
disease, multiple sclerosis and/or as






inhibit production of IL-13
described below), immunodeficiencies






and/or activation of T-cells.
(e.g., as described below), boosting a T






Exemplary assays for IL-13
cell-mediated immune response, and






production that may be used or
suppressing a T cell-mediated immune






routinely modified to test
response.






activity of polypeptides and







antibodies of the invention







(including agonists or







antagonists of the invention)







include, for example, assays







such as disclosed and/or cited







in: Grunig, G, et al.,







“Requirement for IL-13







independently of IL-4 in







Experimental asthma”







Science; 282: 2261-2263







(1998), and Wills-Karp M, et







al., “Interleukin-13: central







mediator of allergic asthma”







Science; 282: 2258-2261







(1998); the contents of each of







which are herein incorporated







by reference in their entirety.







Exemplary cells that may be







used according to these assays







include Th2 cells. IL13, a Th2







type cytokine, is a potent







stimulus for mucus production,







airway hyper-responsiveness







and allergic asthma. Th2 cells







are a class of T cells that







secrete IL4, IL10, IL13, IL5







and IL6. Factors that induce







differentiation and activation of







Th2 cells play a major role in







the initiation and pathogenesis







of allergy and asthma. Primary







T helper 2 cells are generated in







in vitro culture under Th2







polarizing conditions using







peripheral blood lymphocytes







isolated from cord blood.



11
HLHFP03
77
Activation of
Kinase assay. JNK and p38
Preferred indications include neoplastic





T-Cell p38 or
kinase assays for signal
diseases (e.g., as described below under





JNK Signaling
transduction that regulate cell
“Hyperproliferative Disorders”), blood





Pathway.
proliferation, activation, or
disorders (e.g., as described below under






apoptosis are well known in the
“Immune Activity”, “Cardiovascular






art and may be used or
Disorders”, and/or “Blood-Related






routinely modified to assess the
Disorders”), and infection (e.g., an






ability of polypeptides of the
infectious disease as described below under






invention (including antibodies
“Infectious Disease”). Highly preferred






and agonists or antagonists of
indications include autoimmune diseases






the invention) to promote or
(e.g., rheumatoid arthritis, systemic lupus






inhibit immune cell (e.g. T-cell)
erythematosis, multiple sclerosis and/or as






proliferation, activation, and
described below) and immunodeficiencies






apoptosis. Exemplary assays
(e.g., as described below). Additional






for JNK and p38 kinase activity
highly preferred indications include






that may be used or routinely
inflammation and inflammatory disorders.






modified to test JNK and p38
Highly preferred indications also include






kinase-induced activity of
neoplastic diseases (e.g., leukemia,






polypeptides of the invention
lymphoma, and/or as described below






(including antibodies and
under “Hyperproliferative Disorders”).






agonists or antagonists of the
Highly preferred indications include






invention) include the assays
neoplasms and cancers, such as, leukemia,






disclosed in Forrer et al., Biol
lymphoma, prostate, breast, lung, colon,






Chem 379(8-9): 1101-1110
pancreatic, esophageal, stomach, brain,






(1998); Gupta et al., Exp Cell
liver, and urinary cancer. Other preferred






Res 247(2): 495-504 (1999);
indications include benign dysproliferative






Kyriakis J M, Biochem Soc
disorders and pre-neoplastic conditions,






Symp 64: 29-48 (1999); Chang
such as, for example, hyperplasia,






and Karin, Nature
metaplasia, and/or dysplasia. Preferred






410(6824): 37-40 (2001); and
indications include arthritis, asthma, AIDS,






Cobb M H, Prog Biophys Mol
allergy, anemia, pancytopenia, leukopenia,






Biol 71(3-4): 479-500 (1999);
thrombocytopenia, Hodgkin''s disease,






the contents of each of which
acute lymphocytic anemia (ALL),






are herein incorporated by
plasmacytomas, multiple myeloma,






reference in its entirety. T cells
Burkitt''s lymphoma, granulomatous






that may be used according to
disease, inflammatory bowel disease,






these assays are publicly
sepsis, psoriasis, suppression of immune






available (e.g., through the
reactions to transplanted organs and tissues,






ATCC TM). Exemplary mouse T
endocarditis, meningitis, and Lyme






cells that may be used
Disease.






according to these assays







include the CTLL cell line,







which is an IL-2 dependent







suspension-culture cell line







with cytotoxic activity.



11
HLHFP03
79
Activation of
Kinase assay. JNK and p38
Preferred indications include neoplastic





T-Cell p38 or
kinase assays for signal
diseases (e.g., as described below under





JNK Signaling
transduction that regulate cell
“Hyperproliferative Disorders”), blood





Pathway.
proliferation, activation, or
disorders (e.g., as described below under






apoptosis are well known in the
“Immune Activity”, “Cardiovascular






art and may be used or
Disorders”, and/or “Blood-Related






routinely modified to assess the
Disorders”), and infection (e.g., an






ability of polypeptides of the
infectious disease as described below under






invention (including antibodies
“Infectious Disease”). Highly preferred






and agonists or antagonists of
indications include autoimmune diseases






the invention) to promote or
(e.g., rheumatoid arthritis, systemic lupus






inhibit immune cell (e.g. T-cell)
erythematosis, multiple sclerosis and/or as






proliferation, activation, and
described below) and immunodeficiencies






apoptosis. Exemplary assays
(e.g., as described below). Additional






for JNK and p38 kinase activity
highly preferred indications include






that may be used or routinely
inflammation and inflammatory disorders.






modified to test JNK and p38
Highly preferred indications also include






kinase-induced activity of
neoplastic diseases (e.g., leukemia,






polypeptides of the invention
lymphoma, and/or as described below






(including antibodies and
under “Hyperproliferative Disorders”).






agonists or antagonists of the
Highly preferred indications include






invention) include the assays
neoplasms and cancers, such as, leukemia,






disclosed in Forrer et al., Biol
lymphoma, prostate, breast, lung, colon,






Chem 379(8-9): 1101-1110
pancreatic, esophageal, stomach, brain,






(1998); Gupta et al., Exp Cell
liver, and urinary cancer. Other preferred






Res 247(2): 495-504 (1999);
indications include benign dysproliferative






Kyriakis J M, Biochem Soc
disorders and pre-neoplastic conditions,






Symp 64: 29-48 (1999); Chang
such as, for example, hyperplasia,






and Karin, Nature
metaplasia, and/or dysplasia. Preferred






410(6824): 37-40 (2001); and
indications include arthritis, asthma, AIDS,






Cobb M H, Prog Biophys Mol
allergy, anemia, pancytopenia, leukopenia,






Biol 71(3-4): 479-500 (1999);
thrombocytopenia, Hodgkin''s disease,






the contents of each of which
acute lymphocytic anemia (ALL),






are herein incorporated by
plasmacytomas, multiple myeloma,






reference in its entirety. T cells
Burkitt''s lymphoma, granulomatous






that may be used according to
disease, inflammatory bowel disease,






these assays are publicly
sepsis, psoriasis, suppression of immune






available (e.g., through the
reactions to transplanted organs and tissues,






ATCC TM). Exemplary mouse T
endocarditis, meningitis, and Lyme






cells that may be used
Disease.






according to these assays







include the CTLL cell line,







which is an IL-2 dependent







suspension-culture cell line







with cytotoxic activity.



11
HLHFP03
79
SEAP in







HIB/CRE




11
HLHFP03
79
VEGF in







HT1080




11
HLHFP03
79
Production of
Assays for measuring
Highly preferred indications include





VCAM in
expression of VCAM are well-
inflammation (acute and chronic),





endothelial
known in the art and may be
restnosis, atherosclerosis, asthma and





cells (such as
used or routinely modified to
allergy. Highly preferred indications





human
assess the ability of
include inflammation and inflammatory





umbilical vein
polypeptides of the invention
disorders, immunological disorders,





endothelial
(including antibodies and
neoplastic disorders (e.g.





cells
agonists or antagonists of the
cancer/tumorigenesis), and cardiovascular





(HUVEC))
invention) to regulate VCAM
disorders (such as described below under






expression. For example,
“Immune Activity”, “Blood-Related






FMAT may be used to meaure
Disorders”, “Hyperproliferative Disorders”






the upregulation of cell surface
and/or “Cardiovascular Disorders”).






VCAM-1 expresssion in
Highly preferred indications include






endothelial cells. Endothelial
neoplasms and cancers such as, for






cells are cells that line blood
example, leukemia, lymphoma, melanoma,






vessels, and are involved in
renal cell carcinoma, and prostate, breast,






functions that include, but are
lung, colon, pancreatic, esophageal,






not limited to, angiogenesis,
stomach, brain, liver and urinary cancer.






vascular permeability, vascular
Other preferred indications include benign






tone, and immune cell
dysproliferative disorders and pre-






extravasation. Exemplary
neoplastic conditions, such as, for example,






endothelial cells that may be
hyperplasia, metaplasia, and/or dysplasia.






used according to these assays







include human umbilical vein







endothelial cells (HUVEC),







which are available from







commercial sources. The







expression of VCAM (CD106),







a membrane-associated protein,







can be upregulated by cytokines







or other factors, and contributes







to the extravasation of







lymphocytes, leucocytes and







other immune cells from blood







vessels; thus VCAM expression







plays a role in promoting







immune and inflammatory







responses.



11
HLHFP03
79
SEAP in OE-







21




12
HHTLF25
84
Production of
IL-10 FMAT. Assays for
A highly preferred embodiment of the





IL-10 and
immunomodulatory proteins
invention includes a method for stimulating





downregulation
produced by activated T cells,
the production of IL-10. An alternative





of immune
B cells, and monocytes that
preferred embodiment of the invention





responses
exhibit anti-inflammatory
includes a method for inhibiting the






activity and downregulate
production of IL-10. Highly preferred






monocyte/macrophage function
indications include inflammation and






and expression of cytokines are
inflammatory disorders (e.g. inflammatory






well known in the art and may
bowel disease). An additional highly






be used or routinely modified to
preferred indication includes inflammatory






assess the ability of the
bowel disease. Additional highly






polypeptides of the invention
preferred indications include blood






(including antibodies and
disorders (e.g., as described below under






agonists or antagonists of the
“Immune Activity” (e.g. autoimmune






invention) to mediate
disorders), “Blood-Related Disorders”,






immunomodulation, regulate
and/or “Cardiovascular Disorders”). Highly






inflammatory activities, and
preferred indications include autoimmune






modulate immune cell function
diseases (e.g., rheumatoid arthritis,






and cytokine production.
systemic lupus erythematosis, multiple






Exemplary assays that test for
sclerosis and/or as described below) and






immunomodulatory proteins
immunodeficiencies (e.g., as described






evaluate the production of
below). Preferred indications include






cytokines, such as IL-10, and
neoplastic diseases (e.g., leukemia,






the downmodulation of immune
lymphoma, melanoma, and/or as described






responses. Such assays that
below under “Hyperproliferative






may be used or routinely
Disorders”). Preferred indications include






modified to test
neoplasms and cancers, such as, for






immunomodulatory activity of
example, leukemia, lymphoma, melanoma,






polypeptides of the invention
and prostate, breast, lung, colon,






(including antibodies and
pancreatic, esophageal, stomach, brain,






agonists or antagonists of the
liver and urinary cancer. Other preferred






invention) include the assays
indications include benign dysproliferative






disclosed in Miraglia et al., J
disorders and pre-neoplastic conditions,






Biomolecular Screening 4: 193-204
such as, for example, hyperplasia,






(1999); Rowland et al.,
metaplasia, and/or dysplasia. Preferred






“Lymphocytes: a practical
indications include anemia, pancytopenia,






approach” Chapter 6: 138-160
leukopenia, thrombocytopenia, Hodgkin's






(2000); and Koning et al.,
disease, acute lymphocytic anemia (ALL),






Cytokine 9(6): 427-436 (1997),
plasmacytomas, multiple myeloma,






the contents of each of which
Burkitt's lymphoma, Crohn''s disease,






are herein incorporated by
arthritis, AIDS, granulomatous disease,






reference in its entirety.
sepsis, neutropenia, neutrophilia, psoriasis,






Human T cells that may be used
suppression of immune reactions to






according to these assays may
transplanted organs and tissues,






be isolated using techniques
hemophilia, hypercoagulation, diabetes






disclosed herein or otherwise
mellitus, endocarditis, meningitis, Lyme






known in the art. Human T
Disease, asthma and allergy. An






cells are primary human
additional preferred indication is infection






lymphocytes that mature in the
(e.g., as described below under “Infectious






thymus and express a T cell
Disease”).






receptor and CD3, CD4, or







CD8. These cells mediate







humoral or cell-mediated







immunity and may be







preactivated to enhance







responsiveness to







immunomodulatory factors.



13
HTADX17
91
Activation of
Assays for the activation of
Highly preferred indications include





transcription
transcription through the
blood disorders (e.g., as described below





through NFAT
Nuclear Factor of Activated T
under “Immune Activity”, “Blood-Related





response in
cells (NFAT) response element
Disorders”, and/or ““Cardiovascular





immune cells
are well-known in the art and
Disorders””). Highly preferred indications





(such as T-
may be used or routinely
include autoimmune diseases (e.g.,





cells).
modified to assess the ability of
rheumatoid arthritis, systemic lupus






polypeptides of the invention
erythematosis, multiple sclerosis and/or as






(including antibodies and
described below), immunodeficiencies






agonists or antagonists of the
(e.g., as described below), boosting a T






invention) to regulate NFAT
cell-mediated immune response, and






transcription factors and
suppressing a T cell-mediated immune






modulate expression of genes
response. Additional highly preferred






involved in immunomodulatory
indications include inflammation and






functions. Exemplary assays
inflammatory disorders. An additional






for transcription through the
highly preferred indication is infection






NFAT response element that
(e.g., an infectious disease as described






may be used or routinely
below under “Infectious Disease”).






modified to test NFAT-
Preferred indications include neoplastic






response element activity of
diseases (e.g., leukemia, lymphoma, and/or






polypeptides of the invention
as described below under






(including antibodies and
“Hyperproliferative Disorders”). Preferred






agonists or antagonists of the
indications include neoplasms and cancers,






invention) include assays
such as, for example, leukemia, lymphoma,






disclosed in Berger et al., Gene
and prostate, breast, lung, colon,






66: 1-10 (1998); Cullen and
pancreatic, esophageal, stomach, brain,






Malm, Methods in Enzymol
liver and urinary cancer. Other preferred






216: 362-368 (1992); Henthorn
indications include benign dysproliferative






et al., Proc Natl Acad Sci USA
disorders and pre-neoplastic conditions,






85: 6342-6346 (1988); Serfling
such as, for example, hyperplasia,






et al., Biochim Biophys Acta
metaplasia, and/or dysplasia. Preferred






1498(1): 1-18 (2000); De Boer
indications also include anemia,






et al., Int J Biochem Cell Biol
pancytopenia, leukopenia,






31(10): 1221-1236 (1999);
thrombocytopenia, Hodgkin's disease,






Fraser et al., Eur J Immunol
acute lymphocytic anemia (ALL),






29(3): 838-844 (1999); and
plasmacytomas, multiple myeloma,






Yeseen et al., J Biol Chem
Burkitt's lymphoma, arthritis, AIDS,






268(19): 14285-14293 (1993),
granulomatous disease, inflammatory






the contents of each of which
bowel disease, sepsis, neutropenia,






are herein incorporated by
neutrophilia, psoriasis, suppression of






reference in its entirety. T cells
immune reactions to transplanted organs






that may be used according to
and tissues, hemophilia, hypercoagulation,






these assays are publicly
diabetes mellitus, endocarditis, meningitis,






available (e.g., through the
Lyme Disease, asthma and allergy.






ATCC TM). Exemplary human







T cells that may be used







according to these assays







include the JURKAT cell line,







which is a suspension culture of







leukemia cells that produce IL-







2 when stimulated.



13
HTADX17
91
Activation of
Assays for the activation of
Highly preferred indications include





transcription
transcription through the
neoplastic diseases (e.g., leukemia,





through GAS
Gamma Interferon Activation
lymphoma, and/or as described below





response
Site (GAS) response element
under “Hyperproliferative Disorders”).





element in
are well-known in the art and
Highly preferred indications include





immune cells
may be used or routinely
neoplasms and cancers, such as, for





(such as T-
modified to assess the ability of
example, leukemia, lymphoma (e.g., T cell





cells).
polypeptides of the invention
lymphoma, Burkitt's lymphoma, non-






(including antibodies and
Hodgkins lymphoma, Hodgkin's disease),






agonists or antagonists of the
melanoma, and prostate, breast, lung,






invention) to regulate STAT
colon, pancreatic, esophageal, stomach,






transcription factors and
brain, liver and urinary cancer. Other






modulate gene expression
preferred indications include benign






involved in a wide variety of
dysproliferative disorders and pre-






cell functions. Exemplary
neoplastic conditions, such as, for example,






assays for transcription through
hyperplasia, metaplasia, and/or dysplasia.






the GAS response element that
Preferred indications include autoimmune






may be used or routinely
diseases (e.g., rheumatoid arthritis,






modified to test GAS-response
systemic lupus erythematosis, multiple






element activity of polypeptides
sclerosis and/or as described below),






of the invention (including
immunodeficiencies (e.g., as described






antibodies and agonists or
below), boosting a T cell-mediated immune






antagonists of the invention)
response, and suppressing a T cell-






include assays disclosed in
mediated immune response. Additional






Berger et al., Gene 66: 1-10
preferred indications include inflammation






(1998); Cullen and Malm,
and inflammatory disorders. Highly






Methods in Enzymol 216: 362-368
preferred indications include blood






(1992); Henthorn et al.,
disorders (e.g., as described below under






Proc Natl Acad Sci USA
“Immune Activity”, “Blood-Related






85: 6342-6346 (1988);
Disorders”, and/or ““Cardiovascular






Matikainen et al., Blood
Disorders””), and infection (e.g., viral






93(6): 1980-1991 (1999); and
infections, tuberculosis, infections






Henttinen et al., J Immunol
associated with chronic granulomatosus






155(10): 4582-4587 (1995), the
disease and malignant osteoporosis, and/or






contents of each of which are
an infectious disease as described below






herein incorporated by
under “Infectious Disease”). An additional






reference in its entirety.
preferred indication is idiopathic






Exemplary human T cells, such
pulmonary fibrosis. Preferred






as the MOLT4 cell line, that
indications include anemia, pancytopenia,






may be used according to these
leukopenia, thrombocytopenia, acute






assays are publicly available
lymphocytic anemia (ALL),






(e.g., through the ATCC TM).
plasmacytomas, multiple myeloma,







arthritis, AIDS, granulomatous disease,







inflammatory bowel disease, sepsis,







neutropenia, neutrophilia, psoriasis,







suppression of immune reactions to







transplanted organs and tissues,







hemophilia, hypercoagulation, diabetes







mellitus, endocarditis, meningitis, Lyme







Disease, and asthma and allergy.


13
HTADX17
91
Activation of
Assays for the activation of
Highly preferred indications include





transcription
transcription through the NFKB
inflammation and inflammatory disorders.





through NFKB
response element are well-
Highly preferred indications include blood





response
known in the art and may be
disorders (e.g., as described below under





element in
used or routinely modified to
“Immune Activity”, “Blood-Related





immune cells
assess the ability of
Disorders”, and/or ““Cardiovascular





(such as T-
polypeptides of the invention
Disorders””). Highly preferred indications





cells).
(including antibodies and
include autoimmune diseases (e.g.,






agonists or antagonists of the
rheumatoid arthritis, systemic lupus






invention) to regulate NFKB
erythematosis, multiple sclerosis and/or as






transcription factors and
described below), and immunodeficiencies






modulate expression of
(e.g., as described below). An additional






immunomodulatory genes.
highly preferred indication is infection






Exemplary assays for
(e.g., AIDS, and/or an infectious disease as






transcription through the NFKB
described below under “Infectious






response element that may be
Disease”). Highly preferred






used or rountinely modified to
indications include neoplastic diseases






test NFKB-response element
(e.g., melanoma, leukemia, lymphoma,






activity of polypeptides of the
and/or as described below under






invention (including antibodies
“Hyperproliferative Disorders”). Highly






and agonists or antagonists of
preferred indications include neoplasms






the invention) include assays
and cancers, such as, for example,






disclosed in Berger et al., Gene
melanoma, renal cell carcinoma, leukemia,






66: 1-10 (1998); Cullen and
lymphoma, and prostate, breast, lung,






Malm, Methods in Enzymol
colon, pancreatic, esophageal, stomach,






216: 362-368 (1992); Henthorn
brain, liver and urinary cancer. Other






et al., Proc Natl Acad Sci USA
preferred indications include benign






85: 6342-6346 (1988); Black et
dysproliferative disorders and pre-






al., Virus Gnes 15(2): 105-117
neoplastic conditions, such as, for example,






(1997); and Fraser et al.,
hyperplasia, metaplasia, and/or dysplasia.






29(3): 838-844 (1999), the
Preferred indications also include anemia,






contents of each of which are
pancytopenia, leukopenia,






herein incorporated by
thrombocytopenia, Hodgkin's disease,






reference in its entirety.
acute lymphocytic anemia (ALL),






Exemplary human T cells, such
plasmacytomas, multiple myeloma,






as the MOLT4, that may be
Burkitt's lymphoma, arthritis, AIDS,






used according to these assays
granulomatous disease, inflammatory






are publicly available (e.g.,
bowel disease, sepsis, neutropenia,






through the ATCC TM).
neutrophilia, psoriasis, hemophilia,







hypercoagulation, diabetes mellitus,







endocarditis, meningitis, Lyme Disease,







suppression of immune reactions to







transplanted organs, asthma and allergy.


13
HTADX17
93
Activation of
Assays for the activation of
Highly preferred indications include





transcription
transcription through the
blood disorders (e.g., as described below





through NFAT
Nuclear Factor of Activated T
under “Immune Activity”, “Blood-Related





response in
cells (NFAT) response element
Disorders”, and/or “Cardiovascular





immune cells
are well-known in the art and
Disorders”). Highly preferred indications





(such as T-
may be used or routinely
include autoimmune diseases (e.g.,





cells).
modified to assess the ability of
rheumatoid arthritis, systemic lupus






polypeptides of the invention
erythematosis, multiple sclerosis and/or as






(including antibodies and
described below), immunodeficiencies






agonists or antagonists of the
(e.g., as described below), boosting a T






invention) to regulate NFAT
cell-mediated immune response, and






transcription factors and
suppressing a T cell-mediated immune






modulate expression of genes
response. Additional highly preferred






involved in immunomodulatory
indications include inflammation and






functions. Exemplary assays
inflammatory disorders. An additional






for transcription through the
highly preferred indication is infection






NFAT response element that
(e.g., an infectious disease as described






may be used or routinely
below under “Infectious Disease”).






modified to test NFAT-
Preferred indications include neoplastic






response element activity of
diseases (e.g., leukemia, lymphoma, and/or






polypeptides of the invention
as described below under






(including antibodies and
“Hyperproliferative Disorders”). Preferred






agonists or antagonists of the
indications include neoplasms and cancers,






invention) include assays
such as, for example, leukemia, lymphoma,






disclosed in Berger et al., Gene
and prostate, breast, lung, colon,






66: 1-10 (1998); Cullen and
pancreatic, esophageal, stomach, brain,






Malm, Methods in Enzymol
liver and urinary cancer. Other preferred






216: 362-368 (1992); Henthorn
indications include benign dysproliferative






et al., Proc Natl Acad Sci USA
disorders and pre-neoplastic conditions,






85: 6342-6346 (1988); Serfling
such as, for example, hyperplasia,






et al., Biochim Biophys Acta
metaplasia, and/or dysplasia. Preferred






1498(1): 1-18 (2000); De Boer
indications also include anemia,






et al., Int J Biochem Cell Biol
pancytopenia, leukopenia,






31(10): 1221-1236 (1999);
thrombocytopenia, Hodgkin's disease,






Fraser et al., Eur J Immunol
acute lymphocytic anemia (ALL),






29(3): 838-844 (1999); and
plasmacytomas, multiple myeloma,






Yeseen et al., J Biol Chem
Burkitt's lymphoma, arthritis, AIDS,






268(19): 14285-14293 (1993),
granulomatous disease, inflammatory






the contents of each of which
bowel disease, sepsis, neutropenia,






are herein incorporated by
neutrophilia, psoriasis, suppression of






reference in its entirety. T cells
immune reactions to transplanted organs






that may be used according to
and tissues, hemophilia, hypercoagulation,






these assays are publicly
diabetes mellitus, endocarditis, meningitis,






available (e.g., through the
Lyme Disease, asthma and allergy.






ATCC TM). Exemplary human







T cells that may be used







according to these assays







include the JURKAT cell line,







which is a suspension culture of







leukemia cells that produce IL-







2 when stimulated.



13
HTADX17
93
Activation of
Assays for the activation of
Highly preferred indications include





transcription
transcription through the
neoplastic diseases (e.g., leukemia,





through GAS
Gamma Interferon Activation
lymphoma, and/or as described below





response
Site (GAS) response element
under “Hyperproliferative Disorders”).





element in
are well-known in the art and
Highly preferred indications include





immune cells
may be used or routinely
neoplasms and cancers, such as, for





(such as T-
modified to assess the ability of
example, leukemia, lymphoma (e.g., T cell





cells).
polypeptides of the invention
lymphoma, Burkitt's lymphoma, non-






(including antibodies and
Hodgkins lymphoma, Hodgkin''s disease),






agonists or antagonists of the
melanoma, and prostate, breast, lung,






invention) to regulate STAT
colon, pancreatic, esophageal, stomach,






transcription factors and
brain, liver and urinary cancer. Other






modulate gene expression
preferred indications include benign






involved in a wide variety of
dysproliferative disorders and pre-






cell functions. Exemplary
neoplastic conditions, such as, for example,






assays for transcription through
hyperplasia, metaplasia, and/or dysplasia.






the GAS response element that
Preferred indications include autoimmune






may be used or routinely
diseases (e.g., rheumatoid arthritis,






modified to test GAS-response
systemic lupus erythematosis, multiple






element activity of polypeptides
sclerosis and/or as described below),






of the invention (including
immunodeficiencies (e.g., as described






antibodies and agonists or
below), boosting a T cell-mediated immune






antagonists of the invention)
response, and suppressing a T cell-






include assays disclosed in
mediated immune response. Additional






Berger et al., Gene 66: 1-10
preferred indications include inflammation






(1998); Cullen and Malm,
and inflammatory disorders. Highly






Methods in Enzymol 216: 362-368
preferred indications include blood






(1992); Henthorn et al.,
disorders (e.g., as described below under






Proc Natl Acad Sci USA
“Immune Activity”, “Blood-Related






85: 6342-6346 (1988);
Disorders”, and/or “Cardiovascular






Matikainen et al., Blood
Disorders”), and infection (e.g., viral






93(6): 1980-1991 (1999); and
infections, tuberculosis, infections






Henttinen et al., J Immunol
associated with chronic granulomatosus






155(10): 4582-4587 (1995), the
disease and malignant osteoporosis, and/or






contents of each of which are
an infectious disease as described below






herein incorporated by
under “Infectious Disease”). An additional






reference in its entirety.
preferred indication is idiopathic






Exemplary human T cells, such
pulmonary fibrosis. Preferred






as the MOLT4 cell line, that
indications include anemia, pancytopenia,






may be used according to these
leukopenia, thrombocytopenia, acute






assays are publicly available
lymphocytic anemia (ALL),






(e.g., through the ATCC TM).
plasmacytomas, multiple myeloma,







arthritis, AIDS, granulomatous disease,







inflammatory bowel disease, sepsis,







neutropenia, neutrophilia, psoriasis,







suppression of immune reactions to







transplanted organs and tissues,







hemophilia, hypercoagulation, diabetes







mellitus, endocarditis, meningitis, Lyme







Disease, and asthma and allergy.


13
HTADX17
93
Activation of
Assays for the activation of
Highly preferred indications include





transcription
transcription through the NFKB
inflammation and inflammatory disorders.





through NFKB
response element are well-
Highly preferred indications include blood





response
known in the art and may be
disorders (e.g., as described below under





element in
used or routinely modified to
“Immune Activity”, “Blood-Related





immune cells
assess the ability of
Disorders”, and/or “Cardiovascular





(such as T-
polypeptides of the invention
Disorders”). Highly preferred indications





cells).
(including antibodies and
include autoimmune diseases (e.g.,






agonists or antagonists of the
rheumatoid arthritis, systemic lupus






invention) to regulate NFKB
erythematosis, multiple sclerosis and/or as






transcription factors and
described below), and immunodeficiencies






modulate expression of
(e.g., as described below). An additional






immunomodulatory genes.
highly preferred indication is infection






Exemplary assays for
(e.g., AIDS, and/or an infectious disease as






transcription through the NFKB
described below under “Infectious






response element that may be
Disease”). Highly preferred






used or rountinely modified to
indications include neoplastic diseases






test NFKB-response element
(e.g., melanoma, leukemia, lymphoma,






activity of polypeptides of the
and/or as described below under






invention (including antibodies
“Hyperproliferative Disorders”). Highly






and agonists or antagonists of
preferred indications include neoplasms






the invention) include assays
and cancers, such as, for example,






disclosed in Berger et al., Gene
melanoma, renal cell carcinoma, leukemia,






66: 1-10 (1998); Cullen and
lymphoma, and prostate, breast, lung,






Malm, Methods in Enzymol
colon, pancreatic, esophageal, stomach,






216: 362-368 (1992); Henthorn
brain, liver and urinary cancer. Other






et al., Proc Natl Acad Sci USA
preferred indications include benign






85: 6342-6346 (1988); Black et
dysproliferative disorders and pre-






al., Virus Gnes 15(2): 105-117
neoplastic conditions, such as, for example,






(1997); and Fraser et al.,
hyperplasia, metaplasia, and/or dysplasia.






29(3): 838-844 (1999), the
Preferred indications also include anemia,






contents of each of which are
pancytopenia, leukopenia,






herein incorporated by
thrombocytopenia, Hodgkin's disease,






reference in its entirety.
acute lymphocytic anemia (ALL),






Exemplary human T cells, such
plasmacytomas, multiple myeloma,






as the MOLT4, that may be
Burkitt's lymphoma, arthritis, AIDS,






used according to these assays
granulomatous disease, inflammatory






are publicly available (e.g.,
bowel disease, sepsis, neutropenia,






through the ATCC TM).
neutrophilia, psoriasis, hemophilia,







hypercoagulation, diabetes mellitus,







endocarditis, meningitis, Lyme Disease,







suppression of immune reactions to







transplanted organs, asthma and allergy.


13
HTADX17
93
IL-8 in Normal







Human







Bronchial







Epitheliae




14
HJACG02
105
MIP-1a in







HMC




14
HJACG02
105
Proliferation of
Assays for the regulation (i.e.






pre-adipose
increases or decreases) of






cells (such as
viability and proliferation of






3T3-L1 cells)
cells in vitro are well-known in







the art and may be used or







routinely modified to assess the







ability of polypeptides of the







invention (including antibodies







and agonists or antagonists of







the invention) to regulate







viability and proliferation of







pre-adipose cells and cell lines.







For example, the CellTiter-







Gloô Luminescent Cell







Viability Assay (PROMEGA TM







Corp., Madison, WI, USA) can







be used to measure the number







of viable cells in culture based







on quantitation of the ATP







present which signals the







presence of metabolically







active cells. 3T3-L1 is a mouse







preadipocyte cell line. It is a







continuous substrain of 3T3







fibroblast cells developed







through clonal isolation. Cells







were differentiated to an







adipose-like state before being







used in the screen. See Green H







and Meuth M., Cell 3: 127-133







(1974), which is herein







incorporated by reference in its







entirety.



14
HJACG02
105
MCP-1 in







HUVEC




14
HJACG02
105
Activation or
This reporter assay measures






inhibition of
activation or inhibition of the






transcription
NFkB signaling pathway in






through NFKB
Ku812 human basophil cell






response
line. Assays for the activation






element in
or inhibition of transcription






immune cells
through the NFKB response






(such as
element are well-known in the






basophils).
art and may be used or







routinely modified to assess the







ability of polypeptides of the







invention (including antibodies







and agonists or antagonists of







the invention) to regulate







NFKB transcription factors and







modulate expression of







immunomodulatory genes.







NFkB is important in the







pathogenesis of asthma.







Exemplary assays for







transcription through the NFKB







response element that may be







used or rountinely modified to







test NFKB-response element







activity of polypeptides of the







invention (including antibodies







and agonists or antagonists of







the invention) include assays







disclosed in Berger et al., Gene







66: 1-10 (1998); Cullen and







Malm, Methods in Enzymol







216: 362-368 (1992); Henthorn







et al., Proc Natl Acad Sci USA







85: 6342-6346 (1988); Marone







et al, Int Arch Allergy Immunol







114(3): 207-17 (1997), the







contents of each of which are







herein incorporated by







reference in its entirety. Cells







were pretreated with SID







supernatants or controls for 15-18







hours, and then 10 ng/mL of







TNF was added to stimulate the







NFkB reporter. SEAP activity







was measured after 48 hours.







Basophils that may be used







according to these assays are







publicly available (e.g., through







the ATCC TM). Exemplary







human basophil cell lines that







may be used according to these







assays include Ku812,







originally established from a







patient with chronic







myelogenous leukemia. It is an







immature prebasophilic cell







line that can be induced to







differentiate into mature







basophils. See, Kishi et al.,







Leuk Res. 9: 381-390 (1985);







Blom et al., Eur J Immunol.







22: 2025-32 (1992), where the







contents of each are herein







incorporated by reference in its







entirety.



16
HSVAK93
121
Production of
Assays for measuring
Highly preferred indications include





VCAM in
expression of VCAM are well-
inflammation (acute and chronic),





endothelial
known in the art and may be
restnosis, atherosclerosis, asthma and





cells (such as
used or routinely modified to
allergy. Highly preferred indications





human
assess the ability of
include inflammation and inflammatory





umbilical vein
polypeptides of the invention
disorders, immunological disorders,





endothelial
(including antibodies and
neoplastic disorders (e.g.





cells
agonists or antagonists of the
cancer/tumorigenesis), and cardiovascular





(HUVEC))
invention) to regulate VCAM
disorders (such as described below under






expression. For example,
“Immune Activity”, “Blood-Related






FMAT may be used to meaure
Disorders”, “Hyperproliferative Disorders”






the upregulation of cell surface
and/or “Cardiovascular Disorders”).






VCAM-1 expresssion in
Highly preferred indications include






endothelial cells. Endothelial
neoplasms and cancers such as, for






cells are cells that line blood
example, leukemia, lymphoma, melanoma,






vessels, and are involved in
renal cell carcinoma, and prostate, breast,






functions that include, but are
lung, colon, pancreatic, esophageal,






not limited to, angiogenesis,
stomach, brain, liver and urinary cancer.






vascular permeability, vascular
Other preferred indications include benign






tone, and immune cell
dysproliferative disorders and pre-






extravasation. Exemplary
neoplastic conditions, such as, for example,






endothelial cells that may be
hyperplasia, metaplasia, and/or dysplasia.






used according to these assays







include human umbilical vein







endothelial cells (HUVEC),







which are available from







commercial sources. The







expression of VCAM (CD106),







a membrane-associated protein,







can be upregulated by cytokines







or other factors, and contributes







to the extravasation of







lymphocytes, leucocytes and







other immune cells from blood







vessels; thus VCAM expression







plays a role in promoting







immune and inflammatory







responses.



17
HSDEK49
126
Activation of
Assays for the activation of
A preferred embodiment of the





transcription
transcription through the Serum
invention includes a method for inhibiting





through serum
Response Element (SRE) are
(e.g., reducing) TNF alpha production. An





response
well-known in the art and may
alternative preferred embodiment of the





element in
be used or routinely modified to
invention includes a method for stimulating





immune cells
assess the ability of
(e.g., increasing) TNF alpha production.





(such as T-
polypeptides of the invention
Preferred indications include blood





cells).
(including antibodies and
disorders (e.g., as described below under






agonists or antagonists of the
“Immune Activity”, “Blood-Related






invention) to regulate the serum
Disorders”, and/or “Cardiovascular






response factors and modulate
Disorders”), Highly preferred indications






the expression of genes
include autoimmune diseases (e.g.,






involved in growth. Exemplary
rheumatoid arthritis, systemic lupus






assays for transcription through
erythematosis, Crohn''s disease, multiple






the SRE that may be used or
sclerosis and/or as described below),






routinely modified to test SRE
immunodeficiencies (e.g., as described






activity of the polypeptides of
below), boosting a T cell-mediated immune






the invention (including
response, and suppressing a T cell-






antibodies and agonists or
mediated immune response. Additional






antagonists of the invention)
highly preferred indications include






include assays disclosed in
inflammation and inflammatory disorders,






Berger et al., Gene 66: 1-10
and treating joint damage in patients with






(1998); Cullen and Malm,
rheumatoid arthritis. An additional highly






Methods in Enzymol 216: 362-368
preferred indication is sepsis. Highly






(1992); Henthorn et al.,
preferred indications include neoplastic






Proc Natl Acad Sci USA
diseases (e.g., leukemia, lymphoma, and/or






85: 6342-6346 (1988); and
as described below under






Black et al., Virus Genes
“Hyperproliferative Disorders”).






12(2): 105-117 (1997), the
Additionally, highly preferred indications






content of each of which are
include neoplasms and cancers, such as, for






herein incorporated by
example, leukemia, lymphoma, melanoma,






reference in its entirety. T cells
glioma (e.g., malignant glioma), solid






that may be used according to
tumors, and prostate, breast, lung, colon,






these assays are publicly
pancreatic, esophageal, stomach, brain,






available (e.g., through the
liver and urinary cancer. Other preferred






ATCC TMTM). Exemplary
indications include benign dysproliferative






mouse T cells that may be used
disorders and pre-neoplastic conditions,






according to these assays
such as, for example, hyperplasia,






include the CTLL cell line,
metaplasia, and/or dysplasia. Preferred






which is an IL-2 dependent
indications include anemia, pancytopenia,






suspension culture of T cells
leukopenia, thrombocytopenia, Hodgkin's






with cytotoxic activity.
disease, acute lymphocytic anemia (ALL),







plasmacytomas, multiple myeloma,







Burkitt's lymphoma, arthritis, AIDS,







granulomatous disease, inflammatory







bowel disease, neutropenia, neutrophilia,







psoriasis, suppression of immune reactions







to transplanted organs and tissues,







hemophilia, hypercoagulation, diabetes







mellitus, endocarditis, meningitis, Lyme







Disease, cardiac reperfusion injury, and







asthma and allergy. An additional







preferred indication is infection (e.g., an







infectious disease as described below under







“Infectious Disease”).


17
HSDEK49
126
Regulation of
Assays for the regulation of
A highly preferred indication is





transcription of
transcription of Malic Enzyme
diabetes mellitus. An additional





Malic Enzyme
are well-known in the art and
highly preferred indication is a





in adipocytes
may be used or routinely
complication associated with diabetes (e.g.,






modified to assess the ability of
diabetic retinopathy, diabetic nephropathy,






polypeptides of the invention
kidney disease (e.g., renal failure,






(including antibodies and
nephropathy and/or other diseases and






agonists or antagonists of the
disorders as described in the “Renal






invention) to regulate
Disorders” section below), diabetic






transcription of Malic Enzyme,
neuropathy, nerve disease and nerve






a key enzyme in lipogenesis.
damage (e.g., due to diabetic neuropathy),






Malic enzyme is involved in
blood vessel blockage, heart disease,






lipogenesisand its expression is
stroke, impotence (e.g., due to diabetic






stimulted by insulin. ME
neuropathy or blood vessel blockage),






promoter contains two direct
seizures, mental confusion, drowsiness,






repeat (DR1)-like elements
nonketotic hyperglycemic-hyperosmolar






MEp and MEd identified as
coma, cardiovascular disease (e.g., heart






putative PPAR response
disease, atherosclerosis, microvascular






elements. ME promoter may
disease, hypertension, stroke, and other






also responds to AP1 and other
diseases and disorders as described in the






transcription factors.
“Cardiovascular Disorders” section below),






Exemplary assays that may be
dyslipidemia, endocrine disorders (as






used or routinely modified to
described in the “Endocrine Disorders”






test for regulation of
section below), neuropathy, vision






transcription of Malic Enzyme
impairment (e.g., diabetic retinopathy and






(in adipoocytes) by
blindness), ulcers and impaired wound






polypeptides of the invention
healing, and infection (e.g., infectious






(including antibodies and
diseases and disorders as described in the






agonists or antagonists of the
“Infectious Diseases” section below,






invention) include assays
especially of the urinary tract and skin),






disclosed in: Streeper, R. S., et
carpal tunnel syndrome and Dupuytren's






al., Mol Endocrinol,
contracture). An additional highly






12(11): 1778-91 (1998); Garcia-
preferred indication is obesity and/or






Jimenez, C., et al., Mol
complications associated with obesity.






Endocrinol, 8(10): 1361-9
Additional highly preferred indications






(1994); Barroso, I., et al., J Biol
include weight loss or alternatively, weight






Chem, 274(25): 17997-8004
gain. Aditional highly preferred






(1999); Ijpenberg, A., et al., J
indications are complications associated






Biol Chem, 272(32): 20108-20117
with insulin resistance.






(1997); Berger, et al.,







Gene 66: 1-10 (1988); and,







Cullen, B., et al., Methods in







Enzymol. 216: 362-368 (1992),







the contents of each of which is







herein incorporated by







reference in its entirety.







Hepatocytes that may be used







according to these assays are







publicly available (e.g., through







the ATCC TMTM) and/or may be







routinely generated. Exemplary







hepatocytes that may be used







according to these assays







includes the H4IIE rat liver







hepatoma cell line.



17
HSDEK49
126
MIP-1a in







HMC




18
HWBAO62
128
Activation of
Assays for the activation of
A highly preferred embodiment of the





transcription
transcription through the CD28
invention includes a method for stimulating





through CD28
response element are well-
T cell proliferation. An alternative highly





response
known in the art and may be
preferred embodiment of the invention





element in
used or routinely modified to
includes a method for inhibiting T cell





immune cells
assess the ability of
proliferation. A highly preferred





(such as T-
polypeptides of the invention
embodiment of the invention includes a





cells).
(including antibodies and
method for activating T cells. An






agonists or antagonists of the
alternative highly preferred embodiment of






invention) to stimulate IL-2
the invention includes a method for






expression in T cells.
inhibiting the activation of and/or






Exemplary assays for
inactivating T cells. A highly preferred






transcription through the CD28
embodiment of the invention includes a






response element that may be
method for stimulating (e.g., increasing)






used or routinely modified to
IL-2 production. An alternative highly






test CD28-response element
preferred embodiment of the invention






activity of polypeptides of the
includes a method for inhibiting (e.g.,






invention (including antibodies
reducing) IL-2 production. Additional






and agonists or antagonists of
highly preferred indications include






the invention) include assays
inflammation and inflammatory disorders.






disclosed in Berger et al., Gene
Highly preferred indications include






66: 1-10 (1998); Cullen and
autoimmune diseases (e.g., rheumatoid






Malm, Methods in Enzymol
arthritis, systemic lupus erythematosis,






216: 362-368 (1992); Henthorn
multiple sclerosis and/or as described






et al., Proc Natl Acad Sci USA
below), immunodeficiencies (e.g., as






85: 6342-6346 (1988); McGuire
described below), boosting a T cell-






and Iacobelli, J Immunol
mediated immune response, and






159(3): 1319-1327 (1997); Parra
suppressing a T cell-mediated immune






et al., J Immunol 166(4): 2437-2443
response. Highly preferred indications






(2001); and Butscher et
include neoplastic diseases (e.g.,






al., J Biol Chem 3(1): 552-560
melanoma, renal cell carcinoma, leukemia,






(1998), the contents of each of
lymphoma, and/or as described below






which are herein incorporated
under “Hyperproliferative Disorders”).






by reference in its entirety. T
Highly preferred indications include






cells that may be used
neoplasms and cancers, such as, for






according to these assays are
example, melanoma (e.g., metastatic






publicly available (e.g., through
melanoma), renal cell carcinoma (e.g.,






the ATCC TM). Exemplary
metastatic renal cell carcinoma), leukemia,






human T cells that may be used
lymphoma (e.g,. T cell lymphoma), and






according to these assays
prostate, breast, lung, colon, pancreatic,






include the SUPT cell line,
esophageal, stomach, brain, liver and






which is a suspension culture of
urinary cancer. Other preferred indications






IL-2 and IL-4 responsive T
include benign dysproliferative disorders






cells.
and pre-neoplastic conditions, such as, for







example, hyperplasia, metaplasia, and/or







dysplasia. A highly preferred indication







includes infection (e.g., AIDS,







tuberculosis, infections associated with







granulomatous disease, and osteoporosis,







and/or as described below under







“Infectious Disease”). A highly preferred







indication is AIDS. Additional highly







preferred indications include suppression of







immune reactions to transplanted organs







and/or tissues, uveitis, psoriasis, and







tropical spastic paraparesis. Preferred







indications include blood disorders (e.g., as







described below under “Immune Activity”,







“Blood-Related Disorders”, and/or







“Cardiovascular Disorders”). Preferred







indications also include anemia,







pancytopenia, leukopenia,







thrombocytopenia, Hodgkin's disease,







acute lymphocytic anemia (ALL),







plasmacytomas, multiple myeloma,







Burkitt's lymphoma, arthritis,







granulomatous disease, inflammatory







bowel disease, sepsis, neutropenia,







neutrophilia, hemophilia, hypercoagulation,







diabetes mellitus, endocarditis, meningitis,







Lyme Disease, asthma and allergy.


18
HWBAO62
128
Caspase







(+paclitaxel) in







SW480




19
HWHGU54
132
Activation of
Assays for the activation of
Preferred indications include blood





transcription
transcription through the cAMP
disorders (e.g., as described below under





through cAMP
response element are well-
“Immune Activity”, “Blood-Related





response
known in the art and may be
Disorders”, and/or “Cardiovascular





element in
used or routinely modified to
Disorders”), and infection (e.g., an





immune cells
assess the ability of
infectious disease as described below under





(such as T-
polypeptides of the invention
“Infectious Disease”). Preferred





cells).
(including antibodies and
indications include autoimmune diseases






agonists or antagonists of the
(e.g., rheumatoid arthritis, systemic lupus






invention) to increase cAMP
erythematosis, multiple sclerosis and/or as






and regulate CREB
described below), immunodeficiencies






transcription factors, and
(e.g., as described below), boosting a T






modulate expression of genes
cell-mediated immune response, and






involved in a wide variety of
suppressing a T cell-mediated immune






cell functions. Exemplary
response. Additional preferred indications






assays for transcription through
include inflammation and inflammatory






the cAMP response element
disorders. Highly preferred indications






that may be used or routinely
include neoplastic diseases (e.g., leukemia,






modified to test cAMP-
lymphoma, and/or as described below






response element activity of
under “Hyperproliferative Disorders”).






polypeptides of the invention
Highly preferred indications include






(including antibodies and
neoplasms and cancers, such as, for






agonists or antagonists of the
example, leukemia, lymphoma (e.g., T cell






invention) include assays
lymphoma, Burkitt's lymphoma, non-






disclosed in Berger et al., Gene
Hodgkins lymphoma, Hodgkin''s disease),






66: 1-10 (1998); Cullen and
melanoma, and prostate, breast, lung,






Malm, Methods in Enzymol
colon, pancreatic, esophageal, stomach,






216: 362-368 (1992); Henthorn
brain, liver and urinary cancer. Other






et al., Proc Natl Acad Sci USA
preferred indications include benign






85: 6342-6346 (1988); Black et
dysproliferative disorders and pre-






al., Virus Genes 15(2): 105-117
neoplastic conditions, such as, for example,






(1997); and Belkowski et al., J
hyperplasia, metaplasia, and/or dysplasia.






Immunol 161(2): 659-665
Preferred indications include anemia,






(1998), the contents of each of
pancytopenia, leukopenia,






which are herein incorporated
thrombocytopenia, acute lymphocytic






by reference in its entirety. T
anemia (ALL), plasmacytomas, multiple






cells that may be used
myeloma, arthritis, AIDS, granulomatous






according to these assays are
disease, inflammatory bowel disease,






publicly available (e.g., through
sepsis, neutropenia, neutrophilia, psoriasis,






the ATCC TM). Exemplary
suppression of immune reactions to






mouse T cells that may be used
transplanted organs and tissues,






according to these assays
hemophilia, hypercoagulation, diabetes






include the CTLL cell line,
mellitus, endocarditis, meningitis, Lyme






which is a suspension culture of
Disease, and asthma and allergy.






IL-2 dependent cytotoxic T







cells.



19
HWHGU54
132
Production of
Assays measuring production
Highly preferred indications include





IL-8 by by
of IL-8 are well known in the
immunological and inflammatory disorders





endothelial
art and may be used or
(e.g., such as allergy, asthma, leukemia,





cells (such as
routinely modified to assess the
etc. and as described below under “Immune





Human
ability of polypeptides of the
Activity”, and “Blood-Related Disorders”).





Umbilical Cord
invention (including antibodies
Highly preferred indications also includie





Endothelial
and agonists or antagonists of
autoimmune disorders (e.g., rheumatoid





Cells).
the invention) to regulate
arthritis, systemic lupus erythematosis,






production and/or secretion of
Crohn''s disease, multiple sclerosis and/or






IL-8. For example, FMAT may
as described below), neoplastic disorders






be used or routinely modified to
(e.g., organ cancers such as lung, liver,






assess the ability of
colon cancer, and/or as described below






polypeptides of the invention
under “Hyperproliferative Disorders”), and






(including antibodies and
cardiovascular disorders (e.g. such as






agonists or antagonists of the
described below under “Cardiovascular






invention) to regulate
Disorders”). Preferred indications include






production and/or secretion of
thrombosis, bacteremia and sepsis






IL-8 from endothelial cells
syndrome and consequent complications






(such as human umbilical vein
(such as acute respiratory distress






endothelial cells (HUVEC)).
syndrome and systemic ischemia-






HUVECs are endothelial cells
reperfusion resulting from septic shock),






which line venous blood
restnosis and atherosclerosis.






vessels, and are involved in







functions that include, but are







not limited to, angiogenesis,







vascular permeability, vascular







tone, and immune cell







extravasation. Endothelial cells







play a pivotal role in the







initiation and perpetuation of







inflammation and secretion of







IL-8 may play an important role







in recruitment and activation of







immune cells such as







neutrophils, macrophages, and







lymphocytes.



19
HWHGU54
132
SEAP in







HIB/CRE




19
HWHGU54
132
Proliferation of
Assays for the regulation (i.e.






pre-adipose
increases or decreases) of






cells (such as
viability and proliferation of






3T3-L1 cells)
cells in vitro are well-known in







the art and may be used or







routinely modified to assess the







ability of polypeptides of the







invention (including antibodies







and agonists or antagonists of







the invention) to regulate







viability and proliferation of







pre-adipose cells and cell lines.







For example, the CellTiter-







Gloô Luminescent Cell







Viability Assay (PROMEGA TM







Corp., Madison, WI, USA) can







be used to measure the number







of viable cells in culture based







on quantitation of the ATP







present which signals the







presence of metabolically







active cells. 3T3-L1 is a mouse







preadipocyte cell line. It is a







continuous substrain of 3T3







fibroblast cells developed







through clonal isolation. Cells







were differentiated to an







adipose-like state before being







used in the screen. See Green H







and Meuth M., Cell 3: 127-133







(1974), which is herein







incorporated by reference in its







entirety.



19
HWHGU54
132
MCP-1 in







HUVEC




21
HT5GJ57
162
Activation of
Assays for the activation of
Preferred indications include neoplastic





transcription
transcription through the AP1
diseases (e.g., as described below under





through AP1
response element are known in
“Hyperproliferative Disorders”), blood





response
the art and may be used or
disorders (e.g., as described below under





element in
routinely modified to assess the
“Immune Activity”, “Cardiovascular





immune cells
ability of polypeptides of the
Disorders”, and/or “Blood-Related





(such as T-
invention (including antibodies
Disorders”), and infection (e.g., an





cells).
and agonists or antagonists of
infectious disease as described below under






the invention) to modulate
“Infectious Disease”). Highly preferred






growth and other cell functions.
indications include autoimmune diseases






Exemplary assays for
(e.g., rheumatoid arthritis, systemic lupus






transcription through the AP1
erythematosis, multiple sclerosis and/or as






response element that may be
described below) and immunodeficiencies






used or routinely modified to
(e.g., as described below). Additional






test AP1-response element
highly preferred indications include






activity of polypeptides of the
inflammation and inflammatory disorders.






invention (including antibodies
Highly preferred indications also include






and agonists or antagonists of
neoplastic diseases (e.g., leukemia,






the invention) include assays
lymphoma, and/or as described below






disclosed in Berger et al., Gene
under “Hyperproliferative Disorders”).






66: 1-10 (1988); Cullen and
Highly preferred indications include






Malm, Methods in Enzymol
neoplasms and cancers, such as, leukemia,






216: 362-368 (1992); Henthorn
lymphoma, prostate, breast, lung, colon,






et al., Proc Natl Acad Sci USA
pancreatic, esophageal, stomach, brain,






85: 6342-6346 (1988); Rellahan
liver, and urinary cancer. Other preferred






et al., J Biol Chem
indications include benign dysproliferative






272(49): 30806-30811 (1997);
disorders and pre-neoplastic conditions,






Chang et al., Mol Cell Biol
such as, for example, hyperplasia,






18(9): 4986-4993 (1998); and
metaplasia, and/or dysplasia. Preferred






Fraser et al., Eur J Immunol
indications include arthritis, asthma, AIDS,






29(3): 838-844 (1999), the
allergy, anemia, pancytopenia, leukopenia,






contents of each of which are
thrombocytopenia, Hodgkin's disease,






herein incorporated by
acute lymphocytic anemia (ALL),






reference in its entirety. T cells
plasmacytomas, multiple myeloma,






that may be used according to
Burkitt's lymphoma, granulomatous






these assays are publicly
disease, inflammatory bowel disease,






available (e.g., through the
sepsis, psoriasis, suppression of immune






ATCC TM). Exemplary mouse T
reactions to transplanted organs and tissues,






cells that may be used
endocarditis, meningitis, and Lyme






according to these assays
Disease.






include the CTLL cell line,







which is an IL-2 dependent







suspension-culture cell line







with cytotoxic activity.



21
HT5GJ57
162
Production of
MCP-1 FMAT. Assays for
A highly preferred embodiment of the





MCP-1
immunomodulatory proteins
invention includes a method for stimulating






that are produced by a large
(e.g., increasing) MCP-1 production. An






variety of cells and act to
alternative highly preferred embodiment of






induce chemotaxis and
the invention includes a method for






activation of monocytes and T
inhibiting (e.g., reducing) MCP-1






cells are well known in the art
production. A highly preferred indication






and may be used or routinely
is infection (e.g., an infectious disease as






modified to assess the ability of
described below under “Infectious






polypeptides of the invention
Disease”). Additional highly preferred






(including antibodies and
indications include inflammation and






agonists or antagonists of the
inflammatory disorders. Preferred






invention) to mediate
indications include blood disorders (e.g., as






immunomodulation, induce
described below under “Immune Activity”,






chemotaxis, and modulate
“Blood-Related Disorders”, and/or






immune cell activation.
“Cardiovascular Disorders”). Highly






Exemplary assays that test for
preferred indications include autoimmune






immunomodulatory proteins
diseases (e.g., rheumatoid arthritis,






evaluate the production of cell
systemic lupus erythematosis, multiple






surface markers, such as
sclerosis and/or as described below) and






monocyte chemoattractant
immunodeficiencies (e.g., as described






protein (MCP), and the
below). Preferred indications also






activation of monocytes and T
include anemia, pancytopenia, leukopenia,






cells. Such assays that may be
thrombocytopenia, Hodgkin's disease,






used or routinely modified to
acute lymphocytic anemia (ALL),






test immunomodulatory and
plasmacytomas, multiple myeloma,






differentiation activity of
Burkitt's lymphoma, arthritis, AIDS,






polypeptides of the invention
granulomatous disease, inflammatory






(including antibodies and
bowel disease, sepsis, neutropenia,






agonists or antagonists of the
neutrophilia, psoriasis, suppression of






invention) include assays
immune reactions to transplanted organs






disclosed in Miraglia et al., J
and tissues, hemophilia, hypercoagulation,






Biomolecular Screening 4: 193-204
diabetes mellitus, endocarditis, meningitis






(1999); Rowland et al.,
(bacterial and viral), Lyme Disease,






“Lymphocytes: a practical
asthma, and allergy Preferred indications






approach” Chapter 6: 138-160
also include neoplastic diseases (e.g.,






(2000); Satthaporn and Eremin,
leukemia, lymphoma, and/or as described






J R Coll Surg Ednb 45(1): 9-19
below under “Hyperproliferative






(2001); and Verhasselt et al., J
Disorders”). Highly preferred indications






Immunol 158: 2919-2925
include neoplasms and cancers, such as,






(1997), the contents of each of
leukemia, lymphoma, prostate, breast, lung,






which are herein incorporated
colon, pancreatic, esophageal, stomach,






by reference in its entirety.
brain, liver, and urinary cancer. Other






Human dendritic cells that may
preferred indications include benign






be used according to these
dysproliferative disorders and pre-






assays may be isolated using
neoplastic conditions, such as, for example,






techniques disclosed herein or
hyperplasia, metaplasia, and/or dysplasia.






otherwise known in the art.







Human dendritic cells are







antigen presenting cells in







suspension culture, which,







when activated by antigen







and/or cytokines, initiate and







upregulate T cell proliferation







and functional activities.



21
HT5GJ57
162
Inhibition of
Reporter Assay: construct






squalene
contains regulatory and coding






synthetase
sequence of squalene






gene
synthetase, the first specific






transcription.
enzyme in the cholesterol







biosynthetic pathway. See







Jiang, et al., J. Biol. Chem.







268: 12818-128241(993), the







contents of which are herein







incorporated by reference in its







entirety. Cells were treated







with SID supernatants, and







SEAP activity was measured







after 72 hours. HepG2 is a







human hepatocellular







carcinoma cell line (ATCC TM







HB-8065). See Knowles et al.,







Science. 209: 497-9 (1980), the







contents of which are herein







incorporated by reference in its







entirety.



21
HT5GJ57
162
IgG in Human







B cells SAC




21
HT5GJ57
162
IL-10 in







Human T-cell







2B9




21
HT5GJ57
162
TNFa in







Human T-cell







2B9




21
HT5GJ57
162
Caspase







(+paclitaxel) in







SW480




27
HDHMA45
203
Production of
Assays for production of IL-10
Highly preferred indications include allergy





IL-10 and
and activation of T-cells are
and asthma. Additional highly preferred





activation of T-
well known in the art and may
indications include immune and





cells.
be used or routinely modified to
hematopoietic disorders (e.g., as described






assess the ability of
below under “Immune Activity”, and






polypeptides of the invention
“Blood-Related Disorders”), autoimmune






(including antibodies and
diseases (e.g., rheumatoid arthritis,






agonists or antagonists of the
systemic lupus erythematosis, Crohn''s






invention) to stimulate or
disease, multiple sclerosis and/or as






inhibit production of IL-10
described below), immunodeficiencies






and/or activation of T-cells.
(e.g., as described below), boosting a T






Exemplary assays that may be
cell-mediated immune response, and






used or routinely modified to
suppressing a T cell-mediated immune






assess the ability of
response.






polypeptides and antibodies of







the invention (including







agonists or antagonists of the







invention) to modulate IL-10







production and/or T-cell







proliferation include, for







example, assays such as







disclosed and/or cited in:







Robinson, D S, et al., “Th-2







cytokines in allergic disease”







Br Med Bull; 56 (4): 956-968







(2000), and Cohn, et al., “T-







helper type 2 cell-directed







therapy for asthma”







Pharmacology & Therapeutics;







88: 187-196 (2000); the







contents of each of which are







herein incorporated by







reference in their entirety.







Exemplary cells that may be







used according to these assays







include Th2 cells. IL10







secreted from Th2 cells may be







measured as a marker of Th2







cell activation. Th2 cells are a







class of T cells that secrete IL4,







IL10, IL13, IL5 and IL6.







Factors that induce







differentiation and activation of







Th2 cells play a major role in







the initiation and pathogenesis







of allergy and asthma. Primary







T helper 2 cells are generated







via in vitro culture under Th2







polarizing conditions using







peripheral blood lymphocytes







isolated from cord blood.



31
HDPPA04
225
Production of
MIP-1alpha FMAT. Assays for
A highly preferred embodiment of the





MIP1alpha
immunomodulatory proteins
invention includes a method for stimulating






produced by activated dendritic
MIP1a production. An alternative highly






cells that upregulate
preferred embodiment of the invention






monocyte/macrophage and T
includes a method for inhibiting (e.g.,






cell chemotaxis are well known
reducing) MIP1a production. A highly






in the art and may be used or
preferred indication is infection (e.g., an






routinely modified to assess the
infectious disease as described below under






ability of polypeptides of the
“Infectious Disease”). Preferred






invention (including antibodies
indications include blood disorders (e.g., as






and agonists or antagonists of
described below under “Immune Activity”,






the invention) to mediate
“Blood-Related Disorders”, and/or






immunomodulation, modulate
“Cardiovascular Disorders”). Highly






chemotaxis, and modulate T
preferred indications include autoimmune






cell differentiation. Exemplary
diseases (e.g., rheumatoid arthritis,






assays that test for
systemic lupus erythematosis, multiple






immunomodulatory proteins
sclerosis and/or as described below) and






evaluate the production of
immunodeficiencies (e.g., as described






chemokines, such as
below). Additional highly preferred






macrophage inflammatory
indications include inflammation and






protein 1 alpha (MIP-1a), and
inflammatory disorders. Preferred






the activation of
indications also include anemia,






monocytes/macrophages and T
pancytopenia, leukopenia,






cells. Such assays that may be
thrombocytopenia, Hodgkin's disease,






used or routinely modified to
acute lymphocytic anemia (ALL),






test immunomodulatory and
plasmacytomas, multiple myeloma,






chemotaxis activity of
Burkitt's lymphoma, arthritis, AIDS,






polypeptides of the invention
granulomatous disease, inflammatory






(including antibodies and
bowel disease, sepsis, neutropenia,






agonists or antagonists of the
neutrophilia, psoriasis, suppression of






invention) include assays
immune reactions to transplanted organs






disclosed in Miraglia et al., J
and tissues, hemophilia, hypercoagulation,






Biomolecular Screening 4: 193-204
diabetes mellitus, endocarditis, meningitis,






(1999); Rowland et al.,
Lyme Disease, asthma, and allergy.






“Lymphocytes: a practical
Preferred indications also include






approach” Chapter 6: 138-160
neoplastic diseases (e.g., leukemia,






(2000); Satthaporn and Eremin,
lymphoma, and/or as described below






J R Coll Surg Ednb 45(1): 9-19
under “Hyperproliferative Disorders”).






(2001); Drakes et al., Transp
Highly preferred indications include






Immunol 8(1): 17-29 (2000);
neoplasms and cancers, such as, leukemia,






Verhasselt et al., J Immunol
lymphoma, prostate, breast, lung, colon,






158: 2919-2925 (1997); and
pancreatic, esophageal, stomach, brain,






Nardelli et al., J Leukoc Biol
liver, and urinary cancer. Other preferred






65: 822-828 (1999), the contents
indications include benign dysproliferative






of each of which are herein
disorders and pre-neoplastic conditions,






incorporated by reference in its
such as, for example, hyperplasia,






entirety. Human dendritic cells
metaplasia, and/or dysplasia.






that may be used according to







these assays may be isolated







using techniques disclosed







herein or otherwise known in







the art. Human dendritic cells







are antigen presenting cells in







suspension culture, which,







when activated by antigen







and/or cytokines, initiate and







upregulate T cell proliferation







and functional activities.



33
HKABZ65
233
Production of
IL-6 FMAT. IL-6 is produced
A highly preferred embodiment of the





IL-6
by T cells and has strong effects
invention includes a method for stimulating






on B cells. IL-6 participates in
(e.g., increasing) IL-6 production. An






IL-4 induced IgE production
alternative highly preferred embodiment of






and increases IgA production
the invention includes a method for






(IgA plays a role in mucosal
inhibiting (e.g., reducing) IL-6 production.






immunity). IL-6 induces
A highly preferrred indication is the






cytotoxic T cells. Deregulated
stimulation or enhancement of mucosal






expression of IL-6 has been
immunity. Highly preferred indications






linked to autoimmune disease,
include blood disorders (e.g., as described






plasmacytomas, myelomas, and
below under “Immune Activity”, “Blood-






chronic hyperproliferative
Related Disorders”, and/or “Cardiovascular






diseases. Assays for
Disorders”), and infection (e.g., as






immunomodulatory and
described below under “Infectious






differentiation factor proteins
Disease”). Highly preferred indications






produced by a large variety of
include autoimmune diseases (e.g.,






cells where the expression level
rheumatoid arthritis, systemic lupus






is strongly regulated by
erythematosis, multiple sclerosis and/or as






cytokines, growth factors, and
described below) and immunodeficiencies






hormones are well known in the
(e.g., as described below). Highly






art and may be used or
preferred indications also include boosting






routinely modified to assess the
a B cell-mediated immune response and






ability of polypeptides of the
alternatively suppressing a B cell-mediated






invention (including antibodies
immune response. Highly preferred






and agonists or antagonists of
indications include inflammation and






the invention) to mediate
inflammatory disorders. Additional highly






immunomodulation and
preferred indications include asthma and






differentiation and modulate T
allergy. Highly preferred indications






cell proliferation and function.
include neoplastic diseases (e.g., myeloma,






Exemplary assays that test for
plasmacytoma, leukemia, lymphoma,






immunomodulatory proteins
melanoma, and/or as described below






evaluate the production of
under “Hyperproliferative Disorders”).






cytokines, such as IL-6, and the
Highly preferred indications include






stimulation and upregulation of
neoplasms and cancers, such as, myeloma,






T cell proliferation and
plasmacytoma, leukemia, lymphoma,






functional activities. Such
melanoma, and prostate, breast, lung,






assays that may be used or
colon, pancreatic, esophageal, stomach,






routinely modified to test
brain, liver and urinary cancer. Other






immunomodulatory and
preferred indications include benign






differentiation activity of
dysproliferative disorders and pre-






polypeptides of the invention
neoplastic conditions, such as, for example,






(including antibodies and
hyperplasia, metaplasia, and/or dysplasia.






agonists or antagonists of the
Preferred indications include anemia,






invention) include assays
pancytopenia, leukopenia,






disclosed in Miraglia et al., J
thrombocytopenia, Hodgkin's disease,






Biomolecular Screening 4: 193-204
acute lymphocytic anemia (ALL), multiple






(1999); Rowland et al.,
myeloma, Burkitt's lymphoma, arthritis,






“Lymphocytes: a practical
AIDS, granulomatous disease,






approach” Chapter 6: 138-160
inflammatory bowel disease, sepsis,






(2000); and Verhasselt et al., J
neutropenia, neutrophilia, psoriasis,






Immunol 158: 2919-2925
suppression of immune reactions to






(1997), the contents of each of
transplanted organs and tissues,






which are herein incorporated
hemophilia, hypercoagulation, diabetes






by reference in its entirety.
mellitus, endocarditis, meningitis, and






Human dendritic cells that may
Lyme Disease. An additonal preferred






be used according to these
indication is infection (e.g., an infectious






assays may be isolated using
disease as described below under






techniques disclosed herein or
“Infectious Disease”).






otherwise known in the art.







Human dendritic cells are







antigen presenting cells in







suspension culture, which,







when activated by antigen







and/or cytokines, initiate and







upregulate T cell proliferation







and functional activities.



33
HKABZ65
233
Activation of
Kinase assay. JNK and p38
A highly preferred embodiment of the





Endothelial
kinase assays for signal
invention includes a method for stimulating





Cell p38 or
transduction that regulate cell
endothelial cell growth. An alternative





JNK Signaling
proliferation, activation, or
highly preferred embodiment of the





Pathway.
apoptosis are well known in the
invention includes a method for inhibiting






art and may be used or
endothelial cell growth. A highly






routinely modified to assess the
preferred embodiment of the invention






ability of polypeptides of the
includes a method for stimulating






invention (including antibodies
endothelial cell proliferation. An alternative






and agonists or antagonists of
highly preferred embodiment of the






the invention) to promote or
invention includes a method for inhibiting






inhibit cell proliferation,
endothelial cell proliferation. A






activation, and apoptosis.
highly preferred embodiment of the






Exemplary assays for JNK and
invention includes a method for stimulating






p38 kinase activity that may be
apoptosis of endothelial cells. An






used or routinely modified to
alternative highly preferred embodiment of






test JNK and p38 kinase-
the invention includes a method for






induced activity of polypeptides
inhibiting (e.g., decreasing) apoptosis of






of the invention (including
endothelial cells. A highly preferred






antibodies and agonists or
embodiment of the invention includes a






antagonists of the invention)
method for stimulating (e.g., increasing)






include the assays disclosed in
endothelial cell activation. An alternative






Forrer et al., Biol Chem 379(8-9):
highly preferred embodiment of the






1101-1110 (1998); Gupta et
invention includes a method for inhibiting






al., Exp Cell Res 247(2): 495-504
(e.g., decreasing) the activation of and/or






(1999); Kyriakis J M,
inactivating endothelial cells. A






Biochem Soc Symp 64: 29-48
highly preferred embodiment of the






(1999); Chang and Karin,
invention includes a method for stimulating






Nature 410(6824): 37-40
angiogenisis. An alternative highly






(2001); and Cobb M H, Prog
preferred embodiment of the invention






Biophys Mol Biol 71(3-4): 479-500
includes a method for inhibiting






(1999); the contents of
angiogenesis. A highly preferred






each of which are herein
embodiment of the invention includes a






incorporated by reference in its
method for reducing cardiac hypertrophy.






entirety. Endothelial cells that
An alternative highly preferred






may be used according to these
embodiment of the invention includes a






assays are publicly available
method for inducing cardiac hypertrophy.






(e.g., through the ATCC TM).
Highly preferred indications include






Exemplary endothelial cells
neoplastic diseases (e.g., as described






that may be used according to
below under “Hyperproliferative






these assays include human
Disorders”), and disorders of the






umbilical vein endothelial cells
cardiovascular system (e.g., heart disease,






(HUVEC), which are
congestive heart failure, hypertension,






endothelial cells which line
aortic stenosis, cardiomyopathy, valvular






venous blood vessels, and are
regurgitation, left ventricular dysfunction,






involved in functions that
atherosclerosis and atherosclerotic vascular






include, but are not limited to,
disease, diabetic nephropathy, intracardiac






angiogenesis, vascular
shunt, cardiac hypertrophy, myocardial






permeability, vascular tone, and
infarction, chronic hemodynamic overload,






immune cell extravasation.
and/or as described below under







“Cardiovascular Disorders”). Highly







preferred indications include







cardiovascular, endothelial and/or







angiogenic disorders (e.g., systemic







disorders that affect vessels such as







diabetes mellitus, as well as diseases of the







vessels themselves, such as of the arteries,







capillaries, veins and/or lymphatics).







Highly preferred are indications that







stimulate angiogenesis and/or







cardiovascularization. Highly preferred are







indications that inhibit angiogenesis and/or







cardiovascularization. Highly preferred







indications include antiangiogenic activity







to treat solid tumors, leukemias, and







Kaposi''s sarcoma, and retinal disorders.







Highly preferred indications include







neoplasms and cancer, such as, Kaposi''s







sarcoma, hemangioma (capillary and







cavernous), glomus tumors, telangiectasia,







bacillary angiomatosis,







hemangioendothelioma, angiosarcoma,







haemangiopericytoma, lymphangioma,







lymphangiosarcoma. Highly preferred







indications also include cancers such as,







prostate, breast, lung, colon, pancreatic,







esophageal, stomach, brain, liver, and







urinary cancer. Preferred indications







include benign dysproliferative disorders







and pre-neoplastic conditions, such as, for







example, hyperplasia, metaplasia, and/or







dysplasia. Highly preferred indications







also include arterial disease, such as,







atherosclerosis, hypertension, coronary







artery disease, inflammatory vasculitides,







Reynaud''s disease and Reynaud''s







phenomenom, aneurysms, restenosis;







venous and lymphatic disorders such as







thrombophlebitis, lymphangitis, and







lymphedema; and other vascular disorders







such as peripheral vascular disease, and







cancer. Highly preferred indications







also include trauma such as wounds, burns,







and injured tissue (e.g., vascular injury







such as, injury resulting from balloon







angioplasty, and atheroschlerotic lesions),







implant fixation, scarring, ischemia







reperfusion injury, rheumatoid arthritis,







cerebrovascular disease, renal diseases such







as acute renal failure, and osteoporosis.







Additional highly preferred indications







include stroke, graft rejection, diabetic or







other retinopathies, thrombotic and







coagulative disorders, vascularitis, lymph







angiogenesis, sexual disorders, age-related







macular degeneration, and treatment/







prevention of endometriosis and related







conditions. Additional highly preferred







indications include fibromas, heart disease,







cardiac arrest, heart valve disease, and







vascular disease. Preferred indications







include blood disorders (e.g., as described







below under “Immune Activity”, “Blood-







Related Disorders”, and/or “Cardiovascular







Disorders”). Preferred indications include







autoimmune diseases (e.g., rheumatoid







arthritis, systemic lupus erythematosis,







multiple sclerosis and/or as described







below) and immunodeficiencies (e.g., as







described below). Additional preferred







indications include inflammation and







inflammatory disorders (such as acute and







chronic inflammatory diseases, e.g.,







inflammatory bowel disease and Crohn's







disease), and pain management.


33
HKABZ65
233
Regulation of
Caspase Apoptosis. Assays
A highly preferred indication is





apoptosis in
for caspase apoptosis are well
diabetes mellitus. An additional





pancreatic beta
known in the art and may be
highly preferred indication is a





cells.
used or routinely modified to
complication associated with diabetes (e.g.,






assess the ability of
diabetic retinopathy, diabetic nephropathy,






polypeptides of the invention
kidney disease (e.g., renal failure,






(including antibodies and
nephropathy and/or other diseases and






agonists or antagonists of the
disorders as described in the “Renal






invention) to promote caspase
Disorders” section below), diabetic






protease-mediated apoptosis.
neuropathy, nerve disease and nerve






Apoptosis in pancreatic beta is
damage (e.g., due to diabetic neuropathy),






associated with induction and
blood vessel blockage, heart disease,






progression of diabetes.
stroke, impotence (e.g., due to diabetic






Exemplary assays for caspase
neuropathy or blood vessel blockage),






apoptosis that may be used or
seizures, mental confusion, drowsiness,






routinely modified to test
nonketotic hyperglycemic-hyperosmolar






capase apoptosis activity of
coma, cardiovascular disease (e.g., heart






polypeptides of the invention
disease, atherosclerosis, microvascular






(including antibodies and
disease, hypertension, stroke, and other






agonists or antagonists of the
diseases and disorders as described in the






invention) include the assays
“Cardiovascular Disorders” section below),






disclosed in: Loweth, A C, et
dyslipidemia, endocrine disorders (as






al., FEBS Lett, 400(3): 285-8
described in the “Endocrine Disorders”






(1997); Saini, K S, et al.,
section below), neuropathy, vision






Biochem Mol Biol Int,
impairment (e.g., diabetic retinopathy and






39(6): 1229-36 (1996);
blindness), ulcers and impaired wound






Krautheim, A., et al., Br J
healing, and infection (e.g., infectious






Pharmacol, 129(4): 687-94
diseases and disorders as described in the






(2000); Chandra J, et al.,
“Infectious Diseases” section below,






Diabetes, 50 Suppl 1: S44-7
especially of the urinary tract and skin),






(2001); Suk K, et al., J
carpal tunnel syndrome and Dupuytren's






Immunol, 166(7): 4481-9
contracture). An additional highly






(2001); Tejedo J, et al., FEBS
preferred indication is obesity and/or






Lett, 459(2): 238-43 (1999);
complications associated with obesity.






Zhang, S., et al., FEBS Lett,
Additional highly preferred indications






455(3): 315-20 (1999); Lee et
include weight loss or alternatively, weight






al., FEBS Lett 485(2-3): 122-126
gain. Aditional highly preferred






(2000); Nor et al., J Vasc
indications are complications associated






Res 37(3): 209-218 (2000); and
with insulin resistance.






Karsan and Harlan, J







Atheroscler Thromb 3(2): 75-80







(1996); the contents of each







of which are herein







incorporated by reference in its







entirety. Pancreatic cells that







may be used according to these







assays are publicly available







(e.g., through the ATCC TM)







and/or may be routinely







generated. Exemplary







pancreatic cells that may be







used according to these assays







include RIN-m. RIN-m is a







rat adherent pancreatic beta cell







insulinoma cell line derived







from a radiation induced







transplantable rat islet cell







tumor. The cells produce and







secrete islet polypeptide







hormones, and produce insulin,







somatostatin, and possibly







glucagon. ATTC: #CRL-2057







Chick et al. Proc. Natl. Acad.







Sci. 1977 74: 628; AF et al.







Proc. Natl. Acad. Sci. 1980







77: 3519.









Tables 1E.1 and 1E.2 provide information related to biological activities for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Tables 1E.2 also provide information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column of Table 1E.1 (“Gene No.”) provides the gene number in the application for each clone identifier. The second column of Table 1E.1 (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Table 1A through Table 1D. The third column of Table 1E.1 (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column of Table 1E.1 (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides).
In Table 1E.2, each of the biological activities of Table 1E.1 are listed by “Biological Activity Number” and the corresponding “Biological Activity” and are followed by an “Exemplary Activity Assay” column and a “Preferred Indication” column; however, for some biological activities no “Exemplary Activity Assay” or “Preferred Indication” is given. The “Exemplary Activity Assay” column describes the biological activity listed in the column that precedes it and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The “Preferred Indication” column also refers to the biological activity listed in the preceding column and describes disease(s) or disorder(s) that may be detected, diagnosed, prevented, treated, or ameliorated by the nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof).
Tables 1E, 1E.1, and 1E.2 describe the use of, inter alia, FMAT technology for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system which provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound fluorophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for high throughput screening using fluorometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).
Tables 1E, 1E.1, and 1E.2 also describe the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998). Table 1D from U.S. patent application Ser. No. 10/472,532, filed Sep. 20, 2003, is herein incorporated by reference. Table 1D in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 238 to 582 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.










TABLE 1E.1







AA





SEQ



Gene
cDNA
ID



No.
Clone ID
NO: Y
Biological Activity


















3
HTEEB42
10
Biological Activity #119: Regulation of





transcription of Malic Enzyme in





hepatocytes


4
HEQCC55
16
Biological Activity #94: Production of





IL-13 and activation of T-cells.


4
HEQCC55
16
Biological Activity #103: Production 





of MCP-1


11
HLHFP03
79
Biological Activity #12: Activation of





T-Cell p38 or JNK Signaling Pathway.


11
HLHFP03
79
Biological Activity #12: Activation of





T-Cell p38 or JNK Signaling Pathway.


11
HLHFP03
79
Biological Activity #110: Production





of VCAM in endothelial cells (such as 





human umbilical vein endothelial cells





(HUVEC))


12
HHTLF25
84
Biological Activity #92: Production of IL-10





and downregulation of immune responses


13
HTADX17
93
Biological Activity #21: Activation of





transcription through GAS response element





in immune cells (such as T-cells).


13
HTADX17
93
Biological Activity #27: Activation of





transcription through NFAT response in





immune cells (such as T-cells).


13
HTADX17
93
Biological Activity #35: Activation of





transcription through NFKB response 





element in immune cells (such as T-cells).


14
HJACG02
105
Biological Activity #112: Proliferation 





of pre-adipose cells (such as 3T3-L1 cells)


14
HJACG02
105
Biological Activity #48: Activation or





inhibition of transcription through NFKB





response element in immune cells





(such as basophils).


17
HSDEK49
126
Biological Activity #41: Activation of 





transcription through serum response element





in immune cells (such as T-cells).


17
HSDEK49
126
Biological Activity #118: Regulation of 





transcription of Malic Enzyme in adipocytes


17
HSDEK49
126
Biological Activity #41: Activation of 





transcription through serum response element





in immune cells (such as T-cells).


17
HSDEK49
126
Biological Activity #118: Regulation of 





transcription of Malic Enzyme in adipocytes


17
HSDEK49
126
Biological Activity #84: MIP-1a in HMC


18
HWBAO62
128
Biological Activity #17: Activation of 





transcription through CD28 response element





in immune cells (such as T-cells).


19
HWHGU54
132
Biological Activity #16: Activation of 





transcription through cAMP response element





in immune cells (such as T-cells).


19
HWHGU54
132
Biological Activity #100: Production of 





IL-8 by endothelial cells (such as





Human Umbilical Cord Endothelial Cells).


21
HT5GJ57
160
Biological Activity #103: Production of 





MCP-1


21
HT5GJ57
160
Biological Activity #14: Activation of 





transcription through AP1 response





element in immune cells (such as T-cells).


21
HT5GJ57
162
Biological Activity #14: Activation of 





transcription through AP1 response





element in immune cells (such as T-cells).


21
HT5GJ57
162
Biological Activity #103: Production of 





MCP-1


27
HDHMA45
203
Biological Activity #91: Production of IL-10 





and activation of T-cells.


31
HDPPA04
225
Biological Activity #104: Production of 





MIP1alpha


33
HKABZ65
233
Biological Activity #5: Activation of 





Endothelial Cell p38 or JNK





Signaling Pathway.


33
HKABZ65
233
Biological Activity #98: Production of IL-6


33
HKABZ65
233
Biological Activity #115: Regulation of 





apoptosis in pancreatic beta cells.


33
HKABZ65
233
Biological Activity #98: Production of IL-6


33
HKABZ65
233
Biological Activity #5: Activation of





Endothelial Cell p38 or JNK Signaling





Pathway.


33
HKABZ65
233
Biological Activity #115: Regulation of





apoptosis in pancreatic beta cells.



















TABLE 1E.2





Biological





Activity





No.
Biological Activity
Exemplary Activity Assay
Preferred Indication


















1
Activation of
Kinase assay. Kinase assays, for example
A highly preferred embodiment of the invention



Adipocyte ERK
an Elk-1 kinase assay, for ERK signal
includes a method for stimulating adipocyte



Signaling Pathway
transduction that regulate cell proliferation
proliferation. An alternative highly preferred




or differentiation are well known in the art
embodiment of the invention includes a method




and may be used or routinely modified to
for inhibiting adipocyte proliferation. A




assess the ability of polypeptides of the
highly preferred embodiment of the invention




invention (including antibodies and agonists
includes a method for stimulating adipocyte




or antagonists of the invention) to promote
differentiation. An alternative highly preferred




or inhibit cell proliferation, activation, and
embodiment of the invention includes a method




differentiation. Exemplary assays for ERK
for inhibiting adipocyte differentiation. A




kinase activity that may be used or routinely
highly preferred embodiment of the invention




modified to test ERK kinase-induced
includes a method for stimulating (e.g.,




activity of polypeptides of the invention
increasing) adipocyte activation. An alternative




(including antibodies and agonists or
highly preferred embodiment of the invention




antagonists of the invention) include the
includes a method for inhibiting the activation




assays disclosed in Forrer et al., Biol Chem
of (e.g., decreasing) and/or inactivating




379(8-9): 1101-1110 (1998); Le Marchand-
adipocytes. Highly preferred indications




Brustel Y, Exp Clin Endocrinol Diabetes
include endocrine disorders (e.g., as described




107(2): 126-132 (1999); Kyriakis J M,
below under “Endocrine Disorders”).




Biochem Soc Symp 64: 29-48 (1999);
Highly preferred indications also include




Chang and Karin, Nature 410(6824): 37-40
neoplastic diseases (e.g., lipomas, liposarcomas,




(2001); and Cobb M H, Prog Biophys Mol
and/or as described below under




Biol 71(3-4): 479-500 (1999); the contents
“Hyperproliferative Disorders”). Preferred




of each of which are herein incorporated by
indications include blood disorders (e.g.,




reference in its entirety. Mouse adipocyte
hypertension, congestive heart failure, blood




cells that may be used according to these
vessel blockage, heart disease, stroke,




assays are publicly available (e.g., through
impotence and/or as described below under




the ATCC TM). Exemplary mouse adipocyte
“Immune Activity”, “Cardiovascular




cells that may be used according to these
Disorders”, and/or “Blood-Related Disorders”),




assays include 3T3-L1 cells. 3T3-L1 is an
immune disorders (e.g., as described below




adherent mouse preadipocyte cell line that is
under “Immune Activity”), neural disorders




a continuous substrain of 3T3 fibroblast
(e.g., as described below under “Neural Activity




cells developed through clonal isolation and
and Neurological Diseases”), and infection




undergo a pre-adipocyte to adipose-like
(e.g., as described below under “Infectious




conversion under appropriate differentiation
Disease”). A highly preferred




conditions known in the art.
indication is diabetes mellitus. An





additional highly preferred indication is a





complication associated with diabetes (e.g.,





diabetic retinopathy, diabetic nephropathy,





kidney disease (e.g., renal failure, nephropathy





and/or other diseases and disorders as described





in the “Renal Disorders” section below),





diabetic neuropathy, nerve disease and nerve





damage (e.g., due to diabetic neuropathy),





blood vessel blockage, heart disease, stroke,





impotence (e.g., due to diabetic neuropathy or





blood vessel blockage), seizures, mental





confusion, drowsiness, nonketotic





hyperglycemic-hyperosmolar coma,





cardiovascular disease (e.g., heart disease,





atherosclerosis, microvascular disease,





hypertension, stroke, and other diseases and





disorders as described in the “Cardiovascular





Disorders” section below), dyslipidemia,





endocrine disorders (as described in the





“Endocrine Disorders” section below),





neuropathy, vision impairment (e.g., diabetic





retinopathy and blindness), ulcers and impaired





wound healing, infection (e.g., infectious





diseases and disorders as described in the





“Infectious Diseases” section below





(particularly of the urinary tract and skin).





An additional highly preferred indication is





obesity and/or complications associated with





obesity. Additional highly preferred indications





include weight loss or alternatively, weight





gain. Additional highly preferred





indications are complications associated with





insulin resistance. Additional highly





preferred indications are disorders of the





musculoskeletal systems including myopathies,





muscular dystrophy, and/or as described herein.





Additional highly preferred indications include,





hypertension, coronary artery disease,





dyslipidemia, gallstones, osteoarthritis,





degenerative arthritis, eating disorders, fibrosis,





cachexia, and kidney diseases or disorders.





Preferred indications include neoplasms and





cancer, such as, lymphoma, leukemia and





breast, colon, and kidney cancer. Additional





preferred indications include melanoma,





prostate, lung, pancreatic, esophageal,





stomach, brain, liver, and urinary cancer.





Highly preferred indications include lipomas





and liposarcomas. Other preferred indications





include benign dysproliferative disorders and





pre-neoplastic conditions, such as, for example,





hyperplasia, metaplasia, and/or dysplasia.


2
Activation of
Kinase assay. Kinase assays, for example




Adipocyte PI3 Kinase
an GSK-3 assays, for PI3 kinase signal




Signaling Pathway
transduction that regulate glucose





metabolism and cell survival are well-





known in the art and may be used or





routinely modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to promote or inhibit glucose





metabolism and cell survival. Exemplary





assays for PI3 kinase activity that may be





used or routinely modified to test PI3





kinase-induced activity of polypeptides of





the invention (including antibodies and





agonists or antagonists of the invention)





include assays disclosed in Forrer et al.,





Biol Chem 379(8-9): 1101-1110 (1998);





Nikoulina et al., Diabetes 49(2): 263-271





(2000); and Schreyer et al., Diabetes





48(8): 1662-1666 (1999), the contents of





each of which are herein incorporated by





reference in its entirety. Mouse adipocyte





cells that may be used according to these





assays are publicly available (e.g., through





the ATCC TM). Exemplary mouse adipocyte





cells that may be used according to these





assays include 3T3-L1 cells. 3T3-L1 is an





adherent mouse preadipocyte cell line that is





a continuous substrain of 3T3 fibroblast





cells developed through clonal isolation and





undergo a pre-adipocyte to adipose-like





conversion under appropriate differentiation





conditions known in the art.



3
Activation of
Kinase assay. Kinase assays, for example an
A highly preferred embodiment of the invention



Endothelial Cell ERK
Elk-1 kinase assay, for ERK signal
includes a method for stimulating endothelial



Signaling Pathway.
transduction that regulate cell proliferation
cell growth. An alternative highly preferred




or differentiation are well known in the art
embodiment of the invention includes a method




and may be used or routinely modified to
for inhibiting endothelial cell growth. A




assess the ability of polypeptides of the
highly preferred embodiment of the invention




invention (including antibodies and agonists
includes a method for stimulating endothelial




or antagonists of the invention) to promote
cell proliferation. An alternative highly




or inhibit cell proliferation, activation, and
preferred embodiment of the invention includes




differentiation. Exemplary assays for ERK
a method for inhibiting endothelial cell




kinase activity that may be used or routinely
proliferation. A highly preferred




modified to test ERK kinase-induced
embodiment of the invention includes a method




activity of polypeptides of the invention
for stimulating apoptosis of endothelial cells.




(including antibodies and agonists or
An alternative highly preferred embodiment of




antagonists of the invention) include the
the invention includes a method for inhibiting




assays disclosed in Forrer et al., Biol Chem
(e.g., decreasing) apoptosis of endothelial cells.




379(8-9): 1101-1110 (1998); Berra et al.,
A highly preferred embodiment of the invention




Biochem Pharmacol 60(8): 1171-1178
includes a method for stimulating (e.g.,




(2000); Gupta et al., Exp Cell Res
increasing) endothelial cell activation. An




247(2): 495-504 (1999); Chang and Karin,
alternative highly preferred embodiment of the




Nature 410(6824): 37-40 (2001); and Cobb
invention includes a method for inhibiting the




MH, Prog Biophys Mol Biol 71(3-4): 479-500
activation of (e.g., decreasing) and/or




(1999); the contents of each of which
inactivating endothelial cells. A highly




are herein incorporated by reference in its
preferred embodiment of the invention includes




entirety. Endothelial cells that may be used
a method for stimulating endothelial cell




according to these assays are publicly
differentiation. An alternative highly preferred




available (e.g., through the ATCC TM).
embodiment of the invention includes a method




Exemplary endothelial cells that may be
for inhibiting endothelial cell differentiation.




used according to these assays include
A highly preferred embodiment of the invention




human umbilical vein endothelial cells
includes a method for stimulating angiogenisis.




(HUVEC), which are endothelial cells
An alternative highly preferred embodiment of




which line venous blood vessels, and are
the invention includes a method for inhibiting




involved in functions that include, but are
angiogenesis. A highly preferred




not limited to, angiogenesis, vascular
embodiment of the invention includes a method




permeability, vascular tone, and immune
for reducing cardiac hypertrophy. An




cell extravasation.
alternative highly preferred embodiment of the





invention includes a method for inducing





cardiac hypertrophy. Highly preferred





indications include neoplastic diseases (e.g., as





described below under “Hyperproliferative





Disorders”), and disorders of the cardiovascular





system (e.g., heart disease, congestive heart





failure, hypertension, aortic stenosis,





cardiomyopathy, valvular regurgitation, left





ventricular dysfunction, atherosclerosis and





atherosclerotic vascular disease, diabetic





nephropathy, intracardiac shunt, cardiac





hypertrophy, myocardial infarction, chronic





hemodynamic overload, and/or as described





below under “Cardiovascular Disorders”).





Highly preferred indications include





cardiovascular, endothelial and/or angiogenic





disorders (e.g., systemic disorders that affect





vessels such as diabetes mellitus, as well as





diseases of the vessels themselves, such as of





the arteries, capillaries, veins and/or





lymphatics). Highly preferred are indications





that stimulate angiogenesis and/or





cardiovascularization. Highly preferred are





indications that inhibit angiogenesis and/or





cardiovascularization. Highly preferred





indications include antiangiogenic activity to





treat solid tumors, leukemias, and Kaposi's





sarcoma, and retinal disorders. Highly preferred





indications include neoplasms and cancer, such





as, Kaposi's sarcoma, hemangioma (capillary





and cavernous), glomus tumors, telangiectasia,





bacillary angiomatosis, hemangioendothelioma,





angiosarcoma, haemangiopericytoma,





lymphangioma, lymphangiosarcoma. Highly





preferred indications also include cancers such





as, prostate, breast, lung, colon, pancreatic,





esophageal, stomach, brain, liver, and urinary





cancer. Preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia. Highly





preferred indications also include arterial





disease, such as, atherosclerosis, hypertension,





coronary artery disease, inflammatory





vasculitides, Reynaud's disease and Reynaud's





phenomenom, aneurysms, restenosis; venous





and lymphatic disorders such as





thrombophlebitis, lymphangitis, and





lymphedema; and other vascular disorders such





as peripheral vascular disease, and cancer.





Highly preferred indications also include





trauma such as wounds, burns, and injured





tissue (e.g., vascular injury such as, injury





resulting from balloon angioplasty, and





atheroschlerotic lesions), implant fixation,





scarring, ischemia reperfusion injury,





rheumatoid arthritis, cerebrovascular disease,





renal diseases such as acute renal failure, and





osteoporosis. Additional highly preferred





indications include stroke, graft rejection,





diabetic or other retinopathies, thrombotic and





coagulative disorders, vascularitis, lymph





angiogenesis, sexual disorders, age-related





macular degeneration, and treatment/





prevention of endometriosis and related





conditions. Additional highly preferred





indications include fibromas, heart disease,





cardiac arrest, heart valve disease, and vascular





disease. Preferred indications include





blood disorders (e.g., as described below under





“Immune Activity”, “Blood-Related





Disorders”, and/or ““Cardiovascular





Disorders””). Preferred indications include





autoimmune diseases (e.g., rheumatoid arthritis,





systemic lupus erythematosis, multiple sclerosis





and/or as described below) and





immunodeficiencies (e.g., as described below).





Additional preferred indications include





inflammation and inflammatory disorders (such





as acute and chronic inflammatory diseases,





e.g., inflammatory bowel disease and Crohn's





disease), and pain management.


4
Activation of
Kinase assay. JNK kinase assays for signal
A highly preferred embodiment of the invention



Endothelial Cell JNK
transduction that regulate cell proliferation,
includes a method for stimulating endothelial



Signaling Pathway.
activation, or apoptosis are well known in
cell growth. An alternative highly preferred




the art and may be used or routinely
embodiment of the invention includes a method




modified to assess the ability of
for inhibiting endothelial cell growth. A




polypeptides of the invention (including
highly preferred embodiment of the invention




antibodies and agonists or antagonists of the
includes a method for stimulating endothelial




invention) to promote or inhibit cell
cell proliferation. An alternative highly




proliferation, activation, and apoptosis.
preferred embodiment of the invention includes




Exemplary assays for JNK kinase activity
a method for inhibiting endothelial cell




that may be used or routinely modified to
proliferation. A highly preferred




test JNK kinase-induced activity of
embodiment of the invention includes a method




polypeptides of the invention (including
for stimulating apoptosis of endothelial cells.




antibodies and agonists or antagonists of the
An alternative highly preferred embodiment of




invention) include the assays disclosed in
the invention includes a method for inhibiting




Forrer et al., Biol Chem 379(8-9): 1101-1110
apoptosis of endothelial cells. A highly




(1998); Gupta et al., Exp Cell Res
preferred embodiment of the invention includes




247(2): 495-504 (1999); Kyriakis J M,
a method for stimulating endothelial cell




Biochem Soc Symp 64: 29-48 (1999);
activation. An alternative highly preferred




Chang and Karin, Nature 410(6824): 37-40
embodiment of the invention includes a method




(2001); and Cobb M H, Prog Biophys Mol
for inhibiting the activation of and/or




Biol 71(3-4): 479-500 (1999); the contents
inactivating endothelial cells. A highly




of each of which are herein incorporated by
preferred embodiment of the invention includes




reference in its entirety. Endothelial cells
a method for stimulating angiogenisis. An




that may be used according to these assays
alternative highly preferred embodiment of the




are publicly available (e.g., through the
invention includes a method for inhibiting




ATCC TM). Exemplary endothelial cells that
angiogenesis. A highly preferred




may be used according to these assays
embodiment of the invention includes a method




include human umbilical vein endothelial
for reducing cardiac hypertrophy. An




cells (HUVEC), which are endothelial cells
alternative highly preferred embodiment of the




which line venous blood vessels, and are
invention include a method for inducing cardiac




involved in functions that include, but are
hypertrophy. Highly preferred indications




not limited to, angiogenesis, vascular
include neoplastic diseases (e.g., as described




permeability, vascular tone, and immune
below under “Hyperproliferative Disorders”),




cell extravasation.
and disorders of the cardiovascular system (e.g.,





heart disease, congestive heart failure,





hypertension, aortic stenosis, cardiomyopathy,





valvular regurgitation, left ventricular





dysfunction, atherosclerosis and atherosclerotic





vascular disease, diabetic nephropathy,





intracardiac shunt, cardiac hypertrophy,





myocardial infarction, chronic hemodynamic





overload, and/or as described below under





“Cardiovascular Disorders”). Highly preferred





indications include cardiovascular, endothelial





and/or angiogenic disorders (e.g., systemic





disorders that affect vessels such as diabetes





mellitus, as well as diseases of the vessels





themselves, such as of the arteries, capillaries,





veins and/or lymphatics). Highly preferred are





indications that stimulate angiogenesis and/or





cardiovascularization. Highly preferred are





indications that inhibit angiogenesis and/or





cardiovascularization. Highly preferred





indications include antiangiogenic activity to





treat solid tumors, leukemias, and Kaposi''s





sarcoma, and retinal disorders. Highly preferred





indications include neoplasms and cancer, such





as, Kaposi''s sarcoma, hemangioma (capillary





and cavernous), glomus tumors, telangiectasia,





bacillary angiomatosis, hemangioendothelioma,





angiosarcoma, haemangiopericytoma,





lymphangioma, lymphangiosarcoma. Highly





preferred indications also include cancers such





as, prostate, breast, lung, colon, pancreatic,





esophageal, stomach, brain, liver, and urinary





cancer. Preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia. Highly





preferred indications also include arterial





disease, such as, atherosclerosis, hypertension,





coronary artery disease, inflammatory





vasculitides, Reynaud''s disease and Reynaud''s





phenomenom, aneurysms, restenosis; venous





and lymphatic disorders such as





thrombophlebitis, lymphangitis, and





lymphedema; and other vascular disorders such





as peripheral vascular disease, and cancer.





Highly preferred indications also include





trauma such as wounds, burns, and injured





tissue (e.g., vascular injury such as, injury





resulting from balloon angioplasty, and





atheroschlerotic lesions), implant fixation,





scarring, ischemia reperfusion injury,





rheumatoid arthritis, cerebrovascular disease,





renal diseases such as acute renal failure, and





osteoporosis. Additional highly preferred





indications include stroke, graft rejection,





diabetic or other retinopathies, thrombotic and





coagulative disorders, vascularitis, lymph





angiogenesis, sexual disorders, age-related





macular degeneration, and treatment/





prevention of endometriosis and related





conditions. Additional highly preferred





indications include fibromas, heart disease,





cardiac arrest, heart valve disease, and vascular





disease. Preferred indications include





blood disorders (e.g., as described below under





“Immune Activity”, “Blood-Related





Disorders”, and/or “Cardiovascular Disorders”).





Preferred indications include autoimmune





diseases (e.g., rheumatoid arthritis, systemic





lupus erythematosis, multiple sclerosis and/or





as described below) and immunodeficiencies





(e.g., as described below). Additional preferred





indications include inflammation and





inflammatory disorders (such as acute and





chronic inflammatory diseases, e.g.,





inflammatory bowel disease and Crohn's





disease), and pain management.


5
Activation of
Kinase assay. JNK and p38 kinase assays
A highly preferred embodiment of the invention



Endothelial Cell p38
for signal transduction that regulate cell
includes a method for stimulating endothelial



or JNK Signaling
proliferation, activation, or apoptosis are
cell growth. An alternative highly preferred



Pathway.
well known in the art and may be used or
embodiment of the invention includes a method




routinely modified to assess the ability of
for inhibiting endothelial cell growth. A




polypeptides of the invention (including
highly preferred embodiment of the invention




antibodies and agonists or antagonists of the
includes a method for stimulating endothelial




invention) to promote or inhibit cell
cell proliferation. An alternative highly




proliferation, activation, and apoptosis.
preferred embodiment of the invention includes




Exemplary assays for JNK and p38 kinase
a method for inhibiting endothelial cell




activity that may be used or routinely
proliferation. A highly preferred




modified to test JNK and p38 kinase-
embodiment of the invention includes a method




induced activity of polypeptides of the
for stimulating apoptosis of endothelial cells.




invention (including antibodies and agonists
An alternative highly preferred embodiment of




or antagonists of the invention) include the
the invention includes a method for inhibiting




assays disclosed in Forrer et al., Biol Chem
(e.g., decreasing) apoptosis of endothelial cells.




379(8-9): 1101-1110 (1998); Gupta et al.,
A highly preferred embodiment of the invention




Exp Cell Res 247(2): 495-504 (1999);
includes a method for stimulating (e.g.,




Kyriakis J M, Biochem Soc Symp 64: 29-48
increasing) endothelial cell activation. An




(1999); Chang and Karin, Nature
alternative highly preferred embodiment of the




410(6824): 37-40 (2001); and Cobb M H,
invention includes a method for inhibiting (e.g.,




Prog Biophys Mol Biol 71(3-4): 479-500
decreasing) the activation of and/or inactivating




(1999); the contents of each of which are
endothelial cells. A highly preferred




herein incorporated by reference in its
embodiment of the invention includes a method




entirety. Endothelial cells that may be used
for stimulating angiogenisis. An alternative




according to these assays are publicly
highly preferred embodiment of the invention




available (e.g., through the ATCC TMTM).
includes a method for inhibiting angiogenesis.




Exemplary endothelial cells that may be
A highly preferred embodiment of the invention




used according to these assays include
includes a method for reducing cardiac




human umbilical vein endothelial cells
hypertrophy. An alternative highly preferred




(HUVEC), which are endothelial cells
embodiment of the invention includes a method




which line venous blood vessels, and are
for inducing cardiac hypertrophy. Highly




involved in functions that include, but are
preferred indications include neoplastic diseases




not limited to, angiogenesis, vascular
(e.g., as described below under




permeability, vascular tone, and immune
“Hyperproliferative Disorders”), and disorders




cell extravasation.
of the cardiovascular system (e.g., heart disease,





congestive heart failure, hypertension, aortic





stenosis, cardiomyopathy, valvular





regurgitation, left ventricular dysfunction,





atherosclerosis and atherosclerotic vascular





disease, diabetic nephropathy, intracardiac





shunt, cardiac hypertrophy, myocardial





infarction, chronic hemodynamic overload,





and/or as described below under





“Cardiovascular Disorders”). Highly preferred





indications include cardiovascular, endothelial





and/or angiogenic disorders (e.g., systemic





disorders that affect vessels such as diabetes





mellitus, as well as diseases of the vessels





themselves, such as of the arteries, capillaries,





veins and/or lymphatics). Highly preferred are





indications that stimulate angiogenesis and/or





cardiovascularization. Highly preferred are





indications that inhibit angiogenesis and/or





cardiovascularization. Highly preferred





indications include antiangiogenic activity to





treat solid tumors, leukemias, and Kaposi's





sarcoma, and retinal disorders. Highly preferred





indications include neoplasms and cancer, such





as, Kaposi's sarcoma, hemangioma (capillary





and cavernous), glomus tumors, telangiectasia,





bacillary angiomatosis, hemangioendothelioma,





angiosarcoma, haemangiopericytoma,





lymphangioma, lymphangiosarcoma. Highly





preferred indications also include cancers such





as, prostate, breast, lung, colon, pancreatic,





esophageal, stomach, brain, liver, and urinary





cancer. Preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia. Highly





preferred indications also include arterial





disease, such as, atherosclerosis, hypertension,





coronary artery disease, inflammatory





vasculitides, Reynaud's disease and Reynaud's





phenomenom, aneurysms, restenosis; venous





and lymphatic disorders such as





thrombophlebitis, lymphangitis, and





lymphedema; and other vascular disorders such





as peripheral vascular disease, and cancer.





Highly preferred indications also include





trauma such as wounds, burns, and injured





tissue (e.g., vascular injury such as, injury





resulting from balloon angioplasty, and





atheroschlerotic lesions), implant fixation,





scarring, ischemia reperfusion injury,





rheumatoid arthritis, cerebrovascular disease,





renal diseases such as acute renal failure, and





osteoporosis. Additional highly preferred





indications include stroke, graft rejection,





diabetic or other retinopathies, thrombotic and





coagulative disorders, vascularitis, lymph





angiogenesis, sexual disorders, age-related





macular degeneration, and treatment/





prevention of endometriosis and related





conditions. Additional highly preferred





indications include fibromas, heart disease,





cardiac arrest, heart valve disease, and vascular





disease. Preferred indications include





blood disorders (e.g., as described below under





“Immune Activity”, “Blood-Related





Disorders”, and/or ““Cardiovascular





Disorders””). Preferred indications include





autoimmune diseases (e.g., rheumatoid arthritis,





systemic lupus erythematosis, multiple sclerosis





and/or as described below) and





immunodeficiencies (e.g., as described below).





Additional preferred indications include





inflammation and inflammatory disorders (such





as acute and chronic inflammatory diseases,





e.g., inflammatory bowel disease and Crohn's





disease), and pain management.


6
Activation of
Kinase assay. Kinase assays, for example
A highly preferred embodiment of the invention



Hepatocyte ERK
an Elk-1 kinase assay, for ERK signal
includes a method for stimulating hepatocyte



Signaling Pathway
transduction that regulate cell proliferation
cell proliferation. An alternative highly




or differentiation are well known in the art
preferred embodiment of the invention includes




and may be used or routinely modified to
a method for inhibiting hepatocyte cell




assess the ability of polypeptides of the
proliferation. A highly preferred




invention (including antibodies and agonists
embodiment of the invention includes a method




or antagonists of the invention) to promote
for stimulating hepatocyte cell differentiation.




or inhibit cell proliferation, activation, and
An alternative highly preferred embodiment of




differentiation. Exemplary assays for ERK
the invention includes a method for inhibiting




kinase activity that may be used or routinely
hepatocyte cell differentiation. A highly




modified to test ERK kinase-induced
preferred embodiment of the invention includes




activity of polypeptides of the invention
a method for activating hepatocyte cells. An




(including antibodies and agonists or
alternative highly preferred embodiment of the




antagonists of the invention) include the
invention includes a method for inhibiting the




assays disclosed in Forrer et al., Biol Chem
activation of and/or inactivating hepatocyte




379(8-9): 1101-1110 (1998); Kyriakis J M,
cells. Highly preferred indications include




Biochem Soc Symp 64: 29-48 (1999);
disorders of the liver and/or endocrine disorders




Chang and Karin, Nature 410(6824): 37-40
(e.g., as described below under “Endocrine




(2001); and Cobb M H, Prog Biophys Mol
Disorders”). Preferred indications include




Biol 71(3-4): 479-500 (1999); the contents
neoplastic diseases (e.g., as described below




of each of which are herein incorporated by
under “Hyperproliferative Disorders”), blood




reference in its entirety. Rat liver hepatoma
disorders (e.g., as described below under




cells that may be used according to these
“Immune Activity”, “Cardiovascular




assays are publicly available (e.g., through
Disorders”, and/or “Blood-Related Disorders”),




the ATCC TM). Exemplary rat liver
immune disorders (e.g., as described below




hepatoma cells that may be used according
under “Immune Activity”), neural disorders




to these assays include H4lle cells, which
(e.g., as described below under “Neural Activity




are known to respond to glucocorticoids,
and Neurological Diseases”), and infection




insulin, or cAMP derivatives.
(e.g., as described below under “Infectious





Disease”). A highly preferred





indication is diabetes mellitus. An





additional highly preferred indication is a





complication associated with diabetes (e.g.,





diabetic retinopathy, diabetic nephropathy,





kidney disease (e.g., renal failure, nephropathy





and/or other diseases and disorders as described





in the “Renal Disorders” section below),





diabetic neuropathy, nerve disease and nerve





damage (e.g., due to diabetic neuropathy),





blood vessel blockage, heart disease, stroke,





impotence (e.g., due to diabetic neuropathy or





blood vessel blockage), seizures, mental





confusion, drowsiness, nonketotic





hyperglycemic-hyperosmolar coma,





cardiovascular disease (e.g., heart disease,





atherosclerosis, microvascular disease,





hypertension, stroke, and other diseases and





disorders as described in the “Cardiovascular





Disorders” section below), dyslipidemia,





endocrine disorders (as described in the





“Endocrine Disorders” section below),





neuropathy, vision impairment (e.g., diabetic





retinopathy and blindness), ulcers and impaired





wound healing, infection (e.g., infectious





diseases and disorders as described in the





“Infectious Diseases” section below, especially





of the urinary tract and skin), carpal tunnel





syndrome and Dupuytren's contracture). An





additional highly preferred indication is obesity





and/or complications associated with obesity.





Additional highly preferred indications include





weight loss or alternatively, weight gain.





Additional highly preferred indications are





complications associated with insulin





resistance. Additonal highly preferred





indications are disorders of the musculoskeletal





systems including myopathies, muscular





dystrophy, and/or as described herein.





Additional highly preferred indications include,





hepatitis, jaundice, gallstones, cirrhosis of the





liver, degenerative or necrotic liver disease,





alcoholic liver diseases, fibrosis, liver





regeneration, metabolic disease, dyslipidemia





and chlolesterol metabolism.





Additional highly preferred indications include





neoplasms and cancers, such as,





hepatocarcinomas, other liver cancers, and





colon and pancreatic cancer. Preferred





indications also include prostate, breast, lung,





esophageal, stomach, brain, and urinary cancer.





Other preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia.


7
Activation of JNK
Kinase assay. JNK kinase assays for signal
Highly preferred indications include asthma,



Signaling Pathway in
transduction that regulate cell proliferation,
allergy, hypersensitivity reactions,



immune cells (such
activation, or apoptosis are well known in
inflammation, and inflammatory disorders.



as eosinophils).
the art and may be used or routinely
Additional highly preferred indications include




modified to assess the ability of
immune and hematopoietic disorders (e.g., as




polypeptides of the invention (including
described below under “Immune Activity”, and




antibodies and agonists or antagonists of the
“Blood-Related Disorders”), autoimmune




invention) to promote or inhibit cell
diseases (e.g., rheumatoid arthritis, systemic




proliferation, activation, and apoptosis.
lupus erythematosis, Crohn''s disease, multiple




Exemplary assays for JNK kinase activity
sclerosis and/or as described below),




that may be used or routinely modified to
immunodeficiencies (e.g., as described below).




test JNK kinase-induced activity of
Highly preferred indications also include




polypeptides of the invention (including
boosting or inhibiting immune cell




antibodies and agonists or antagonists of the
proliferation. Preferred indications include




invention) include the assays disclosed in
neoplastic diseases (e.g., leukemia, lymphoma,




Forrer et al., Biol Chem 379(8-9): 1101-1110
and/or as described below under




(1998); Gupta et al., Exp Cell Res
“Hyperproliferative Disorders”). Highly




247(2): 495-504 (1999); Kyriakis J M,
preferred indications include boosting an




Biochem Soc Symp 64: 29-48 (1999);
eosinophil-mediated immune response, and




Chang and Karin, Nature 410(6824): 37-40
suppressing an eosinophil-mediated immune




(2001); and Cobb M H, Prog Biophys Mol
response.




Biol 71(3-4): 479-500 (1999); the contents





of each of which are herein incorporated by





reference in its entirety. Exemplary cells





that may be used according to these assays





include eosinophils. Eosinophils are





important in the late stage of allergic





reactions; they are recruited to tissues and





mediate the inflammatory response of late





stage allergic reaction. Moreover,





exemplary assays that may be used or





routinely modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to modulate signal transduction,





cell proliferation, activation, or apoptosis in





eosinophils include assays disclosed and/or





cited in: Zhang J P, et al., “Role of caspases





in dexamethasone-induced apoptosis and





activation of c-Jun NH2-terminal kinase and





p38 mitogen-activated protein kinase in





human eosinophils” Clin Exp Immunol;





October; 122(1): 20-7 (2000); Hebestreit H, et al.,





“Disruption of fas receptor signaling by





nitric oxide in eosinophils” J Exp Med; February





2; 187(3): 415-25 (1998); J Allergy Clin





Immunol 1999 September; 104(3 Pt 1): 565-74; and,





Sousa A R, et al., “In vivo resistance to





corticosteroids in bronchial asthma is





associated with enhanced phosyphorylation





of JUN N-terminal kinase and failure of





prednisolone to inhibit JUN N-terminal





kinase phosphorylation” J Allergy Clin





Immunol; September; 104(3 Pt 1): 565-74 (1999);





the contents of each of which are herein





incorporated by reference in its entirety.



8
Activation of Natural
Kinase assay. Kinase assays, for example
A highly preferred embodiment of the invention



Killer Cell ERK
an Elk-1 kinase assay, for ERK signal
includes a method for stimulating natural killer



Signaling Pathway.
transduction that regulate cell proliferation
cell proliferation. An alternative highly




or differentiation are well known in the art
preferred embodiment of the invention includes




and may be used or routinely modified to
a method for inhibiting natural killer cell




assess the ability of polypeptides of the
proliferation. A highly preferred




invention (including antibodies and agonists
embodiment of the invention includes a method




or antagonists of the invention) to promote
for stimulating natural killer cell differentiation.




or inhibit cell proliferation, activation, and
An alternative highly preferred embodiment of




differentiation. Exemplary assays for ERK
the invention includes a method for inhibiting




kinase activity that may be used or routinely
natural killer cell differentiation. Highly




modified to test ERK kinase-induced
preferred indications include neoplastic diseases




activity of polypeptides of the invention
(e.g., as described below under




(including antibodies and agonists or
“Hyperproliferative Disorders”), blood




antagonists of the invention) include the
disorders (e.g., as described below under




assays disclosed in Forrer et al., Biol Chem
“Immune Activity”, “Cardiovascular




379(8-9): 1101-1110 (1998); Kyriakis J M,
Disorders”, and/or “Blood-Related Disorders”),




Biochem Soc Symp 64: 29-48 (1999);
immune disorders (e.g., as described below




Chang and Karin, Nature 410(6824): 37-40
under “Immune Activity”) and infections (e.g.,




(2001); and Cobb M H, Prog Biophys Mol
as described below under “Infectious Disease”).




Biol 71(3-4): 479-500 (1999); the contents
Preferred indications include blood disorders




of each of which are herein incorporated by
(e.g., as described below under “Immune




reference in its entirety. Natural killer cells
Activity”, “Blood-Related Disorders”, and/or




that may be used according to these assays
“Cardiovascular Disorders”). Highly preferred




are publicly available (e.g., through the
indications include autoimmune diseases (e.g.,




ATCC TM). Exemplary natural killer cells
rheumatoid arthritis, systemic lupus




that may be used according to these assays
erythematosis, multiple sclerosis and/or as




include the human natural killer cell lines
described below) and immunodeficiencies (e.g.,




(for example, NK-YT cells which have
as described below). Additional highly




cytolytic and cytotoxic activity) or primary
preferred indications include inflammation and




NK cells.
inflammatory disorders. Highly preferred





indications also include cancers such as, kidney,





melanoma, prostate, breast, lung, colon,





pancreatic, esophageal, stomach, brain, liver,





urinary cancer, lymphoma and leukemias. Other





preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia. Other highly





preferred indications include, pancytopenia,





leukopenia, leukemias, Hodgkin's disease,





acute lymphocytic anemia (ALL), arthritis,





asthma, AIDS, granulomatous disease,





inflammatory bowel disease, sepsis, psoriasis,





immune reactions to transplanted organs and





tissues, endocarditis, meningitis, Lyme Disease,





and allergies.


9
Activation of Skeletal
Kinase assay. Kinase assays, for example
A highly preferred embodiment of the invention



Mucle Cell PI3
an GSK-3 kinase assay, for PI3 kinase
includes a method for increasing muscle cell



Kinase Signalling
signal transduction that regulate glucose
survival An alternative highly preferred



Pathway
metabolism and cell survivial are well-
embodiment of the invention includes a method




known in the art and may be used or
for decreasing muscle cell survival. A




routinely modified to assess the ability of
preferred embodiment of the invention includes




polypeptides of the invention (including
a method for stimulating muscle cell




antibodies and agonists or antagonists of the
proliferation. In a specific embodiment,




invention) to promote or inhibit glucose
skeletal muscle cell proliferation is stimulated.




metabolism and cell survival. Exemplary
An alternative highly preferred embodiment of




assays for PI3 kinase activity that may be
the invention includes a method for inhibiting




used or routinely modified to test PI3
muscle cell proliferation. In a specific




kinase-induced activity of polypeptides of
embodiment, skeletal muscle cell proliferation




the invention (including antibodies and
is inhibited. A preferred embodiment of the




agonists or antagonists of the invention)
invention includes a method for stimulating




include assays disclosed in Forrer et al.,
muscle cell differentiation. In a specific




Biol Chem 379(8-9): 1101-1110 (1998);
embodiment, skeletal muscle cell differentiation




Nikoulina et al., Diabetes 49(2): 263-271
is stimulated. An alternative highly preferred




(2000); and Schreyer et al., Diabetes
embodiment of the invention includes a method




48(8): 1662-1666 (1999), the contents of
for inhibiting muscle cell differentiation. In a




each of which are herein incorporated by
specific embodiment, skeletal muscle cell




reference in its entirety. Rat myoblast cells
differentiation is inhibited. Highly preferred




that may be used according to these assays
indications include disorders of the




are publicly available (e.g., through the
musculoskeletal system. Preferred indications




ATCC TM). Exemplary rat myoblast cells
include neoplastic diseases (e.g., as described




that may be used according to these assays
below under “Hyperproliferative Disorders”),




include L6 cells. L6 is an adherent rat
endocrine disorders (e.g., as described below




myoblast cell line, isolated from primary
under ““Endocrine Disorders””), neural




cultures of rat thigh muscle, that fuses to
disorders (e.g., as described below under




form multinucleated myotubes and striated
““Neural Activity and Neurological




fibers after culture in differentiation media.
Diseases””), blood disorders (e.g., as described





below under “Immune Activity”,





“Cardiovascular Disorders”, and/or “Blood-





Related Disorders”), immune disorders (e.g., as





described below under ““Immune Activity””),





and infection (e.g., as described below under





“Infectious Disease”). A highly preferred





indication is diabetes mellitus. An





additional highly preferred indication is a





complication associated with diabetes (e.g.,





diabetic retinopathy, diabetic nephropathy,





kidney disease (e.g., renal failure, nephropathy





and/or other diseases and disorders as described





in the ““Renal Disorders”” section below),





diabetic neuropathy, nerve disease and nerve





damage (e.g, due to diabetic neuropathy), blood





vessel blockage, heart disease, stroke,





impotence (e.g., due to diabetic neuropathy or





blood vessel blockage), seizures, mental





confusion, drowsiness, nonketotic





hyperglycemic-hyperosmolar coma,





cardiovascular disease (e.g., heart disease,





atherosclerosis, microvascular disease,





hypertension, stroke, and other diseases and





disorders as described in the ““Cardiovascular





Disorders”” section below), dyslipidemia,





endocrine disorders (as described in the





““Endocrine Disorders”” section below),





neuropathy, vision impairment (e.g., diabetic





retinopathy and blindness), ulcers and impaired





wound healing, infections (e.g., infectious





diseases and disorders as described in the





““Infectious Diseases”” section below,





especially of the urinary tract and skin), carpal





tunnel syndrome and Dupuytren's contracture).





An additional highly preferred indication is





obesity and/or complications associated with





obesity. Additional highly preferred indications





include weight loss or alternatively, weight





gain. Additional highly preferred





indications are complications associated with





insulin resistance. Additonal highly





preferred indications are disorders of the





musculoskeletal system including myopathies,





muscular dystrophy, and/or as described herein.





Additional highly preferred indications include:





myopathy, atrophy, congestive heart failure,





cachexia, myxomas, fibromas, congenital





cardiovascular abnormalities, heart disease,





cardiac arrest, heart valve disease, and vascular





disease. Highly preferred indications





include neoplasms and cancer, such as,





rhabdomyoma, rhabdosarcoma, stomach,





esophageal, prostate, and urinary cancer.





Preferred indications also include breast, lung,





colon, pancreatic, brain, and liver cancer. Other





preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, hyperplasia, metaplasia,





and/or dysplasia.


10
Activation of Skeletal
Kinase assay. Kinase assays, for example




Muscle Cell ERK
Elk-1 kinase assays, for ERK signal




Signaling Pathway
transduction that regulate cell proliferation





or differentiation are well known in the art





and may be used or routinely modified to





assess the ability of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) to promote





or inhibit cell proliferation, activation, and





differentiation. Exemplary assays for ERK





kinase activity that may be used or routinely





modified to test ERK kinase-induced





activity of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) include the





assays disclosed in Forrer et al., Biol Chem





379(8-9): 1101-1110 (1998); Le Marchand-





Brustel Y, Exp Clin Endocrinol Diabetes





107(2): 126-132 (1999); Kyriakis J M,





Biochem Soc Symp 64: 29-48 (1999);





Chang and Karin, Nature 410(6824): 37-40





(2001); and Cobb M H, Prog Biophys Mol





Biol 71(3-4): 479-500 (1999); the contents





of each of which are herein incorporated by





reference in its entirety. Rat myoblast cells





that may be used according to these assays





are publicly available (e.g., through the





ATCC TM). Exemplary rat myoblast cells





that may be used according to these assays





include L6 cells. L6 is an adherent rat





myoblast cell line, isolated from primary





cultures of rat thigh muscle, that fuses to





form multinucleated myotubes and striated





fibers after culture in differentiation media.



11
Activation of Skeletal
Kinase assay. Kinase assays, for example




Muscle Cell PI3
an GSK-3 kinase assay, for PI3 kinase




Kinase Signaling
signal transduction that regulate glucose




Pathway
metabolism and cell survival are well-





known in the art and may be used or





routinely modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to promote or inhibit glucose





metabolism and cell survival. Exemplary





assays for PI3 kinase activity that may be





used or routinely modified to test PI3





kinase-induced activity of polypeptides of





the invention (including antibodies and





agonists or antagonists of the invention)





include assays disclosed in Forrer et al.,





Biol Chem 379(8-9): 1101-1110 (1998);





Nikoulina et al., Diabetes 49(2): 263-271





(2000); and Schreyer et al., Diabetes





48(8): 1662-1666 (1999), the contents of





each of which are herein incorporated by





reference in its entirety. Rat myoblast cells





that may be used according to these assays





are publicly available (e.g., through the





ATCC TM). Exemplary rat myoblast cells





that may be used according to these assays





include L6 cells. L6 is an adherent rat





myoblast cell line, isolated from primary





cultures of rat thigh muscle, that fuses to





form multinucleated myotubes and striated





fibers after culture in differentiation media.



12
Activation of T-Cell
Kinase assay. JNK and p38 kinase assays
Preferred indications include neoplastic



p38 or JNK Signaling
for signal transduction that regulate cell
diseases (e.g., as described below under



Pathway.
proliferation, activation, or apoptosis are
“Hyperproliferative Disorders”), blood




well known in the art and may be used or
disorders (e.g., as described below under




routinely modified to assess the ability of
“Immune Activity”, “Cardiovascular




polypeptides of the invention (including
Disorders”, and/or “Blood-Related Disorders”),




antibodies and agonists or antagonists of the
and infection (e.g., an infectious disease as




invention) to promote or inhibit immune
described below under “Infectious Disease”).




cell (e.g. T-cell) proliferation, activation,
Highly preferred indications include




and apoptosis. Exemplary assays for JNK
autoimmune diseases (e.g., rheumatoid arthritis,




and p38 kinase activity that may be used or
systemic lupus erythematosis, multiple sclerosis




routinely modified to test JNK and p38
and/or as described below) and




kinase-induced activity of polypeptides of
immunodeficiencies (e.g., as described below).




the invention (including antibodies and
Additional highly preferred indications include




agonists or antagonists of the invention)
inflammation and inflammatory disorders.




include the assays disclosed in Forrer et al.,
Highly preferred indications also include




Biol Chem 379(8-9): 1101-1110 (1998);
neoplastic diseases (e.g., leukemia, lymphoma,




Gupta et al., Exp Cell Res 247(2): 495-504
and/or as described below under




(1999); Kyriakis J M, Biochem Soc Symp
“Hyperproliferative Disorders”). Highly




64: 29-48 (1999); Chang and Karin, Nature
preferred indications include neoplasms and




410(6824): 37-40 (2001); and Cobb M H,
cancers, such as, leukemia, lymphoma, prostate,




Prog Biophys Mol Biol 71(3-4): 479-500
breast, lung, colon, pancreatic, esophageal,




(1999); the contents of each of which are
stomach, brain, liver, and urinary cancer. Other




herein incorporated by reference in its
preferred indications include benign




entirety. T cells that may be used according
dysproliferative disorders and pre-neoplastic




to these assays are publicly available (e.g.,
conditions, such as, for example, hyperplasia,




through the ATCC TM). Exemplary mouse T
metaplasia, and/or dysplasia. Preferred




cells that may be used according to these
indications include arthritis, asthma, AIDS,




assays include the CTLL cell line, which is
allergy, anemia, pancytopenia, leukopenia,




an IL-2 dependent suspension-culture cell
thrombocytopenia, Hodgkin''s disease, acute




line with cytotoxic activity.
lymphocytic anemia (ALL), plasmacytomas,





multiple myeloma, Burkitt''s lymphoma,





granulomatous disease, inflammatory bowel





disease, sepsis, psoriasis, suppression of





immune reactions to transplanted organs and





tissues, endocarditis, meningitis, and Lyme





Disease.


13
Activation of
Assays for activation of transcription are




Transcription
well-known in the art and may be used and





routinely modified to assess ability of





polypeptides of the invention to inhibit or





activate transcription. An example of such





an assay follows: Cells were pretreated with





SID supernatants or controls for 15-18





hours. SEAP activity was measured after





48 hours. LS174T is an epithelial colon





adenocarcinoma cell line. Its





tumourigenicity in nude mice make cell line





LS174T a model for studies on the





mechanism of synthesis and secretion of





specific tumoral markers in colon cancer.





See, Patan et al., Circ Res, 89(8): 732-39





(2001), the contents of which are herein





incorporated by reference in its entirety.



14
Activation of
Assays for the activation of transcription
Preferred indications include neoplastic



transcription through
through the AP1 response element are
diseases (e.g., as described below under



AP1 response
known in the art and may be used or
“Hyperproliferative Disorders”), blood



element in immune
routinely modified to assess the ability of
disorders (e.g., as described below under



cells (such as T-
polypeptides of the invention (including
“Immune Activity”, “Cardiovascular



cells).
antibodies and agonists or antagonists of the
Disorders”, and/or “Blood-Related Disorders”),




invention) to modulate growth and other
and infection (e.g., an infectious disease as




cell functions. Exemplary assays for
described below under “Infectious Disease”).




transcription through the AP1 response
Highly preferred indications include




element that may be used or routinely
autoimmune diseases (e.g., rheumatoid arthritis,




modified to test AP1-response element
systemic lupus erythematosis, multiple sclerosis




activity of polypeptides of the invention
and/or as described below) and




(including antibodies and agonists or
immunodeficiencies (e.g., as described below).




antagonists of the invention) include assays
Additional highly preferred indications include




disclosed in Berger et al., Gene 66: 1-10
inflammation and inflammatory disorders.




(1988); Cullen and Malm, Methods in
Highly preferred indications also include




Enzymol 216: 362-368 (1992); Henthorn et
neoplastic diseases (e.g., leukemia, lymphoma,




al., Proc Natl Acad Sci USA 85: 6342-6346
and/or as described below under




(1988); Rellahan et al., J Biol Chem
“Hyperproliferative Disorders”). Highly




272(49): 30806-30811 (1997); Chang et al.,
preferred indications include neoplasms and




Mol Cell Biol 18(9): 4986-4993 (1998); and
cancers, such as, leukemia, lymphoma, prostate,




Fraser et al., Eur J Immunol 29(3): 838-844
breast, lung, colon, pancreatic, esophageal,




(1999), the contents of each of which are
stomach, brain, liver, and urinary cancer. Other




herein incorporated by reference in its
preferred indications include benign




entirety. T cells that may be used according
dysproliferative disorders and pre-neoplastic




to these assays are publicly available (e.g.,
conditions, such as, for example, hyperplasia,




through the ATCC TM). Exemplary mouse T
metaplasia, and/or dysplasia. Preferred




cells that may be used according to these
indications include arthritis, asthma, AIDS,




assays include the CTLL cell line, which is
allergy, anemia, pancytopenia, leukopenia,




an IL-2 dependent suspension-culture cell
thrombocytopenia, Hodgkin's disease, acute




line with cytotoxic activity. Additional
lymphocytic anemia (ALL), plasmacytomas,




exemplary mouse T cells that may be used
multiple myeloma, Burkitt's lymphoma,




according to these assays include the HT2
granulomatous disease, inflammatory bowel




cell line, which is an IL-2 dependent
disease, sepsis, psoriasis, suppression of




suspension culture cell line that also
immune reactions to transplanted organs and




responds to IL-4. Exemplary human T cells
tissues, endocarditis, meningitis, and Lyme




that may be used according to these assays
Disease.




include the SUPT cell line, which is an IL-2





and IL-4 responsive suspension-culture cell





line.



15
Activation of
Assays for the activation of transcription
A highly preferred indication is obesity and/or



transcription through
through the cAMP response element are
complications associated with obesity.



cAMP response
well-known in the art and may be used or
Additional highly preferred indications include



element (CRE) in
routinely modified to assess the ability of
weight loss or alternatively, weight gain. An



pre-adipocytes.
polypeptides of the invention (including
additional highly preferred indication is




antibodies and agonists or antagonists of the
diabetes mellitus. An additional highly




invention) to increase cAMP, regulate
preferred indication is a complication




CREB transcription factors, and modulate
associated with diabetes (e.g., diabetic




expression of genes involved in a wide
retinopathy, diabetic nephropathy, kidney




variety of cell functions. For example, a
disease (e.g., renal failure, nephropathy and/or




3T3-L1/CRE reporter assay may be used to
other diseases and disorders as described in the




identify factors that activate the cAMP
“Renal Disorders” section below), diabetic




signaling pathway. CREB plays a major
neuropathy, nerve disease and nerve damage




role in adipogenesis, and is involved in
(e.g., due to diabetic neuropathy), blood vessel




differentiation into adipocytes. CRE
blockage, heart disease, stroke, impotence (e.g.,




contains the binding sequence for the
due to diabetic neuropathy or blood vessel




transcription factor CREB (CRE binding
blockage), seizures, mental confusion,




protein). Exemplary assays for transcription
drowsiness, nonketotic hyperglycemic-




through the cAMP response element that
hyperosmolar coma, cardiovascular disease




may be used or routinely modified to test
(e.g., heart disease, atherosclerosis,




cAMP-response element activity of
microvascular disease, hypertension, stroke,




polypeptides of the invention (including
and other diseases and disorders as described in




antibodies and agonists or antagonists of the
the “Cardiovascular Disorders” section below),




invention) include assays disclosed in
dyslipidemia, endocrine disorders (as described




Berger et al., Gene 66: 1-10 (1998); Cullen
in the “Endocrine Disorders” section below),




and Malm, Methods in Enzymol 216: 362-368
neuropathy, vision impairment (e.g., diabetic




(1992); Henthorn et al., Proc Natl Acad
retinopathy and blindness), ulcers and impaired




Sci USA 85: 6342-6346 (1988); Reusch et
wound healing, and infection (e.g., infectious




al., Mol Cell Biol 20(3): 1008-1020 (2000);
diseases and disorders as described in the




and Klemm et al., J Biol Chem 273: 917-923
“Infectious Diseases” section below, especially




(1998), the contents of each of which are
of the urinary tract and skin), carpal tunnel




herein incorporated by reference in its
syndrome and Dupuytren's contracture).




entirety. Pre-adipocytes that may be used
Additional highly preferred indications are




according to these assays are publicly
complications associated with insulin




available (e.g., through the ATCC TM) and/or
resistance.




may be routinely generated. Exemplary





mouse adipocyte cells that may be used





according to these assays include 3T3-L1





cells. 3T3-L1 is an adherent mouse





preadipocyte cell line that is a continuous





substrain of 3T3 fibroblast cells developed





through clonal isolation and undergo a pre-





adipocyte to adipose-like conversion under





appropriate differentiation conditions





known in the art.



16
Activation of
Assays for the activation of transcription
Preferred indications include blood disorders



transcription through
through the cAMP response element are
(e.g., as described below under “Immune



cAMP response
well-known in the art and may be used or
Activity”, “Blood-Related Disorders”, and/or



element in immune
routinely modified to assess the ability of
“Cardiovascular Disorders”), and infection



cells (such as T-
polypeptides of the invention (including
(e.g., an infectious disease as described below



cells).
antibodies and agonists or antagonists of the
under “Infectious Disease”). Preferred




invention) to increase cAMP, bind to CREB
indications include autoimmune diseases (e.g.,




transcription factor, and modulate
rheumatoid arthritis, systemic lupus




expression of genes involved in a wide
erythematosis, multiple sclerosis and/or as




variety of cell functions. Exemplary assays
described below), immunodeficiencies (e.g., as




for transcription through the cAMP
described below), boosting a T cell-mediated




response element that may be used or
immune response, and suppressing a T cell-




routinely modified to test cAMP-response
mediated immune response. Additional




element activity of polypeptides of the
preferred indications include inflammation and




invention (including antibodies and agonists
inflammatory disorders. Highly preferred




or antagonists of the invention) include
indications include neoplastic diseases (e.g.,




assays disclosed in Berger et al., Gene 66: 1-10
leukemia, lymphoma, and/or as described




(1998); Cullen and Malm, Methods in
below under “Hyperproliferative Disorders”).




Enzymol 216: 362-368 (1992); Henthorn et
Highly preferred indications include neoplasms




al., Proc Natl Acad Sci USA 85: 6342-6346
and cancers, such as, leukemia, lymphoma (e.g.,




(1988); Black et al., Virus Genes 15(2): 105-117
T cell lymphoma, Burkitt's lymphoma, non-




(1997); and Belkowski et al., J
Hodgkins lymphoma, Hodgkin''s disease),




Immunol 161(2): 659-665 (1998), the
melanoma, and prostate, breast, lung, colon,




contents of each of which are herein
pancreatic, esophageal, stomach, brain, liver




incorporated by reference in its entirety. T
and urinary cancer. Other preferred indications




cells that may be used according to these
include benign dysproliferative disorders and




assays are publicly available (e.g., through
pre-neoplastic conditions, such as, for example,




the ATCC TM). Exemplary human T cells
hyperplasia, metaplasia, and/or dysplasia.




that may be used according to these assays
Preferred indications include anemia,




include the JURKAT cell line, which is a
pancytopenia, leukopenia, thrombocytopenia,




suspension culture of leukemia cells that
acute lymphocytic anemia (ALL),




produce IL-2 when stimulated. Exemplary
plasmacytomas, multiple myeloma, arthritis,




mouse T cells that may be used according to
AIDS, granulomatous disease, inflammatory




these assays include the CTLL cell line,
bowel disease, sepsis, neutropenia, neutrophilia,




which is a suspension culture of IL-2
psoriasis, suppression of immune reactions to




dependent cytotoxic T cells. Aditional
transplanted organs and tissues, hemophilia,




exemplary mouse T cells that may be used
hypercoagulation, diabetes mellitus,




according to these assays include the HT2
endocarditis, meningitis, Lyme Disease, and




cell line, which is a suspension culture of
asthma and allergy.




IL-2 dependent T cells that also respond to





IL-4.



17
Activation of
Assays for the activation of transcription
A highly preferred embodiment of the invention



transcription through
through the CD28 response element are
includes a method for stimulating T cell



CD28 response
well-known in the art and may be used or
proliferation. An alternative highly preferred



element in immune
routinely modified to assess the ability of
embodiment of the invention includes a method



cells (such as T-
polypeptides of the invention (including
for inhibiting T cell proliferation. A highly



cells).
antibodies and agonists or antagonists of the
preferred embodiment of the invention includes




invention) to stimulate IL-2 expression in T
a method for activating T cells. An alternative




cells. Exemplary assays for transcription
highly preferred embodiment of the invention




through the CD28 response element that
includes a method for inhibiting the activation




may be used or routinely modified to test
of and/or inactivating T cells. A highly




CD28-response element activity of
preferred embodiment of the invention includes




polypeptides of the invention (including
a method for stimulating (e.g., increasing) IL-2




antibodies and agonists or antagonists of the
production. An alternative highly preferred




invention) include assays disclosed in
embodiment of the invention includes a method




Berger et al., Gene 66: 1-10 (1998); Cullen
for inhibiting (e.g., reducing) IL-2 production.




and Malm, Methods in Enzymol 216: 362-368
Additional highly preferred indications include




(1992); Henthorn et al., Proc Natl Acad
inflammation and inflammatory disorders.




Sci USA 85: 6342-6346 (1988); McGuire
Highly preferred indications include




and Iacobelli, J Immunol 159(3): 1319-1327
autoimmune diseases (e.g., rheumatoid arthritis,




(1997); Parra et al., J Immunol 166(4): 2437-2443
systemic lupus erythematosis, multiple sclerosis




(2001); and Butscher et al., J Biol
and/or as described below),




Chem 3(1): 552-560 (1998), the contents of
immunodeficiencies (e.g., as described below),




each of which are herein incorporated by
boosting a T cell-mediated immune response,




reference in its entirety. T cells that may be
and suppressing a T cell-mediated immune




used according to these assays are publicly
response. Highly preferred indications




available (e.g., through the ATCC TM).
include neoplastic diseases (e.g., melanoma,




Exemplary human T cells that may be used
renal cell carcinoma, leukemia, lymphoma,




according to these assays include the SUPT
and/or as described below under




cell line, which is a suspension culture of
“Hyperproliferative Disorders”). Highly




IL-2 and IL-4 responsive T cells. Additional
preferred indications include neoplasms and




exemplary human T cells that may be used
cancers, such as, for example, melanoma (e.g.,




according to these assays include the
metastatic melanoma), renal cell carcinoma




JURKAT cell line, which is a suspension
(e.g., metastatic renal cell carcinoma),




culture of leukemia cells that produce IL-2
leukemia, lymphoma (e.g,. T cell lymphoma),




when stimulated.
and prostate, breast, lung, colon, pancreatic,





esophageal, stomach, brain, liver and urinary





cancer. Other preferred indications include





benign dysproliferative disorders and pre-





neoplastic conditions, such as, for example,





hyperplasia, metaplasia, and/or dysplasia. A





highly preferred indication includes infection





(e.g., AIDS, tuberculosis, infections associated





with granulomatous disease, and osteoporosis,





and/or as described below under “Infectious





Disease”). A highly preferred indication is





AIDS. Additional highly preferred indications





include suppression of immune reactions to





transplanted organs and/or tissues, uveitis,





psoriasis, and tropical spastic paraparesis.





Preferred indications include blood disorders





(e.g., as described below under “Immune





Activity”, “Blood-Related Disorders”, and/or





“Cardiovascular Disorders”). Preferred





indications also include anemia, pancytopenia,





leukopenia, thrombocytopenia, Hodgkin's





disease, acute lymphocytic anemia (ALL),





plasmacytomas, multiple myeloma, Burkitt's





lymphoma, arthritis, granulomatous disease,





inflammatory bowel disease, sepsis,





neutropenia, neutrophilia, hemophilia,





hypercoagulation, diabetes mellitus,





endocarditis, meningitis, Lyme Disease, asthma





and allergy.


18
Activation of
Assays for the activation of transcription
A highly preferred embodiment of the invention



transcription through
through the Gamma Interferon Activation
includes a method for stimulating endothelial



GAS response
Site (GAS) response element are well-
cell growth. An alternative highly preferred



element in epithelial
known in the art and may be used or
embodiment of the invention includes a method



cells (such as HELA
routinely modified to assess the ability of
for inhibiting endothelial cell growth. A



cells).
polypeptides of the invention (including
highly preferred embodiment of the invention




antibodies and agonists or antagonists of the
includes a method for stimulating endothelial




invention) to regulate STAT transcription
cell proliferation. An alternative highly




factors and modulate gene expression
preferred embodiment of the invention includes




involved in a wide variety of cell functions.
a method for inhibiting endothelial cell




Exemplary assays for transcription through
proliferation. A highly preferred




the GAS response element that may be used
embodiment of the invention includes a method




or routinely modified to test GAS-response
for stimulating apoptosis of endothelial cells.




element activity of polypeptides of the
An alternative highly preferred embodiment of




invention (including antibodies and agonists
the invention includes a method for inhibiting




or antagonists of the invention) include
(e.g., decreasing) apoptosis of endothelial cells.




assays disclosed in: You M, et al, J Biol
A highly preferred embodiment of the invention




Chem, 272(37): 23376-23381(1997); Min W,
includes a method for stimulating (e.g.,




et al., Circ Res, 83(8): 815-823 (1998);
increasing) endothelial cell activation. An




Berger et al., Gene 66: 1-10 (1998); Cullen
alternative highly preferred embodiment of the




and Malm, Methods in Enzymol 216: 362-368
invention includes a method for inhibiting (e.g.,




(1992); Henthorn et al., Proc Natl Acad
decreasing) the activation of and/or inactivating




Sci USA 85: 6342-6346 (1988); Matikainen
endothelial cells. A highly preferred




et al., Blood 93(6): 1980-1991 (1999); and
embodiment of the invention includes a method




Henttinen et al., J Immunol 155(10): 4582-4587
for stimulating angiogenisis. An alternative




(1995), the contents of each of which
highly preferred embodiment of the invention




are herein incorporated by reference in its
includes a method for inhibiting angiogenesis.




entirety. Epithelial cells that may be used
A highly preferred embodiment of the invention




according to these assays are publicly
includes a method for reducing cardiac




available (e.g., through the ATCC TM).
hypertrophy. An alternative highly preferred




Exemplary epithelial cells that may be used
embodiment of the invention includes a method




according to these assays include the HELA
for inducing cardiac hypertrophy. Preferred




cell line.
embodiments of the invention include using





polypeptides of the invention (or antibodies,





agonists, or antagonists thereof) in detection,





diagnosis, prevention, and/or treatment of





Cancer, Wound Healing, and Inflamation.





Highly preferred indications include neoplastic





diseases (e.g., as described below under





“Hyperproliferative Disorders”), and disorders





of the cardiovascular system (e.g., heart disease,





congestive heart failure, hypertension, aortic





stenosis, cardiomyopathy, valvular





regurgitation, left ventricular dysfunction,





atherosclerosis and atherosclerotic vascular





disease, diabetic nephropathy, intracardiac





shunt, cardiac hypertrophy, myocardial





infarction, chronic hemodynamic overload,





and/or as described below under





“Cardiovascular Disorders”). Highly preferred





indications include cardiovascular, endothelial





and/or angiogenic disorders (e.g., systemic





disorders that affect vessels such as diabetes





mellitus, as well as diseases of the vessels





themselves, such as of the arteries, capillaries,





veins and/or lymphatics). Highly preferred are





indications that stimulate angiogenesis and/or





cardiovascularization. Highly preferred are





indications that inhibit angiogenesis and/or





cardiovascularization. Highly preferred





indications include antiangiogenic activity to





treat solid tumors, leukemias, and Kaposi's





sarcoma, and retinal disorders. Highly preferred





indications include neoplasms and cancers, such





as, Kaposi's sarcoma, hemangioma (capillary





and cavernous), glomus tumors, telangiectasia,





bacillary angiomatosis, hemangioendothelioma,





angiosarcoma, haemangiopericytoma,





lymphangioma, lymphangiosarcoma. Highly





preferred indications also include cancers such





as, melanoma and prostate, breast, lung, colon,





pancreatic, esophageal, stomach, brain, liver,





and urinary cancer. Other preferred indications





include benign dysproliferative disorders and





pre-neoplastic conditions, such as, for example,





hyperplasia, metaplasia, and/or dysplasia.





Highly preferred indications also include





arterial disease, such as, atherosclerosis,





hypertension, coronary artery disease,





inflammatory vasculitides, Reynaud's disease





and Reynaud's phenomenom, aneurysms,





restenosis; venous and lymphatic disorders such





as thrombophlebitis, lymphangitis, and





lymphedema; and other vascular disorders such





as peripheral vascular disease, and cancer.





Highly preferred indications also include





trauma such as wounds, burns, and injured





tissue (e.g., vascular injury such as, injury





resulting from balloon angioplasty, and





atheroschlerotic lesions), implant fixation,





scarring, ischemia reperfusion injury,





rheumatoid arthritis, cerebrovascular disease,





renal diseases such as acute renal failure, and





osteoporosis. Additional highly preferred





indications include stroke, graft rejection,





diabetic or other retinopathies, thrombotic and





coagulative disorders, vascularitis, lymph





angiogenesis, sexual disorders, age-related





macular degeneration, and treatment/





prevention of endometriosis and related





conditions. Additional highly preferred





indications include fibromas, heart disease,





cardiac arrest, heart valve disease, and vascular





disease. Preferred indications include





blood disorders (e.g., as described below under





“Immune Activity”, “Blood-Related





Disorders”, and/or ““Cardiovascular





Disorders””). Preferred indications include





autoimmune diseases (e.g., rheumatoid arthritis,





systemic lupus erythematosis, multiple sclerosis





and/or as described below) and





immunodeficiencies (e.g., as described below).





Additional preferred indications include





inflammation and inflammatory disorders (such





as acute and chronic inflammatory diseases,





e.g., inflammatory bowel disease and Crohn's





disease), and pain management.


19
Activation of
Assays for the activation of transcription
Highly preferred indications include asthma,



transcription through
through the Gamma Interferon Activation
allergy, hypersensitivity reactions,



GAS response
Site (GAS) response element are well-
inflammation, and inflammatory disorders.



element in immune
known in the art and may be used or
Additional highly preferred indications include



cells (such as
routinely modified to assess the ability of
immune and hematopoietic disorders (e.g., as



eosinophils).
polypeptides of the invention (including
described below under “Immune Activity”, and




antibodies and agonists or antagonists of the
“Blood-Related Disorders”), autoimmune




invention) to modulate gene expression
diseases (e.g., rheumatoid arthritis, systemic




(commonly via STAT transcription factors)
lupus erythematosis, Crohn''s disease, multiple




involved in a wide variety of cell functions.
sclerosis and/or as described below),




Exemplary assays for transcription through
immunodeficiencies (e.g., as described below),




the GAS response element that may be used
boosting an eosinophil-mediated immune




or routinely modified to test GAS-response
response and, alternatively, suppressing an




element activity of polypeptides of the
eosinophil-mediated immune response.




invention (including antibodies and agonists





or antagonists of the invention) include





assays disclosed in Berger et al., Gene 66: 1-10





(1998); Cullen and Malm, Methods in





Enzymol 216: 362-368 (1992); Henthorn et





al., Proc Natl Acad Sci USA 85: 6342-6346





(1988); Matikainen et al., Blood





93(6): 1980-1991 (1999); and Henttinen et





al., J Immunol 155(10): 4582-4587 (1995);





the contents of each of which are herein





incorporated by reference in its entirety.





Moreover, exemplary assays that may be





used or routinely modified to assess the





ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to activate or





inhibit activation of immune cells include





assays disclosed and/or cited in: Mayumi M.,





“EoL-1, a human eosinophilic cell line”





Leuk Lymphoma; June; 7(3): 243-50 (1992);





Bhattacharya S, “Granulocyte macrophage





colony-stimulating factor and interleukin-5





activate STAT5 and induce CIS1 mRNA in





human peripheral blood eosinophils” Am J





Respir Cell Mol Biol; March; 24(3): 312-6





(2001); and, Du J, et al., “Engagement of





the CrkL adapter in interleukin-5 signaling





in eosinophils” J Biol Chem; October





20; 275(42): 33167-75 (2000); the contents





of each of which are herein incorporated by





reference in its entirety. Exemplary cells





that may be used according to these assays





include eosinophils. Eosinophils are a type





of immune cell important in the late stage of





allergic reactions; they are recruited to





tissues and mediate the inflammatory





response of late stage allergic reaction.





Increases in GAS mediated transcription in





eosinophils is typically a result of STAT





activation, normally a direct consequence of





interleukin or other cytokine receptor





stimulation (e.g. IL3, IL5 or GMCSF).



20
Activation of
Assays for the activation of transcription
Preferred embodiments of the invention include



transcription through
through the Gamma Interferon Activation
using polypeptides of the invention (or



GAS response
Site (GAS) response element are well-
antibodies, agonists, or antagonists thereof) in



element in immune
known in the art and may be used or
detection, diagnosis, prevention, and/or



cells (such as
routinely modified to assess the ability of
treatment of Inflammation, Infection, Cancer,



monocytes).
polypeptides of the invention (including
Hypersensitivity, and Atherosclerosis.




antibodies and agonists or antagonists of the





invention) to regulate STAT transcription





factors and modulate gene expression





involved in a wide variety of cell functions.





Exemplary assays for transcription through





the GAS response element that may be used





or routinely modified to test GAS-response





element activity of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) include





assays disclosed in: Gustafson K S, et al., J





Biol Chem, 271(33): 20035-20046 (1996);





Eilers A, et al., Immunobiology, 193(2-4):





328-333 (1995); Berger et al., Gene 66: 1-10





(1998); Cullen and Malm, Methods in





Enzymol 216: 362-368 (1992); Henthorn et





al., Proc Natl Acad Sci USA 85: 6342-6346





(1988); Matikainen et al., Blood





93(6): 1980-1991 (1999); and Henttinen et





al., J Immunol 155(10): 4582-4587 (1995),





the contents of each of which are herein





incoporated by reference in its entirety.





Exemplary immune cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM).





Exemplary immune cells that may be used





according to these assays include the U937





cell line, which is a monocytic cell line.



21
Activation of
Assays for the activation of transcription
Highly preferred indications include neoplastic



transcription through
through the Gamma Interferon Activation
diseases (e.g., leukemia, lymphoma, and/or as



GAS response
Site (GAS) response element are well-
described below under “Hyperproliferative



element in immune
known in the art and may be used or
Disorders”). Highly preferred indications



cells (such as T-
routinely modified to assess the ability of
include neoplasms and cancers, such as, for



cells).
polypeptides of the invention (including
example, leukemia, lymphoma (e.g., T cell




antibodies and agonists or antagonists of the
lymphoma, Burkitt's lymphoma, non-Hodgkins




invention) to regulate STAT transcription
lymphoma, Hodgkin's disease), melanoma, and




factors and modulate gene expression
prostate, breast, lung, colon, pancreatic,




involved in a wide variety of cell functions.
esophageal, stomach, brain, liver and urinary




Exemplary assays for transcription through
cancer. Other preferred indications include




the GAS response element that may be used
benign dysproliferative disorders and pre-




or routinely modified to test GAS-response
neoplastic conditions, such as, for example,




element activity of polypeptides of the
hyperplasia, metaplasia, and/or dysplasia.




invention (including antibodies and agonists
Preferred indications include autoimmune




or antagonists of the invention) include
diseases (e.g., rheumatoid arthritis, systemic




assays disclosed in Berger et al., Gene 66: 1-10
lupus erythematosis, multiple sclerosis and/or




(1998); Cullen and Malm, Methods in
as described below), immunodeficiencies (e.g.,




Enzymol 216: 362-368 (1992); Henthorn et
as described below), boosting a T cell-mediated




al., Proc Natl Acad Sci USA 85: 6342-6346
immune response, and suppressing a T cell-




(1988); Matikainen et al., Blood
mediated immune response. Additional




93(6): 1980-1991 (1999); and Henttinen et
preferred indications include inflammation and




al., J Immunol 155(10): 4582-4587 (1995),
inflammatory disorders. Highly preferred




the contents of each of which are herein
indications include blood disorders (e.g., as




incorporated by reference in its entirety.
described below under “Immune Activity”,




Exemplary mouse T cells that may be used
“Blood-Related Disorders”, and/or




according to these assays are publicly
““Cardiovascular Disorders””), and infection




available (e.g., through the ATCC TM).
(e.g., viral infections, tuberculosis, infections




Exemplary human T cells, such as the
associated with chronic granulomatosus disease




MOLT4 cell line, that may be used
and malignant osteoporosis, and/or an




according to these assays are publicly
infectious disease as described below under




available (e.g., through the ATCC TM).
“Infectious Disease”). An additional preferred




Additional exemplary human T cells, such
indication is idiopathic pulmonary fibrosis.




as the SUPT cell line, that may be used
Preferred indications include anemia,




according to these assays are publicly
pancytopenia, leukopenia, thrombocytopenia,




available (e.g., through the ATCC TM).
acute lymphocytic anemia (ALL),




Additional exemplary T cells that may be
plasmacytomas, multiple myeloma, arthritis,




used according to these assays include the
AIDS, granulomatous disease, inflammatory




CTLL cell line, which is a suspension
bowel disease, sepsis, neutropenia, neutrophilia,




culture of IL-2 dependent cytotoxic T cells.
psoriasis, suppression of immune reactions to





transplanted organs and tissues, hemophilia,





hypercoagulation, diabetes mellitus,





endocarditis, meningitis, Lyme Disease, and





asthma and allergy.


22
Activation of
This reporter assay measures activation of
Highly preferred indications include allergy,



transcription through
the GATA-3 signaling pathway in HMC-1
asthma, and rhinitis. Additional preferred



GATA-3 response
human mast cell line. Activation of GATA-
indications include infection (e.g., an infectious



element in immune
3 in mast cells has been linked to cytokine
disease as described below under “Infectious



cells (such as mast
and chemokine production. Assays for the
Disease”), and inflammation and inflammatory



cells).
activation of transcription through the
disorders. Preferred indications also include




GATA3 response element are well-known
blood disorders (e.g., as described below under




in the art and may be used or routinely
“Immune Activity”, “Blood-Related




modified to assess the ability of
Disorders”, and/or “Cardiovascular Disorders”).




polypeptides of the invention (including
Preferred indications include autoimmune




antibodies and agonists or antagonists of the
diseases (e.g., rheumatoid arthritis, systemic




invention) to regulate GATA3 transcription
lupus erythematosis, multiple sclerosis and/or




factors and modulate expression of mast cell
as described below) and immunodeficiencies




genes important for immune response
(e.g., as described below). Preferred indications




development. Exemplary assays for
include neoplastic diseases (e.g., leukemia,




transcription through the GATA3 response
lymphoma, melanoma, prostate, breast, lung,




element that may be used or routinely
colon, pancreatic, esophageal, stomach, brain,




modified to test GATA3-response element
liver, and urinary tract cancers and/or as




activity of polypeptides of the invention
described below under “Hyperproliferative




(including antibodies and agonists or
Disorders”). Other preferred indications




antagonists of the invention) include assays
include benign dysproliferative disorders and




disclosed in Berger et al., Gene 66: 1-10
pre-neoplastic conditions, such as, for example,




(1998); Cullen and Malm, Methods in
hyperplasia, metaplasia, and/or dysplasia.




Enzymol 216: 362-368 (1992); Henthorn et
Preferred indications include anemia,




al., Proc Natl Acad Sci USA 85: 6342-6346
pancytopenia, leukopenia, thrombocytopenia,




(1988); Flavell et al., Cold Spring Harb
leukemias, Hodgkin's disease, acute




Symp Quant Biol 64: 563-571 (1999);
lymphocytic anemia (ALL), plasmacytomas,




Rodriguez-Palmero et al., Eur J Immunol
multiple myeloma, Burkitt's lymphoma,




29(12): 3914-3924 (1999); Zheng and
arthritis, AIDS, granulomatous disease,




Flavell, Cell 89(4): 587-596 (1997); and
inflammatory bowel disease, sepsis,




Henderson et al., Mol Cell Biol 14(6): 4286-4294
neutropenia, neutrophilia, psoriasis, suppression




(1994), the contents of each of which
of immune reactions to transplanted organs and




are herein incorporated by reference in its
tissues, hemophilia, hypercoagulation, diabetes




entirety. Mast cells that may be used
mellitus, endocarditis, meningitis, and Lyme




according to these assays are publicly
Disease.




available (e.g., through the ATCC TM).





Exemplary human mast cells that may be





used according to these assays include the





HMC-1 cell line, which is an immature





human mast cell line established from the





peripheral blood of a patient with mast cell





leukemia, and exhibits many characteristics





of immature mast cells.



23
Activation of
Assays for the activation of transcription




transcription through
through the GATA3 response element are




GATA-3 response
well-known in the art and may be used or




element in immune
routinely modified to assess the ability of




cells (such as T-
polypeptides of the invention (including




cells).
antibodies and agonists or antagonists of the





invention) to regulate GATA3 transcription





factors and modulate expression of genes





important for Th2 immune response





development. Exemplary assays for





transcription through the GATA3 response





element that may be used or routinely





modified to test GATA3-response element





activity of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) include assays





disclosed in Berger et al., Gene 66: 1-10





(1998); Cullen and Malm, Methods in





Enzymol 216: 362-368 (1992); Henthorn et





al., Proc Natl Acad Sci USA 85: 6342-6346





(1988); Flavell et al., Cold Spring Harb





Symp Quant Biol 64: 563-571 (1999);





Rodriguez-Palmero et al., Eur J Immunol





29(12): 3914-3924 (1999); Zheng and





Flavell, Cell 89(4): 587-596 (1997); and





Henderson et al., Mol Cell Biol 14(6): 4286-4294





(1994), the contents of each of which





are herein incorporated by reference in its





entirety. T cells that may be used according





to these assays are publicly available (e.g.,





through the ATCC TM). Exemplary mouse T





cells that may be used according to these





assays include the HT2 cell line, which is a





suspension culture of IL-2 dependent T cells





that also respond to IL-4.



24
Activation of
This reporter assay measures activation of
Highly preferred indications include allergy,



transcription through
the NFAT signaling pathway in HMC-1
asthma, and rhinitis. Additional preferred



NFAT response
human mast cell line. Activation of NFAT
indications include infection (e.g., an infectious



element in immune
in mast cells has been linked to cytokine
disease as described below under “Infectious



cells (such as mast
and chemokine production. Assays for the
Disease”), and inflammation and inflammatory



cells).
activation of transcription through the
disorders. Preferred indications also include




Nuclear Factor of Activated T cells (NFAT)
blood disorders (e.g., as described below under




response element are well-known in the art
“Immune Activity”, “Blood-Related




and may be used or routinely modified to
Disorders”, and/or “Cardiovascular Disorders”).




assess the ability of polypeptides of the
Preferred indications include autoimmune




invention (including antibodies and agonists
diseases (e.g., rheumatoid arthritis, systemic




or antagonists of the invention) to regulate
lupus erythematosis, multiple sclerosis and/or




NFAT transcription factors and modulate
as described below) and immunodeficiencies




expression of genes involved in
(e.g., as described below). Preferred indications




immunomodulatory functions. Exemplary
include neoplastic diseases (e.g., leukemia,




assays for transcription through the NFAT
lymphoma, melanoma, prostate, breast, lung,




response element that may be used or
colon, pancreatic, esophageal, stomach, brain,




routinely modified to test NFAT-response
liver, and urinary tract cancers and/or as




element activity of polypeptides of the
described below under “Hyperproliferative




invention (including antibodies and agonists
Disorders”). Other preferred indications




or antagonists of the invention) include
include benign dysproliferative disorders and




assays disclosed in Berger et al., Gene 66: 1-10
pre-neoplastic conditions, such as, for example,




(1998); Cullen and Malm, Methods in
hyperplasia, metaplasia, and/or dysplasia.




Enzymol 216: 362-368 (1992); Henthorn et
Preferred indications include anemia,




al., Proc Natl Acad Sci USA 85: 6342-6346
pancytopenia, leukopenia, thrombocytopenia,




(1988); De Boer et al., Int J Biochem Cell
leukemias, Hodgkin's disease, acute




Biol 31(10): 1221-1236 (1999); Ali et al., J
lymphocytic anemia (ALL), plasmacytomas,




Immunol 165(12): 7215-7223 (2000);
multiple myeloma, Burkitt's lymphoma,




Hutchinson and McCloskey, J Biol Chem
arthritis, AIDS, granulomatous disease,




270(27): 16333-16338 (1995), and Turner et
inflammatory bowel disease, sepsis,




al., J Exp Med 188: 527-537 (1998), the
neutropenia, neutrophilia, psoriasis, suppression




contents of each of which are herein
of immune reactions to transplanted organs and




incorporated by reference in its entirety.
tissues, hemophilia, hypercoagulation, diabetes




Mast cells that may be used according to
mellitus, endocarditis, meningitis, and Lyme




these assays are publicly available (e.g.,
Disease.




through the ATCC TM). Exemplary human





mast cells that may be used according to





these assays include the HMC-1 cell line,





which is an immature human mast cell line





established from the peripheral blood of a





patient with mast cell leukemia, and





exhibits many characteristics of immature





mast cells.



25
Activation of
Assays for the activation of transcription
Highly preferred indications include blood



transcription through
through the Nuclear Factor of Activated T
disorders (e.g., as described below under



NFAT response
cells (NFAT) response element are well-
“Immune Activity”, “Blood-Related



element in immune
known in the art and may be used or
Disorders”, and/or “Cardiovascular Disorders”).



cells (such as natural
routinely modified to assess the ability of
Highly preferred indications include



killer cells).
polypeptides of the invention (including
autoimmune diseases (e.g., rheumatoid arthritis,




antibodies and agonists or antagonists of the
systemic lupus erythematosis, multiple sclerosis




invention) to regulate NFAT transcription
and/or as described below),




factors and modulate expression of genes
immunodeficiencies (e.g., as described below),




involved in immunomodulatory functions.
boosting a T cell-mediated immune response,




Exemplary assays for transcription through
and suppressing a T cell-mediated immune




the NFAT response element that may be
response. Additional highly preferred




used or routinely modified to test NFAT-
indications include inflammation and




response element activity of polypeptides of
inflammatory disorders. An additional highly




the invention (including antibodies and
preferred indication is infection (e.g., an




agonists or antagonists of the invention)
infectious disease as described below under




include assays disclosed in Berger et al.,
“Infectious Disease”). Preferred indications




Gene 66: 1-10 (1998); Cullen and Malm,
include neoplastic diseases (e.g., leukemia,




Methods in Enzymol 216: 362-368 (1992);
lymphoma, and/or as described below under




Henthorn et al., Proc Natl Acad Sci USA
“Hyperproliferative Disorders”). Preferred




85: 6342-6346 (1988); Aramburu et al., J
indications include neoplasms and cancers, such




Exp Med 182(3): 801-810 (1995); De Boer
as, for example, leukemia, lymphoma, and




et al., Int J Biochem Cell Biol 31(10): 1221-1236
prostate, breast, lung, colon, pancreatic,




(1999); Fraser et al., Eur J Immunol
esophageal, stomach, brain, liver and urinary




29(3): 838-844 (1999); and Yeseen et al., J
cancer. Other preferred indications include




Biol Chem 268(19): 14285-14293 (1993),
benign dysproliferative disorders and pre-




the contents of each of which are herein
neoplastic conditions, such as, for example,




incorporated by reference in its entirety.
hyperplasia, metaplasia, and/or dysplasia.




NK cells that may be used according to
Preferred indications also include anemia,




these assays are publicly available (e.g.,
pancytopenia, leukopenia, thrombocytopenia,




through the ATCC TM). Exemplary human
Hodgkin's disease, acute lymphocytic anemia




NK cells that may be used according to
(ALL), plasmacytomas, multiple myeloma,




these assays include the NK-YT cell line,
Burkitt's lymphoma, arthritis, AIDS,




which is a human natural killer cell line
granulomatous disease, inflammatory bowel




with cytolytic and cytotoxic activity.
disease, sepsis, neutropenia, neutrophilia,





psoriasis, suppression of immune reactions to





transplanted organs and tissues, hemophilia,





hypercoagulation, diabetes mellitus,





endocarditis, meningitis, Lyme Disease, asthma





and allergy.


26
Activation of
Assays for the activation of transcription
Highly preferred indications include blood



transcription through
through the Nuclear Factor of Activated T
disorders (e.g., as described below under



NFAT response
cells (NFAT) response element are well-
“Immune Activity”, “Blood-Related



element in immune
known in the art and may be used or
Disorders”, and/or “Cardiovascular Disorders”).



cells (such as T-
routinely modified to assess the ability of
Highly preferred indications include



cells).
polypeptides of the invention (including
autoimmune diseases (e.g., rheumatoid arthritis,




antibodies and agonists or antagonists of the
systemic lupus erythematosis, multiple sclerosis




invention) to regulate NFAT transcription
and/or as described below),




factors and modulate expression of genes
immunodeficiencies (e.g., as described below),




involved in immunomodulatory functions.
boosting a T cell-mediated immune response,




Exemplary assays for transcription through
and suppressing a T cell-mediated immune




the NFAT response element that may be
response. Additional highly preferred




used or routinely modified to test NFAT-
indications include inflammation and




response element activity of polypeptides of
inflammatory disorders. An additional highly




the invention (including antibodies and
preferred indication is infection (e.g., an




agonists or antagonists of the invention)
infectious disease as described below under




include assays disclosed in Berger et al.,
“Infectious Disease”). Preferred indications




Gene 66: 1-10 (1998); Cullen and Malm,
include neoplastic diseases (e.g., leukemia,




Methods in Enzymol 216: 362-368 (1992);
lymphoma, and/or as described below under




Henthorn et al., Proc Natl Acad Sci USA
“Hyperproliferative Disorders”). Preferred




85: 6342-6346 (1988); Serfling et al.,
indications include neoplasms and cancers, such




Biochim Biophys Acta 1498(1): 1-18
as, for example, leukemia, lymphoma, and




(2000); De Boer et al., Int J Biochem Cell
prostate, breast, lung, colon, pancreatic,




Biol 31(10): 1221-1236 (1999); Fraser et al.,
esophageal, stomach, brain, liver and urinary




Eur J Immunol 29(3): 838-844 (1999); and
cancer. Other preferred indications include




Yeseen et al., J Biol Chem 268(19): 14285-14293
benign dysproliferative disorders and pre-




(1993), the contents of each of which
neoplastic conditions, such as, for example,




are herein incorporated by reference in its
hyperplasia, metaplasia, and/or dysplasia.




entirety. T cells that may be used according
Preferred indications also include anemia,




to these assays are publicly available (e.g.,
pancytopenia, leukopenia, thrombocytopenia,




through the ATCC TM). Exemplary human T
Hodgkin's disease, acute lymphocytic anemia




cells that may be used according to these
(ALL), plasmacytomas, multiple myeloma,




assays include the SUPT cell line, which is
Burkitt's lymphoma, arthritis, AIDS,




a suspension culture of IL-2 and IL-4
granulomatous disease, inflammatory bowel




responsive T cells.
disease, sepsis, neutropenia, neutrophilia,





psoriasis, suppression of immune reactions to





transplanted organs and tissues, hemophilia,





hypercoagulation, diabetes mellitus,





endocarditis, meningitis, Lyme Disease, asthma





and allergy.


27
Activation of
Assays for the activation of transcription
Highly preferred indications include blood



transcription through
through the Nuclear Factor of Activated T
disorders (e.g., as described below under



NFAT response in
cells (NFAT) response element are well-
“Immune Activity”, “Blood-Related



immune cells (such
known in the art and may be used or
Disorders”, and/or “Cardiovascular Disorders”).



as T-cells).
routinely modified to assess the ability of
Highly preferred indications include




polypeptides of the invention (including
autoimmune diseases (e.g., rheumatoid arthritis,




antibodies and agonists or antagonists of the
systemic lupus erythematosis, multiple sclerosis




invention) to regulate NFAT transcription
and/or as described below),




factors and modulate expression of genes
immunodeficiencies (e.g., as described below),




involved in immunomodulatory functions.
boosting a T cell-mediated immune response,




Exemplary assays for transcription through
and suppressing a T cell-mediated immune




the NFAT response element that may be
response. Additional highly preferred




used or routinely modified to test NFAT-
indications include inflammation and




response element activity of polypeptides of
inflammatory disorders. An additional highly




the invention (including antibodies and
preferred indication is infection (e.g., an




agonists or antagonists of the invention)
infectious disease as described below under




include assays disclosed in Berger et al.,
“Infectious Disease”). Preferred indications




Gene 66: 1-10 (1998); Cullen and Malm,
include neoplastic diseases (e.g., leukemia,




Methods in Enzymol 216: 362-368 (1992);
lymphoma, and/or as described below under




Henthorn et al., Proc Natl Acad Sci USA
“Hyperproliferative Disorders”). Preferred




85: 6342-6346 (1988); Serfling et al.,
indications include neoplasms and cancers, such




Biochim Biophys Acta 1498(1): 1-18
as, for example, leukemia, lymphoma, and




(2000); De Boer et al., Int J Biochem Cell
prostate, breast, lung, colon, pancreatic,




Biol 31(10): 1221-1236 (1999); Fraser et al.,
esophageal, stomach, brain, liver and urinary




Eur J Immunol 29(3): 838-844 (1999); and
cancer. Other preferred indications include




Yeseen et al., J Biol Chem 268(19): 14285-14293
benign dysproliferative disorders and pre-




(1993), the contents of each of which
neoplastic conditions, such as, for example,




are herein incorporated by reference in its
hyperplasia, metaplasia, and/or dysplasia.




entirety. T cells that may be used according
Preferred indications also include anemia,




to these assays are publicly available (e.g.,
pancytopenia, leukopenia, thrombocytopenia,




through the ATCC TM). Exemplary human T
Hodgkin's disease, acute lymphocytic anemia




cells that may be used according to these
(ALL), plasmacytomas, multiple myeloma,




assays include the JURKAT cell line, which
Burkitt's lymphoma, arthritis, AIDS,




is a suspension culture of leukemia cells
granulomatous disease, inflammatory bowel




that produce IL-2 when stimulated.
disease, sepsis, neutropenia, neutrophilia,





psoriasis, suppression of immune reactions to





transplanted organs and tissues, hemophilia,





hypercoagulation, diabetes mellitus,





endocarditis, meningitis, Lyme Disease, asthma





and allergy.


28
Activation of
Assays for the activation of transcription
Preferred embodiments of the invention include



transcription through
through the NFKB response element are
using polypeptides of the invention (or



NFKB response
well-known in the art and may be used or
antibodies, agonists, or antagonists thereof) in



element in epithelial
routinely modified to assess the ability of
detection, diagnosis, prevention, and/or



cells (such as HELA
polypeptides of the invention (including
treatment of Cancer, Wound Healing, and



cells).
antibodies and agonists or antagonists of the
Inflamation. Highly preferred indications




invention) to regulate NFKB transcription
include neoplastic diseases (e.g., as described




factors and modulate expression of
below under “Hyperproliferative Disorders”).




epithhelial genes. Exemplary assays for
Highly preferred indications include neoplasms




transcription through the NFKB response
and cancers, such as, for example, melanoma,




element that may be used or routinely
and prostate, breast, lung, colon, pancreatic,




modified to test NFKB-response element
esophageal, stomach, brain, liver and urinary




activity of polypeptides of the invention
cancer. Other preferred indications include




(including antibodies and agonists or
benign dysproliferative disorders and pre-




antagonists of the invention) include assays
neoplastic conditions, such as, for example,




disclosed in: Kaltschmidt B, et al.,
hyperplasia, metaplasia, and/or dysplasia.




Oncogene, 18(21): 3213-3225 (1999); Beetz A,
Preferred indications include include




et al., Int J Radiat Biol, 76(11): 1443-1453
inflammation and inflammatory disorders.




(2000); Berger et al., Gene 66: 1-10





(1998); Cullen and Malm, Methods in





Enzymol 216: 362-368 (1992); Henthorn et





al., Proc Natl Acad Sci USA 85: 6342-6346





(1988); Valle Blazquez et al, Immunology





90(3): 455-460 (1997); Aramburau et al., J





Exp Med 82(3): 801-810 (1995); and Fraser





et al., 29(3): 838-844 (1999), the contents of





each of which are herein incorporated by





reference in its entirety. Epithelial cells that





may be used according to these assays are





publicly available (e.g., through the





ATCC TM). Exemplary epithelial cells that





may be used according to these assays





include the HELA cell line.



29
Activation of
Assays for the activation of transcription




transcription through
through the NFKB response element are




NFKB response
well-known in the art and may be used or




element in immune
routinely modified to assess the ability of




cells (such as B-
polypeptides of the invention (including




cells).
antibodies and agonists or antagonists of the





invention) to regulate NFKB transcription





factors and modulate expression of





immunomodulatory genes. Exemplary





assays for transcription through the NFKB





response element that may be used or





routinely modified to test NFKB-response





element activity of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) include





assays disclosed in: Gri G, et al., Biol





Chem, 273(11): 6431-6438 (1998); Pyatt D W,





et al., Cell Biol Toxicol





2000; 16(1): 41-51 (2000); Berger et al.,





Gene 66: 1-10 (1998); Cullen and Malm,





Methods in Enzymol 216: 362-368 (1992);





Henthorn et al., Proc Natl Acad Sci USA





85: 6342-6346 (1988); Valle Blazquez et al,





Immunology 90(3): 455-460 (1997);





Aramburau et al., J Exp Med 82(3): 801-810





(1995); and Fraser et al., 29(3): 838-844





(1999), the contents of each of which are





herein incorporated by reference in its





entirety. Immune cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM).





Exemplary immune cells that may be used





according to these assays include the Reh





B-cell line.



30
Activation of
Assays for the activation of transcription
Preferred embodiments of the invention include



transcription through
through the NFKB response element are
using polypeptides of the invention (or



NFKB response
well-known in the art and may be used or
antibodies, agonists, or antagonists thereof) in



element in immune
routinely modified to assess the ability of
detection, diagnosis, prevention, and/or



cells (such as B-
polypeptides of the invention (including
treatment of Cancer, Autoimmunity, Allergy



cells).
antibodies and agonists or antagonists of the
and Asthma




invention) to regulate NFKB transcription





factors and modulate expression of





immunomodulatory genes. Exemplary





assays for transcription through the NFKB





response element that may be used or





rountinely modified to test NFKB-response





element activity of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) include





assays disclosed in: Gri G, et al., Biol





Chem, 273(11): 6431-6438 (1998); Pyatt D W,





et al., Cell Biol Toxicol





2000; 16(1): 41-51 (2000); Berger et al.,





Gene 66: 1-10 (1998); Cullen and Malm,





Methods in Enzymol 216: 362-368 (1992);





Henthorn et al., Proc Natl Acad Sci USA





85: 6342-6346 (1988); Valle Blazquez et al,





Immunology 90(3): 455-460 (1997);





Aramburau et al., J Exp Med 82(3): 801-810





(1995); and Fraser et al., 29(3): 838-844





(1999), the contents of each of which are





herein incorporated by reference in its





entirety. Immune cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM).





Exemplary immune cells that may be used





according to these assays include the Reh





B-cell line.



31
Activation of
This reporter assay measures activation of
Highly preferred indication includes allergy,



transcription through
the NFkB signaling pathway in Ku812
asthma, and rhinitis. Additional highly



NFKB response
human basophil cell line. Assays for the
preferred indications include infection (e.g., an



element in immune
activation of transcription through the
infectious disease as described below under



cells (such as
NFKB response element are well-known in
“Infectious Disease”), and inflammation and



basophils).
the art and may be used or routinely
inflammatory disorders. Preferred indications




modified to assess the ability of
include immunological and hempatopoietic




polypeptides of the invention (including
disorders (e.g., as described below under




antibodies and agonists or antagonists of the
“Immune Activity”, and “Blood-Related




invention) to regulate NFKB transcription
Disorders”). Preferred indications also include




factors and modulate expression of
autoimmune diseases (e.g., rheumatoid arthritis,




immunomodulatory genes. Exemplary
systemic lupus erythematosis, multiple sclerosis




assays for transcription through the NFKB
and/or as described below) and




response element that may be used or
immunodeficiencies (e.g., as described below).




rountinely modified to test NFKB-response
Preferred indications also include neoplastic




element activity of polypeptides of the
diseases (e.g., leukemia, lymphoma, melanoma,




invention (including antibodies and agonists
and/or as described below under




or antagonists of the invention) include
“Hyperproliferative Disorders”). Preferred




assays disclosed in Berger et al., Gene 66: 1-10
indications include neoplasms and cancer, such




(1998); Cullen and Malm, Methods in
as, for example, leukemia, lymphoma,




Enzymol 216: 362-368 (1992); Henthorn et
melanoma, and prostate, breast, lung, colon,




al., Proc Natl Acad Sci USA 85: 6342-6346
pancreatic, esophageal, stomach, brain, liver,




(1988); Marone et al, Int Arch Allergy
urinary tract cancers and as described below




Immunol 114(3): 207-17 (1997), the
under “Hyperproliferative Disorders”.




contents of each of which are herein





incorporated by reference in its entirety.





Basophils that may be used according to





these assays are publicly available (e.g.,





through the ATCC TM). Exemplary human





basophil cell lines that may be used





according to these assays include Ku812,





originally established from a patient with





chronic myelogenous leukemia. It is an





immature prebasophilic cell line that can be





induced to differentiate into mature





basophils.



32
Activation of
Assays for the activation of transcription
Highly preferred indications include asthma,



transcription through
through the NFKB response element are
allergy, hypersensitivity reactions, and



NFKB response
well-known in the art and may be used or
inflammation. Preferred indications include



element in immune
routinely modified to assess the ability of
infection (e.g., an infectious disease as



cells (such as EOL1
polypeptides of the invention (including
described below under “Infectious Disease”),



cells).
antibodies and agonists or antagonists of the
immunological disorders, inflammation and




invention) to regulate NFKB transcription
inflammatory disorders (e.g., as described




factors and modulate expression of
below under “Immune Activity”, and “Blood-




immunomodulatory genes. Exemplary
Related Disorders”). Preferred indications




assays for transcription through the NFKB
include autoimmune diseases (e.g., rheumatoid




response element that may be used or
arthritis, systemic lupus erythematosis, multiple




rountinely modified to test NFKB-response
sclerosis and/or as described below) and




element activity of polypeptides of the
immunodeficiencies (e.g., as described below).




invention (including antibodies and agonists





or antagonists of the invention) include





assays disclosed in Berger et al., Gene 66: 1-10





(1998); Cullen and Malm, Methods in





Enzymol 216: 362-368 (1992); Henthorn et





al., Proc Natl Acad Sci USA 85: 6342-6346





(1988); Valle Blazquez et al, Immunology





90(3): 455-460 (1997); Aramburau et al., J





Exp Med 82(3): 801-810 (1995); and Fraser





et al., 29(3): 838-844 (1999), the contents of





each of which are herein incorporated by





reference in its entirety. For example, a





reporter assay (which measures increases in





transcription inducible from a NFkB





responsive element in EOL-1 cells) may





link the NFKB element to a repeorter gene





and binds to the NFKB transcription factor,





which is upregulated by cytokines and other





factors. Exemplary immune cells that may





be used according to these assays include





eosinophils such as the human EOL-1 cell





line of eosinophils. Eosinophils are a type





of immune cell important in the allergic





responses; they are recruited to tissues and





mediate the inflammtory response of late





stage allergic reaction. Eol-1 is a human





eosinophil cell line.



33
Activation of
This reporter assay measures activation of
Highly preferred indication includes allergy,



transcription through
the NFkB signaling pathway in HMC-1
asthma, and rhinitis. Additional highly



NFKB response
human mast cell line. Activation of NFkB in
preferred indications include infection (e.g., an



element in immune
mast cells has been linked to production of
infectious disease as described below under



cells (such as mast
certain cytokines, such as IL-6 and IL-9.
“Infectious Disease”), and inflammation and



cells).
Assays for the activation of transcription
inflammatory disorders. Preferred indications




through the NFKB response element are
include immunological and hempatopoietic




well-known in the art and may be used or
disorders (e.g., as described below under




routinely modified to assess the ability of
“Immune Activity”, and “Blood-Related




polypeptides of the invention (including
Disorders”). Preferred indications also include




antibodies and agonists or antagonists of the
autoimmune diseases (e.g., rheumatoid arthritis,




invention) to regulate NFKB transcription
systemic lupus erythematosis, multiple sclerosis




factors and modulate expression of
and/or as described below) and




immunomodulatory genes. Exemplary
immunodeficiencies (e.g., as described below).




assays for transcription through the NFKB
Preferred indications also include neoplastic




response element that may be used or
diseases (e.g., leukemia, lymphoma, melanoma,




rountinely modified to test NFKB-response
and/or as described below under




element activity of polypeptides of the
“Hyperproliferative Disorders”). Preferred




invention (including antibodies and agonists
indications include neoplasms and cancer, such




or antagonists of the invention) include
as, for example, leukemia, lymphoma,




assays disclosed in Berger et al., Gene 66: 1-10
melanoma, and prostate, breast, lung, colon,




(1998); Cullen and Malm, Methods in
pancreatic, esophageal, stomach, brain, liver,




Enzymol 216: 362-368 (1992); Henthorn et
urinary tract cancers and as described below




al., Proc Natl Acad Sci USA 85: 6342-6346
under “Hyperproliferative Disorders”.




(1988); Stassen et al, J Immunol





166(7): 4391-8 (2001); and Marquardt and





Walker, J Allergy Clin Immunol





105(3): 500-5 (2000), the contents of each of





which are herein incorporated by reference





in its entirety. Mast cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM).





Exemplary human mast cells that may be





used according to these assays include the





HMC-1 cell line, which is an immature





human mast cell line established from the





peripheral blood of a patient with mast cell





leukemia, and exhibits many characteristics





of immature mast cells.



34
Activation of
Assays for the activation of transcription
Highly preferred indications include



transcription through
through the NFKB response element are
inflammation and inflammatory disorders.



NFKB response
well-known in the art and may be used or
Highly preferred indications include blood



element in immune
routinely modified to assess the ability of
disorders (e.g., as described below under



cells (such as natural
polypeptides of the invention (including
“Immune Activity”, “Blood-Related



killer cells).
antibodies and agonists or antagonists of the
Disorders”, and/or “Cardiovascular Disorders”).




invention) to regulate NFKB transcription
Highly preferred indications include




factors and modulate expression of
autoimmune diseases (e.g., rheumatoid arthritis,




immunomodulatory genes. Exemplary
systemic lupus erythematosis, multiple sclerosis




assays for transcription through the NFKB
and/or as described below), and




response element that may be used or
immunodeficiencies (e.g., as described below).




rountinely modified to test NFKB-response
An additional highly preferred indication is




element activity of polypeptides of the
infection (e.g., AIDS, and/or an infectious




invention (including antibodies and agonists
disease as described below under “Infectious




or antagonists of the invention) include
Disease”). Highly preferred indications




assays disclosed in Berger et al., Gene 66: 1-10
include neoplastic diseases (e.g., melanoma,




(1998); Cullen and Malm, Methods in
leukemia, lymphoma, and/or as described




Enzymol 216: 362-368 (1992); Henthorn et
below under “Hyperproliferative Disorders”).




al., Proc Natl Acad Sci USA 85: 6342-6346
Highly preferred indications include neoplasms




(1988); Valle Blazquez et al, Immunology
and cancers, such as, for example, melanoma,




90(3): 455-460 (1997); Aramburau et al., J
renal cell carcinoma, leukemia, lymphoma, and




Exp Med 82(3): 801-810 (1995); and Fraser
prostate, breast, lung, colon, pancreatic,




et al., 29(3): 838-844 (1999), the contents of
esophageal, stomach, brain, liver and urinary




each of which are herein incorporated by
cancer. Other preferred indications include




reference in its entirety. NK cells that may
benign dysproliferative disorders and pre-




be used according to these assays are
neoplastic conditions, such as, for example,




publicly available (e.g., through the
hyperplasia, metaplasia, and/or dysplasia.




ATCC TM). Exemplary NK cells that may be
Preferred indications also include anemia,




used according to these assays include the
pancytopenia, leukopenia, thrombocytopenia,




NK-YT cell line, which is a human natural
Hodgkin's disease, acute lymphocytic anemia




killer cell line with cytolytic and cytotoxic
(ALL), plasmacytomas, multiple myeloma,




activity.
Burkitt's lymphoma, arthritis, AIDS,





granulomatous disease, inflammatory bowel





disease, sepsis, neutropenia, neutrophilia,





psoriasis, hemophilia, hypercoagulation,





diabetes mellitus, endocarditis, meningitis,





Lyme Disease, suppression of immune





reactions to transplanted organs, asthma and





allergy.


35
Activation of
Assays for the activation of transcription
Highly preferred indications include



transcription through
through the NFKB response element are
inflammation and inflammatory disorders.



NFKB response
well-known in the art and may be used or
Highly preferred indications include blood



element in immune
routinely modified to assess the ability of
disorders (e.g., as described below under



cells (such as T-
polypeptides of the invention (including
“Immune Activity”, “Blood-Related



cells).
antibodies and agonists or antagonists of the
Disorders”, and/or “Cardiovascular Disorders”).




invention) to regulate NFKB transcription
Highly preferred indications include




factors and modulate expression of
autoimmune diseases (e.g., rheumatoid arthritis,




immunomodulatory genes. Exemplary
systemic lupus erythematosis, multiple sclerosis




assays for transcription through the NFKB
and/or as described below), and




response element that may be used or
immunodeficiencies (e.g., as described below).




rountinely modified to test NFKB-response
An additional highly preferred indication is




element activity of polypeptides of the
infection (e.g., AIDS, and/or an infectious




invention (including antibodies and agonists
disease as described below under “Infectious




or antagonists of the invention) include
Disease”). Highly preferred indications




assays disclosed in Berger et al., Gene 66: 1-10
include neoplastic diseases (e.g., melanoma,




(1998); Cullen and Malm, Methods in
leukemia, lymphoma, and/or as described




Enzymol 216: 362-368 (1992); Henthorn et
below under “Hyperproliferative Disorders”).




al., Proc Natl Acad Sci USA 85: 6342-6346
Highly preferred indications include neoplasms




(1988); Black et al., Virus Gnes 15(2): 105-117
and cancers, such as, for example, melanoma,




(1997); and Fraser et al., 29(3): 838-844
renal cell carcinoma, leukemia, lymphoma, and




(1999), the contents of each of which are
prostate, breast, lung, colon, pancreatic,




herein incorporated by reference in its
esophageal, stomach, brain, liver and urinary




entirety. T cells that may be used according
cancer. Other preferred indications include




to these assays are publicly available (e.g.,
benign dysproliferative disorders and pre-




through the ATCC TM). Exemplary human T
neoplastic conditions, such as, for example,




cells that may be used according to these
hyperplasia, metaplasia, and/or dysplasia.




assays include the SUPT cell line, which is
Preferred indications also include anemia,




a suspension culture of IL-2 and IL-4
pancytopenia, leukopenia, thrombocytopenia,




responsive T cells. Additional exemplary
Hodgkin's disease, acute lymphocytic anemia




human T cells, such as the MOLT4, that
(ALL), plasmacytomas, multiple myeloma,




may be used according to these assays are
Burkitt's lymphoma, arthritis, AIDS,




publicly available (e.g., through the
granulomatous disease, inflammatory bowel




ATCC TM).
disease, sepsis, neutropenia, neutrophilia,





psoriasis, hemophilia, hypercoagulation,





diabetes mellitus, endocarditis, meningitis,





Lyme Disease, suppression of immune





reactions to transplanted organs, asthma and





allergy.


36
Activation of
Assays for the activation of transcription
Highly preferred indications include



transcription through
through the NFKB response element are
inflammation and inflammatory disorders.



NFKB response
well-known in the art and may be used or
Highly preferred indications include



element in immune
routinely modified to assess the ability of
immunological and hematopoietic disorders



cells (such as the
polypeptides of the invention (including
(e.g., as described below under “Immune



Jurkat human T cell
antibodies and agonists or antagonists of the
Activity”, “Blood-Related Disorders”, and/or



line).
invention) to regulate NFKB transcription
“Cardiovascular Disorders”). Highly preferred




factors and modulate expression of
indications include autoimmune diseases (e.g.,




immunomodulatory genes. Exemplary
rheumatoid arthritis, systemic lupus




assays for transcription through the NFKB
erythematosis, multiple sclerosis and/or as




response element that may be used or
described below), and immunodeficiencies




rountinely modified to test NFKB-response
(e.g., as described below). An additional highly




element activity of polypeptides of the
preferred indication is infection (e.g., AIDS,




invention (including antibodies and agonists
and/or an infectious disease as described below




or antagonists of the invention) include
under “Infectious Disease”). Highly preferred




assays disclosed in Berger et al., Gene 66: 1-10
indications include neoplastic diseases (e.g.,




(1998); Cullen and Malm, Methods in
melanoma, leukemia, lymphoma, and/or as




Enzymol 216: 362-368 (1992); Henthorn et
described below under “Hyperproliferative




al., Proc Natl Acad Sci USA 85: 6342-6346
Disorders”). Highly preferred indications




(1988); Valle Blazquez et al, Immunology
include neoplasms and cancers, such




90(3): 455-460 (1997); Aramburau et al., J
as, melanoma, renal cell carcinoma, leukemia,




Exp Med 82(3): 801-810 (1995); and Fraser
lymphoma, and prostate, breast, lung, colon,




et al., 29(3): 838-844 (1999), the contents of
pancreatic, esophageal, stomach, brain, liver




each of which are herein incorporated by
and urinary cancer. Other preferred indications




reference in its entirety. T cells that may be
include benign dysproliferative disorders and




used according to these assays are publicly
pre-neoplastic conditions, such as, for example,




available (e.g., through the ATCC TM). T
hyperplasia, metaplasia, and/or dysplasia.




cells that may be used according to these
Preferred indications also include anemia,




assays are publicly available (e.g., through
pancytopenia, leukopenia, thrombocytopenia,




the ATCC TM). Exemplary human T cells
Hodgkin's disease, acute lymphocytic anemia




that may be used according to these assays
(ALL), plasmacytomas, multiple myeloma,




include the JURKAT cell line, which is a
Burkitt's lymphoma, arthritis, AIDS,




suspension culture of leukemia cells that
granulomatous disease, inflammatory bowel




produce IL-2 when stimulated.
disease, sepsis, neutropenia, neutrophilia,





psoriasis, hemophilia, hypercoagulation,





diabetes mellitus, endocarditis, meningitis,





Lyme Disease, suppression of immune





reactions to transplanted organs, asthma and





allergy.


37
Activation of
This assay uses a NFKB response element
Highly preferred indications include



transcription through
(which will bind NFKB transcription
inflammation and inflammatory disorders.



NFKB response
factors) linked to a reporter gene to measure
Highly preferred indications include



element in immune
NFKB mediated transcription in the human
immunological and hematopoietic disorders



cells (such as the
monocyte cell line U937. NFKB is
(e.g., as described below under “Immune



U937 human
upregulated by cytokines and other factors
Activity”, “Blood-Related Disorders”, and/or



monocyte cell line).
and NFKB element activation leads to
“Cardiovascular Disorders”). Highly preferred




expression of immunomodulatory genes.
indications include autoimmune diseases (e.g.,




Activation of NFKB in monocytes can play
rheumatoid arthritis, systemic lupus




a role in immune responses. Exemplary
erythematosis, multiple sclerosis and/or as




assays for transcription through the NFKB
described below), and immunodeficiencies




response element that may be used or
(e.g., as described below). An additional highly




rountinely modified to test NFKB-response
preferred indication is infection (e.g., AIDS,




element activity of polypeptides of the
and/or an infectious disease as described below




invention (including antibodies and agonists
under “Infectious Disease”). Highly preferred




or antagonists of the invention) include
indications include neoplastic diseases (e.g.,




assays disclosed in Berger et al., Gene 66: 1-10
melanoma, leukemia, lymphoma, and/or as




(1998); Cullen and Malm, Methods in
described below under “Hyperproliferative




Enzymol 216: 362-368 (1992); Henthorn et
Disorders”). Highly preferred indications




al., Proc Natl Acad Sci USA 85: 6342-6346
include neoplasms and cancers, such




(1988); Valle Blazquez et al, Immunology
as, melanoma, renal cell carcinoma, leukemia,




90(3): 455-460 (1997); Aramburau et al., J
lymphoma, and prostate, breast, lung, colon,




Exp Med 82(3): 801-810 (1995); and Fraser
pancreatic, esophageal, stomach, brain, liver




et al., 29(3): 838-844 (1999), the contents of
and urinary cancer. Other preferred indications




each of which are herein incorporated by
include benign dysproliferative disorders and




reference in its entirety. Monocytic cells
pre-neoplastic conditions, such as, for example,




that may be used according to these assays
hyperplasia, metaplasia, and/or dysplasia.




are publicly available (e.g., through the
Preferred indications also include anemia,




ATCC TM). Exemplary human monocyte
pancytopenia, leukopenia, thrombocytopenia,




cells that may be used according to these
Hodgkin's disease, acute lymphocytic anemia




assays include the U937 cell line, which is
(ALL), plasmacytomas, multiple myeloma,




cell line derived by Sundstrom and Nilsson
Burkitt's lymphoma, arthritis, AIDS,




in 1974 from malignant cells obtained from
granulomatous disease, inflammatory bowel




the pleural effusion of a patient with
disease, sepsis, neutropenia, neutrophilia,




histiocytic lymphoma.
psoriasis, hemophilia, hypercoagulation,





diabetes mellitus, endocarditis, meningitis,





Lyme Disease, suppression of immune





reactions to transplanted organs, asthma and





allergy.


38
Activation of
Assays for the activation of transcription




transcription through
through the NFKB response element are




NFKB response
well-known in the art and may be used or




element in neuronal
routinely modified to assess the ability of




cells (such as
polypeptides of the invention (including




SKNMC cells).
antibodies and agonists or antagonists of the





invention) to regulate NFKB transcription





factors and modulate expression of neuronal





genes. Exemplary assays for transcription





through the NFKB response element that





may be used or routinely modified to test





NFKB-response element activity of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) include assays disclosed in: Gill J S,





et al., Neurobiol Dis, 7(4): 448-461





(2000); Tamatani M, et al., J Biol Chem,





274(13): 8531-8538 (1999); Berger et al.,





Gene 66: 1-10 (1998); Cullen and Malm,





Methods in Enzymol 216: 362-368 (1992);





Henthorn et al., Proc Natl Acad Sci USA





85: 6342-6346 (1988); Valle Blazquez et al,





Immunology 90(3): 455-460 (1997);





Aramburau et al., J Exp Med 82(3): 801-810





(1995); and Fraser et al., 29(3): 838-844





(1999), the contents of each of which are





herein incorporated by reference in its





entirety. Neuronal cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM).





Exemplary neuronal cells that may be used





according to these assays include the





SKNMC neuronal cell line.



39
Activation of
Assays for the activation of transcription
Preferred embodiments of the invention include



transcription through
through the NFKB response element are
using polypeptides of the invention (or



NFKB response
well-known in the art and may be used or
antibodies, agonists, or antagonists thereof) in



element in neuronal
routinely modified to assess the ability of
detection, diagnosis, prevention, and/or



cells (such as
polypeptides of the invention (including
treatment of Neurological Diseases and



SKNMC cells).
antibodies and agonists or antagonists of the
Disorders (e.g. Alzheimer''s Disease,




invention) to regulate NFKB transcription
Parkinson''s Disease, Brain Cancer, Seizures).




factors and modulate expression of neuronal





genes. Exemplary assays for transcription





through the NFKB response element that





may be used or routinely modified to test





NFKB-response element activity of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) include assays disclosed in: Gill J S,





et al., Neurobiol Dis, 7(4): 448-461





(2000); Tamatani M, et al., J Biol Chem,





274(13): 8531-8538 (1999); Berger et al.,





Gene 66: 1-10 (1998); Cullen and Malm,





Methods in Enzymol 216: 362-368 (1992);





Henthorn et al., Proc Natl Acad Sci USA





85: 6342-6346 (1988); Valle Blazquez et al,





Immunology 90(3): 455-460 (1997);





Aramburau et al., J Exp Med 82(3): 801-810





(1995); and Fraser et al., 29(3): 838-844





(1999), the contents of each of which are





herein incorporated by reference in its





entirety. Neuronal cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM).





Exemplary neuronal cells that may be used





according to these assays include the





SKNMC neuronal cell line.



40
Activation of
Assays for the activation of transcription
A preferred embodiment of the invention



transcription through
through the Serum Response Element
includes a method for inhibiting (e.g., reducing)



serum response
(SRE) are well-known in the art and may be
TNF alpha production. An alternative highly



element in immune
used or routinely modified to assess the
preferred embodiment of the invention includes



cells (such as natural
ability of polypeptides of the invention
a method for stimulating (e.g., increasing) TNF



killer cells).
(including antibodies and agonists or
alpha production. Preferred indications




antagonists of the invention) to regulate
include blood disorders (e.g., as described




serum response factors and modulate the
below under “Immune Activity”, “Blood-




expression of genes involved in growth and
Related Disorders”, and/or “Cardiovascular




upregulate the function of growth-related
Disorders”), Highly preferred indications




genes in many cell types. Exemplary assays
include autoimmune diseases (e.g., rheumatoid




for transcription through the SRE that may
arthritis, systemic lupus erythematosis, Crohn''s




be used or routinely modified to test SRE
disease, multiple sclerosis and/or as described




activity of the polypeptides of the invention
below), immunodeficiencies (e.g., as described




(including antibodies and agonists or
below), boosting a T cell-mediated immune




antagonists of the invention) include assays
response, and suppressing a T cell-mediated




disclosed in Berger et al., Gene 66: 1-10
immune response. Additional highly preferred




(1998); Cullen and Malm, Methods in
indications include inflammation and




Enzymol 216: 362-368 (1992); Henthorn et
inflammatory disorders, and treating joint




al., Proc Natl Acad Sci USA 85: 6342-6346
damage in patients with rheumatoid arthritis.




(1988); Benson et al., J Immunol
An additional highly preferred indication is




153(9): 3862-3873 (1994); and Black et al.,
sepsis. Highly preferred indications include




Virus Genes 12(2): 105-117 (1997), the
neoplastic diseases (e.g., leukemia, lymphoma,




content of each of which are herein
and/or as described below under




incorporated by reference in its entirety. T
“Hyperproliferative Disorders”). Additionally,




cells that may be used according to these
highly preferred indications include neoplasms




assays are publicly available (e.g., through
and cancers, such as, for example, leukemia,




the ATCC TM). Exemplary T cells that may
lymphoma, melanoma, glioma (e.g., malignant




be used according to these assays include
glioma), solid tumors, and prostate, breast,




the NK-YT cell line, which is a human
lung, colon, pancreatic, esophageal, stomach,




natural killer cell line with cytolytic and
brain, liver and urinary cancer. Other preferred




cytotoxic activity.
indications include benign dysproliferative





disorders and pre-neoplastic conditions, such





as, for example, hyperplasia, metaplasia, and/or





dysplasia. Preferred indications include





anemia, pancytopenia, leukopenia,





thrombocytopenia, Hodgkin's disease, acute





lymphocytic anemia (ALL), plasmacytomas,





multiple myeloma, Burkitt's lymphoma,





arthritis, AIDS, granulomatous disease,





inflammatory bowel disease, neutropenia,





neutrophilia, psoriasis, suppression of immune





reactions to transplanted organs and tissues,





hemophilia, hypercoagulation, diabetes





mellitus, endocarditis, meningitis, Lyme





Disease, cardiac reperfusion injury, and asthma





and allergy. An additional preferred





indication is infection (e.g., an infectious





disease as described below under “Infectious





Disease”).


41
Activation of
Assays for the activation of transcription
A preferred embodiment of the invention



transcription through
through the Serum Response Element
includes a method for inhibiting (e.g., reducing)



serum response
(SRE) are well-known in the art and may be
TNF alpha production. An alternative preferred



element in immune
used or routinely modified to assess the
embodiment of the invention includes a method



cells (such as T-
ability of polypeptides of the invention
for stimulating (e.g., increasing) TNF alpha



cells).
(including antibodies and agonists or
production. Preferred indications include




antagonists of the invention) to regulate the
blood disorders (e.g., as described below under




serum response factors and modulate the
“Immune Activity”, “Blood-Related




expression of genes involved in growth.
Disorders”, and/or ““Cardiovascular




Exemplary assays for transcription through
Disorders””), Highly preferred indications




the SRE that may be used or routinely
include autoimmune diseases (e.g., rheumatoid




modified to test SRE activity of the
arthritis, systemic lupus erythematosis, Crohn's




polypeptides of the invention (including
disease, multiple sclerosis and/or as described




antibodies and agonists or antagonists of the
below), immunodeficiencies (e.g., as described




invention) include assays disclosed in
below), boosting a T cell-mediated immune




Berger et al., Gene 66: 1-10 (1998); Cullen
response, and suppressing a T cell-mediated




and Malm, Methods in Enzymol 216: 362-368
immune response. Additional highly preferred




(1992); Henthorn et al., Proc Natl Acad
indications include inflammation and




Sci USA 85: 6342-6346 (1988); and Black et
inflammatory disorders, and treating joint




al., Virus Genes 12(2): 105-117 (1997), the
damage in patients with rheumatoid arthritis.




content of each of which are herein
An additional highly preferred indication is




incorporated by reference in its entirety. T
sepsis. Highly preferred indications include




cells that may be used according to these
neoplastic diseases (e.g., leukemia, lymphoma,




assays are publicly available (e.g., through
and/or as described below under




the ATCC TM). Exemplary mouse T cells
“Hyperproliferative Disorders”). Additionally,




that may be used according to these assays
highly preferred indications include neoplasms




include the CTLL cell line, which is an IL-2
and cancers, such as, for example, leukemia,




dependent suspension culture of T cells with
lymphoma, melanoma, glioma (e.g., malignant




cytotoxic activity. Exemplary human T
glioma), solid tumors, and prostate, breast,




cells, such as the MOLT4, that may be used
lung, colon, pancreatic, esophageal, stomach,




according to these assays are publicly
brain, liver and urinary cancer. Other preferred




available (e.g., through the ATCC TM).
indications include benign dysproliferative




Additional exemplary human T cells that
disorders and pre-neoplastic conditions, such




may be used according to these assays
as, for example, hyperplasia, metaplasia, and/or




include the JURKAT cell line, which is a
dysplasia. Preferred indications include




suspension culture of leukemia cells that
anemia, pancytopenia, leukopenia,




produce IL-2 when stimulated.
thrombocytopenia, Hodgkin's disease, acute





lymphocytic anemia (ALL), plasmacytomas,





multiple myeloma, Burkitt's lymphoma,





arthritis, AIDS, granulomatous disease,





inflammatory bowel disease, neutropenia,





neutrophilia, psoriasis, suppression of immune





reactions to transplanted organs and tissues,





hemophilia, hypercoagulation, diabetes





mellitus, endocarditis, meningitis, Lyme





Disease, cardiac reperfusion injury, and asthma





and allergy. An additional preferred indication





is infection (e.g., an infectious disease as





described below under “Infectious Disease”).


42
Activation of
Assays for the activation of transcription
A highly preferred indication is obesity and/or



transcription through
through the Serum Response Element
complications associated with obesity.



serum response
(SRE) are well-known in the art and may be
Additional highly preferred indications include



element in pre-
used or routinely modified to assess the
weight loss or alternatively, weight gain. An



adipocytes.
ability of polypeptides of the invention
additional highly preferred indication is




(including antibodies and agonists or
diabetes mellitus. An additional highly




antagonists of the invention) to regulate the
preferred indication is a complication




serum response factors and modulate the
associated with diabetes (e.g., diabetic




expression of genes involved in growth.
retinopathy, diabetic nephropathy, kidney




Exemplary assays for transcription through
disease (e.g., renal failure, nephropathy and/or




the SRE that may be used or routinely
other diseases and disorders as described in the




modified to test SRE activity of the
“Renal Disorders” section below), diabetic




polypeptides of the invention (including
neuropathy, nerve disease and nerve damage




antibodies and agonists or antagonists of the
(e.g., due to diabetic neuropathy), blood vessel




invention) include assays disclosed in
blockage, heart disease, stroke, impotence (e.g.,




Berger et al., Gene 66: 1-10 (1998); Cullen
due to diabetic neuropathy or blood vessel




and Malm, Methods in Enzymol 216: 362-368
blockage), seizures, mental confusion,




(1992); Henthorn et al., Proc Natl Acad
drowsiness, nonketotic hyperglycemic-




Sci USA 85: 6342-6346 (1988); and Black et
hyperosmolar coma, cardiovascular disease




al., Virus Genes 12(2): 105-117 (1997), the
(e.g., heart disease, atherosclerosis,




content of each of which are herein
microvascular disease, hypertension, stroke,




incorporated by reference in its entirety.
and other diseases and disorders as described in




Pre-adipocytes that may be used according
the “Cardiovascular Disorders” section below),




to these assays are publicly available (e.g.,
dyslipidemia, endocrine disorders (as described




through the ATCC TM) and/or may be
in the “Endocrine Disorders” section below),




routinely generated. Exemplary mouse
neuropathy, vision impairment (e.g., diabetic




adipocyte cells that may be used according
retinopathy and blindness), ulcers and impaired




to these assays include 3T3-L1 cells. 3T3-
wound healing, and infection (e.g., infectious




L1 is an adherent mouse preadipocyte cell
diseases and disorders as described in the




line that is a continuous substrain of 3T3
“Infectious Diseases” section below).




fibroblast cells developed through clonal
Additional highly preferred indications are




isolation and undergo a pre-adipocyte to
complications associated with insulin




adipose-like conversion under appropriate
resistance.




differentiation conditions known in the art.



43
Activation of
Assays for the activation of transcription
Highly preferred indications include allergy,



transcription through
through the Signal Transducers and
asthma, and rhinitis. Additional highly



STAT6 response
Activators of Transcription (STAT6)
preferred indications include infection (e.g., an



element in immune
response element in immune cells (such as
infectious disease as described below under



cells (such as mast
in the human HMC-1 mast cell line) are
“Infectious Disease”), and inflammation and



cells).
well-known in the art and may be used or
inflammatory disorders. Preferred indications




routinely modified to assess the ability of
also include hematopoietic and immunological




polypeptides of the invention (including
disorders (e.g., as described below under




antibodies and agonists or antagonists of the
“Immune Activity”, “Blood-Related




invention) to regulate STAT6 transcription
Disorders”, and/or “Cardiovascular Disorders”),




factors and modulate the expression of
autoimmune diseases (e.g., rheumatoid arthritis,




multiple genes. Exemplary assays for
systemic lupus erythematosis, multiple sclerosis




transcription through the STAT6 response
and/or as described below), and




element that may be used or routinely
immunodeficiencies (e.g., as described below).




modified to test STAT6 response element
Preferred indications include neoplastic




activity of the polypeptides of the invention
diseases (e.g., leukemia, lymphoma, melanoma,




(including antibodies and agonists or
and/or as described below under




antagonists of the invention) include assays
“Hyperproliferative Disorders”). Preferred




disclosed in Berger et al., Gene 66: 1-10
indications include neoplasms and cancer, such




(1998); Cullen and Malm, Methods in
as, for example, leukemia, lymphoma,




Enzymol 216: 362-368 (1992); Henthorn et
melanoma, and prostate, breast, lung, colon,




al., Proc Natl Acad Sci USA 85: 6342-6346
pancreatic, esophageal, stomach, brain, liver




(1988); Sherman, Immunol Rev 179: 48-56
and urinary cancer. Other preferred indications




(2001); Malaviya and Uckun, J Immunol
include benign dysproliferative disorders and




168: 421-426 (2002); Masuda et al., J Biol
pre-neoplastic conditions, such as, for example,




Chem 275(38): 29331-29337 (2000); and
hyperplasia, metaplasia, and/or dysplasia.




Masuda et al., J Biol Chem 276: 26107-26113
Preferred indications include hematopoietic and




(2001), the contents of each of which
immunological disorders such as arthritis,




are herein incorporated by reference in its
AIDS, granulomatous disease, inflammatory




entirety. Mast cells that may be used
bowel disease, sepsis, neutropenia, neutrophilia,




according to these assays are publicly
psoriasis, suppression of immune reactions to




available (e.g., through the ATCC TM).
transplanted organs and tissues, hemophilia,




Exemplary human mast cells that may be
hypercoagulation, diabetes mellitus,




used according to these assays include the
endocarditis, meningitis, and Lyme Disease.




HMC-1 cell line, which is an immature





human mast cell line established from the





peripheral blood of a patient with mast cell





leukemia, and exhibits many characteristics





of immature mast cells.



44
Activation of
Assays for the activation of transcription




transcription through
through the Signal Transducers and




STAT6 response
Activators of Transcription (STAT6)




element in immune
response element are well-known in the art




cells (such as natural
and may be used or routinely modified to




killer cells).
assess the ability of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) to regulate





STAT6 transcription factors and modulate





the expression of multiple genes.





Exemplary assays for transcription through





the STAT6 response element that may be





used or routinely modified to test STAT6





response element activity of the





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) include assays disclosed in





Berger et al., Gene 66: 1-10 (1998); Cullen





and Malm, Methods in Enzymol 216: 362-368





(1992); Henthorn et al., Proc Natl Acad





Sci USA 85: 6342-6346 (1988); Georas et





al., Blood 92(12): 4529-4538 (1998);





Moffatt et al., Transplantation 69(7): 1521-1523





(2000); Curiel et al., Eur J Immunol





27(8): 1982-1987 (1997); and Masuda et al.,





J Biol Chem 275(38): 29331-29337 (2000),





the contents of each of which are herein





incorporated by reference in its entirety. T





cells that may be used according to these





assays are publicly available (e.g., through





the ATCC TM). Exemplary rat natural killer





cells that may be used according to these





assays are publicly available (e.g., through





the ATCC TM).



45
Activation of
Assays for the activation of transcription
A highly preferred indication is allergy.



transcription through
through the Signal Transducers and
Another highly preferred indication is asthma.



STAT6 response
Activators of Transcription (STAT6)
Additional highly preferred indications include



element in immune
response element are well-known in the art
inflammation and inflammatory disorders.



cells (such as T-
and may be used or routinely modified to
Preferred indications include blood disorders



cells).
assess the ability of polypeptides of the
(e.g., as described below under “Immune




invention (including antibodies and agonists
Activity”, “Blood-Related Disorders”, and/or




or antagonists of the invention) to regulate
“Cardiovascular Disorders”). Preferred




STAT6 transcription factors and modulate
indications include autoimmune diseases (e.g.,




the expression of multiple genes.
rheumatoid arthritis, systemic lupus




Exemplary assays for transcription through
erythematosis, multiple sclerosis and/or as




the STAT6 response element that may be
described below) and immunodeficiencies (e.g.,




used or routinely modified to test STAT6
as described below). Preferred




response element activity of the
indications include neoplastic diseases (e.g.,




polypeptides of the invention (including
leukemia, lymphoma, melanoma, and/or as




antibodies and agonists or antagonists of the
described below under “Hyperproliferative




invention) include assays disclosed in
Disorders”). Preferred indications include




Berger et al., Gene 66: 1-10 (1998); Cullen
neoplasms and cancers, such as, leukemia,




and Malm, Methods in Enzymol 216: 362-368
lymphoma, melanoma, and prostate, breast,




(1992); Henthorn et al., Proc Natl Acad
lung, colon, pancreatic, esophageal, stomach,




Sci USA 85: 6342-6346 (1988); Georas et
brain, liver and urinary cancer. Other preferred




al., Blood 92(12): 4529-4538 (1998);
indications include benign dysproliferative




Moffatt et al., Transplantation 69(7): 1521-1523
disorders and pre-neoplastic conditions, such




(2000); Curiel et al., Eur J Immunol
as, for example, hyperplasia, metaplasia, and/or




27(8): 1982-1987 (1997); and Masuda et al.,
dysplasia. Preferred indications




J Biol Chem 275(38): 29331-29337 (2000),
include anemia, pancytopenia, leukopenia,




the contents of each of which are herein
thrombocytopenia, Hodgkin's disease, acute




incorporated by reference in its entirety. T
lymphocytic anemia (ALL), plasmacytomas,




cells that may be used according to these
multiple myeloma, Burkitt's lymphoma,




assays are publicly available (e.g., through
arthritis, AIDS, granulomatous disease,




the ATCC TM). Exemplary T cells that may
inflammatory bowel disease, sepsis,




be used according to these assays include
neutropenia, neutrophilia, psoriasis, suppression




the SUPT cell line, which is a suspension
of immune reactions to transplanted organs and




culture of IL-2 and IL-4 responsive T cells.
tissues, hemophilia, hypercoagulation, diabetes





mellitus, endocarditis, meningitis, and Lyme





Disease. An additional preferred





indication is infection (e.g., an infectious





disease as described below under “Infectious





Disease”).


46
Activation of
Assays for the activation of transcription




transcription through
through the EGR response element are well-




the EGR (Early
known in the art and may be used or




Growth Response)
routinely modified to assess the ability of




element in immune
polypeptides of the invention (including




cells (such as B-
antibodies and agonists or antagonists of the




cells).
invention) to regulate EGR transcription





factors and modulate expression of





immunomodulatory genes. Exemplary





assays for transcription through the EGR





response element that may be used or





routinely modified to test EGR response





element activity of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) include





assays disclosed in: Richards J D, et al., J





Immunol, 166(6): 3855-3864 (2001);





Dinkel, A, et al., J Exp Med, 188(12): 2215-2224





(1998); and, Newton, J S, et al., Eur J





Immunol 1996 April; 26(4): 811-816 (1996),





the contents of each of which are herein





incorporated by reference in its entirety.





Immune cells that may be used according to





these assays are publicly available (e.g.,





through the ATCC TM). Exemplary





epithelial cells that may be used according





to these assays include the Raji cell line.



47
Activation of
Assays for the activation of transcription
Preferred embodiments of the invention include



transcription through
through the EGR response element are well-
using polypeptides of the invention (or



the EGR (Early
known in the art and may be used or
antibodies, agonists, or antagonists thereof) in



Growth Response)
routinely modified to assess the ability of
detection, diagnosis, prevention, and/or



element in immune
polypeptides of the invention (including
treatment of Cancer, Autoimmunity, Allergy



cells (such as B-
antibodies and agonists or antagonists of the
and Asthma.



cells).
invention) to regulate EGR transcription





factors and modulate expression of





immunomodulatory genes. Exemplary





assays for transcription through the EGR





response element that may be used or





routinely modified to test EGR response





element activity of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) include





assays disclosed in: Richards J D, et al., J





Immunol, 166(6): 3855-3864 (2001);





Dinkel, A, et al., J Exp Med, 188(12): 2215-2224





(1998); and, Newton, J S, et al., Eur J





Immunol 1996 April; 26(4): 811-816 (1996),





the contents of each of which are herein





incorporated by reference in its entirety.





Immune cells that may be used according to





these assays are publicly available (e.g.,





through the ATCC TM). Exemplary





epithelial cells that may be used according





to these assays include the Raji cell line.



48
Activation or
This reporter assay measures activation or




inhibition of
inhibition of the NFkB signaling pathway in




transcription through
Ku812 human basophil cell line. Assays for




NFKB response
the activation or inhibition of transcription




element in immune
through the NFKB response element are




cells (such as
well-known in the art and may be used or




basophils).
routinely modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to regulate NFKB transcription





factors and modulate expression of





immunomodulatory genes. NFkB is





important in the pathogenesis of asthma.





Exemplary assays for transcription through





the NFKB response element that may be





used or rountinely modified to test NFKB-





response element activity of polypeptides of





the invention (including antibodies and





agonists or antagonists of the invention)





include assays disclosed in Berger et al.,





Gene 66: 1-10 (1998); Cullen and Malm,





Methods in Enzymol 216: 362-368 (1992);





Henthorn et al., Proc Natl Acad Sci USA





85: 6342-6346 (1988); Marone et al, Int





Arch Allergy Immunol 114(3): 207-17





(1997), the contents of each of which are





herein incorporated by reference in its





entirety. Cells were pretreated with SID





supernatants or controls for 15-18 hours,





and then 10 ng/mL of TNF was added to





stimulate the NFkB reporter. SEAP activity





was measured after 48 hours. Basophils





that may be used according to these assays





are publicly available (e.g., through the





ATCC TM). Exemplary human basophil cell





lines that may be used according to these





assays include Ku812, originally established





from a patient with chronic myelogenous





leukemia. It is an immature prebasophilic





cell line that can be induced to differentiate





into mature basophils. See, Kishi et al.,





Leuk Res. 9: 381-390 (1985); Blom et al.,





Eur J Immunol. 22: 2025-32 (1992), where





the contents of each are herein incorporated





by reference in its entirety.



49
ATF-2 in CTLL-2
final_assay_desc
final_assay_embodiment


50
Bone marrow cell
Assay for measuring regulation of




proliferation
proliferation of mouse bone marrow cells




(fibronectin
(in the presence or absence of exogenous




enhanced)
Stem Cell Factor (SCF)) on a fibronectin





extracellular matrix. Mouse bone marrow





cells are plated onto 96-well fibronectin





fragment coated plates in 0.2 ml of serum-





free medium. Secreted protein factors (test





factors) are tested with appropriate negative





controls in the presence and absence of SCF





(5.0 ng/ml), where secreted test factor





supernates represent 10% of the total assay





volume. The cells are grown for 7 days.





The number of proliferating cells within the





wells is quantitated by measuring thymidine





incorporation into cellular DNA. This and





similar assays may be used or routinely





modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to regulate proliferation of bone





marrow cells. Interactions between





adhesion receptors on progenitor cells and





their extracellular matrix ligands are





essential for the control of hematopoiesis in





bone marrow stroma. These interactions





may help retain CD34+ hematopoietic





progenitor cells within the an appropriate





bone marrow environment, and adhesive





interactions can also provide important





costimulatory signals. As the ability of





stem cells to undergo self-renewal in vitro is





dependent upon their interaction with the





stromal cells and the extracellular matrix





(ECM), this assay identify factors which





integrate with the ECM environment and





are important for stimulating stem cell self-





renewal.



51
Calcium flux in
Assays for measuring calcium flux are well-
Preferred embodiments of the invention include



chondrocytes
known in the art and may be used or
using polypeptides of the invention (or




routinely modified to assess the ability of
antibodies, agonists, or antagonists thereof) in




polypeptides of the invention (including
detection, diagnosis, prevention, and/or




antibodies and agonists or antagonists of the
treatment of Bone and Cartilage Diseases,




invention) to mobilize calcium. Cells
including but not limited to Arthritis, Cartilige




normally have very low concentrations of
repair, Bone Repair, Osteoporosis, and related




cytosolic calcium compared to much higher
tumors including chondrosarcomas,




extracellular calcium. Extracellular factors
chondroblastomas, and chondromas.




can cause an influx of calcium, leading to





activation of calcium responsive signaling





pathways and alterations in cell functions.





Exemplary assays that may be used or





routinely modified to measure calcium flux





in chondrocytes include assays disclosed in:





Asada S, et al., Inflamm Res, 50(1): 19-23





(2001); Schwartz Z, et al., J Bone Miner





Res, 6(7): 709-718 (1991); Iannotti J P, et al.,





J Bone Joint Surg Am, 67(1): 113-120





(1985); Sullivan E., et al., Methods Mol





Biol 1999; 114: 125-133 (1999), the





contents of each of which is herein





incorporated by reference in its entirety.





Cells that may be used according to these





assays are publicly available (e.g., through





the ATCC TM) and/or may be routinely





generated. Exemplary cells that may be





used according to these assays include





bovine chondrocytes.



52
Calcium flux in
Assays for measuring calcium flux are well-
Preferred embodiments of the invention include



immune cells (such
known in the art and may be used or
using polypeptides of the invention (or



as monocytes)
routinely modified to assess the ability of
antibodies, agonists, or antagonists thereof) in




polypeptides of the invention (including
detection, diagnosis, prevention, and/or




antibodies and agonists or antagonists of the
treatment of Infection, Inflammation,




invention) to mobilize calcium. Cells
Atherosclerosis, Hypersensitivity, and




normally have very low concentrations of
Leukemias




cytosolic calcium compared to much higher





extracellular calcium. Extracellular factors





can cause an influx of calcium, leading to





activation of calcium responsive signaling





pathways and alterations in cell functions.





Exemplary assays that may be used or





routinely modified to measure calcium flux





in immune cells (such as monocytes)





include assays disclosed in: Chan, C C, et





al., J Pharmacol Exp Ther, 269(3): 891-896





(1994); Andersson, K, et al., Cytokine,





12(12): 1784-1787 (2000); Scully, S P, et al.,





J Clin Invest, 74(2) 589-599 (1984); and,





Sullivan, E, et al., Methods Mol Biol,





114: 125-133 (1999), the contents of each of





which is herein incorporated by reference in





its entirety. Cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM) and/or





may be routinely generated. Exemplary





cells that may be used according to these





assays include the THP-1 monocyte cell





line.



53
Caspase





(+camptothecin) in





SW480




54
Caspase (+paclitaxel)





in SW480




55
CD152 in Human T





cells




56
CD69 in Human T





cells




57
CXCR4 in HT1080




58
CXCR4 in SW480




59
Endothelial Cell
Caspase Apoptosis. Assays for caspase
A highly preferred embodiment of the invention



Apoptosis
apoptosis are well known in the art and may
includes a method for stimulating endothelial




be used or routinely modified to assess the
cell growth. An alternative highly preferred




ability of polypeptides of the invention
embodiment of the invention includes a method




(including antibodies and agonists or
for inhibiting endothelial cell growth. A




antagonists of the invention) to promote
highly preferred embodiment of the invention




caspase protease-mediated apoptosis.
includes a method for stimulating endothelial




Induction of apoptosis in endothelial cells
cell proliferation. An alternative highly




supporting the vasculature of tumors is
preferred embodiment of the invention includes




associated with tumor regression due to loss
a method for inhibiting endothelial cell




of tumor blood supply. Exemplary assays
proliferation. A highly preferred




for caspase apoptosis that may be used or
embodiment of the invention includes a method




routinely modified to test capase apoptosis
for stimulating apoptosis of endothelial cells.




activity of polypeptides of the invention
An alternative highly preferred embodiment of




(including antibodies and agonists or
the invention includes a method for inhibiting




antagonists of the invention) include the
(e.g., decreasing) apoptosis of endothelial cells.




assays disclosed in Lee et al., FEBS Lett
A highly preferred embodiment of the invention




485(2-3): 122-126 (2000); Nor et al., J Vasc
includes a method for stimulating angiogenisis.




Res 37(3): 209-218 (2000); and Karsan and
An alternative highly preferred embodiment of




Harlan, J Atheroscler Thromb 3(2): 75-80
the invention includes a method for inhibiting




(1996); the contents of each of which are
angiogenesis. A highly preferred




herein incorporated by reference in its
embodiment of the invention includes a method




entirety. Endothelial cells that may be used
for reducing cardiac hypertrophy. An




according to these assays are publicly
alternative highly preferred embodiment of the




available (e.g., through commercial
invention includes a method for inducing




sources). Exemplary endothelial cells that
cardiac hypertrophy. Highly preferred




may be used according to these assays
indications include neoplastic diseases (e.g., as




include bovine aortic endothelial cells
described below under “Hyperproliferative




(bAEC), which are an example of
Disorders”), and disorders of the cardiovascular




endothelial cells which line blood vessels
system (e.g., heart disease, congestive heart




and are involved in functions that include,
failure, hypertension, aortic stenosis,




but are not limited to, angiogenesis,
cardiomyopathy, valvular regurgitation, left




vascular permeability, vascular tone, and
ventricular dysfunction, atherosclerosis and




immune cell extravasation.
atherosclerotic vascular disease, diabetic





nephropathy, intracardiac shunt, cardiac





hypertrophy, myocardial infarction, chronic





hemodynamic overload, and/or as described





below under “Cardiovascular Disorders”).





Highly preferred indications include





cardiovascular, endothelial and/or angiogenic





disorders (e.g., systemic disorders that affect





vessels such as diabetes mellitus, as well as





diseases of the vessels themselves, such as of





the arteries, capillaries, veins and/or





lymphatics). Highly preferred are indications





that stimulate angiogenesis and/or





cardiovascularization. Highly preferred are





indications that inhibit angiogenesis and/or





cardiovascularization. Highly preferred





indications include antiangiogenic activity to





treat solid tumors, leukemias, and Kaposi''s





sarcoma, and retinal disorders. Highly preferred





indications include neoplasms and cancer, such





as, Kaposi''s sarcoma, hemangioma (capillary





and cavernous), glomus tumors, telangiectasia,





bacillary angiomatosis, hemangioendothelioma,





angiosarcoma, haemangiopericytoma,





lymphangioma, lymphangiosarcoma. Highly





preferred indications also include cancers such





as, prostate, breast, lung, colon, pancreatic,





esophageal, stomach, brain, liver, and urinary





cancer. Preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia. Highly





preferred indications also include arterial





disease, such as, atherosclerosis, hypertension,





coronary artery disease, inflammatory





vasculitides, Reynaud''s disease and Reynaud''s





phenomenom, aneurysms, restenosis; venous





and lymphatic disorders such as





thrombophlebitis, lymphangitis, and





lymphedema; and other vascular disorders such





as peripheral vascular disease, and cancer.





Highly preferred indications also include





trauma such as wounds, burns, and injured





tissue (e.g., vascular injury such as, injury





resulting from balloon angioplasty, and





atheroschlerotic lesions), implant fixation,





scarring, ischemia reperfusion injury,





rheumatoid arthritis, cerebrovascular disease,





renal diseases such as acute renal failure, and





osteoporosis. Additional highly preferred





indications include stroke, graft rejection,





diabetic or other retinopathies, thrombotic and





coagulative disorders, vascularitis, lymph





angiogenesis, sexual disorders, age-related





macular degeneration, and treatment/





prevention of endometriosis and related





conditions. Additional highly preferred





indications include fibromas, heart disease,





cardiac arrest, heart valve disease, and vascular





disease. Preferred indications include





blood disorders (e.g., as described below under





“Immune Activity”, “Blood-Related





Disorders”, and/or “Cardiovascular Disorders”).





Preferred indications include autoimmune





diseases (e.g., rheumatoid arthritis, systemic





lupus erythematosis, multiple sclerosis and/or





as described below) and immunodeficiencies





(e.g., as described below). Additional preferred





indications include inflammation and





inflammatory disorders (such as acute and





chronic inflammatory diseases, e.g.,





inflammatory bowel disease and Crohn's





disease), and pain management.


60
Glucose Production





in H4IIE




61
Hexosaminidase in





RBL-2H3




62
HLA-DR in Human





T cells




63
ICAM in Normal





Human Bronchial





Epitheliae




64
ICAM in OE19




65
IFNg in Human T-





cell 293T




66
IFNg in Human T-





cell 2B9




67
IgG in Human B cells




68
IgG in Human B cells





SAC




69
IL-10 in Human T-





cell 293T




70
IL-10 in Human T-





cell 2B9




71
IL-13 in HMC




72
IL-13 in Human T





cells




73
IL-2 in Human T





cells




74
IL-2 in Human T-cell





293T




75
IL-4 in HMC




76
IL-6 in HUVEC




77
IL-8 in Normal





Human Bronchial





Epitheliae




78
IL-8 in SW480




79
Inhibition of
Kinase assay: measures the phosphorylation




adipocyte ERK
of Elk-1, an indication of activation of




signaling pathway.
extracellular signal regulated kinase (ERK).





ERK pathway regulates cell growth,





proliferation and differentiation. Cells were





pretreated with SID supernatants for 15-18





hours, and then 100 nM of insulin was





added to stimulate ERK kinase.





Phosphorylation of Elk-1 was measured





after a 20 minute incubation. Pre-





adipocytes that may be used according to





these assays are publicly available (e.g.,





through the ATCC TM) and/or may be





routinely generated. Exemplary mouse





adipocyte cells that may be used according





to these assays include 3T3-L1 cells. 3T3-





L1 is an adherent mouse preadipocyte cell





line that is a continuous substrain of 3T3





fibroblast cells developed through clonal





isolation and undergo a pre-adipocyte to





adipose-like conversion under appropriate





differentiation conditions known in the art.





Cells were differentiated to an adipose-like





state before being used in the screen. See





Green et al., Cell 3: 127-133 (1974), the





contents of which are herein incorporated





by reference in its entirety.



80
Inhibition of squalene
Reporter Assay: construct contains




synthetase gene
regulatory and coding sequence of squalene




transcription.
synthetase, the first specific enzyme in the





cholesterol biosynthetic pathway. See





Jiang, et al., J. Biol. Chem. 268: 12818-128241





(993), the contents of which are





herein incorporated by reference in its





entirety. Cells were treated with SID





supernatants, and SEAP activity was





measured after 72 hours. HepG2 is a





human hepatocellular carcinoma cell line





(ATCC TM HB-8065). See Knowles et al.,





Science. 209: 497-9 (1980), the contents of





which are herein incorporated by reference





in its entirety.



81
Insulin Secretion
Assays for measuring secretion of insulin
A highly preferred indication is diabetes




are well-known in the art and may be used
mellitus. An additional highly preferred




or routinely modified to assess the ability of
indication is a complication associated with




polypeptides of the invention (including
diabetes (e.g., diabetic retinopathy, diabetic




antibodies and agonists or antagonists of the
nephropathy, kidney disease (e.g., renal failure,




invention) to stimulate insulin secretion.
nephropathy and/or other diseases and disorders




For example, insulin secretion is measured
as described in the “Renal Disorders” section




by FMAT using anti-rat insulin antibodies.
below), diabetic neuropathy, nerve disease and




Insulin secretion from pancreatic beta cells
nerve damage (e.g., due to diabetic neuropathy),




is upregulated by glucose and also by
blood vessel blockage, heart disease, stroke,




certain proteins/peptides, and disregulation
impotence (e.g., due to diabetic neuropathy or




is a key component in diabetes. Exemplary
blood vessel blockage), seizures, mental




assays that may be used or routinely
confusion, drowsiness, nonketotic




modified to test for stimulation of insulin
hyperglycemic-hyperosmolar coma,




secretion (from pancreatic cells) by
cardiovascular disease (e.g., heart disease,




polypeptides of the invention (including
atherosclerosis, microvascular disease,




antibodies and agonists or antagonists of the
hypertension, stroke, and other diseases and




invention) include assays disclosed in:
disorders as described in the “Cardiovascular




Shimizu, H., et al., Endocr J, 47(3): 261-9
Disorders” section below), dyslipidemia,




(2000); Salapatek, A. M., et al., Mol
endocrine disorders (as described in the




Endocrinol, 13(8): 1305-17 (1999);
“Endocrine Disorders” section below),




Filipsson, K., et al., Ann N Y Acad Sci,
neuropathy, vision impairment (e.g., diabetic




865: 441-4 (1998); Olson, L. K., et al., J Biol
retinopathy and blindness), ulcers and impaired




Chem, 271(28): 16544-52 (1996); and,
wound healing, and infection (e.g., infectious




Miraglia S et. al., Journal of Biomolecular
diseases and disorders as described in the




Screening, 4: 193-204 (1999), the contents
“Infectious Diseases” section below, especially




of each of which is herein incorporated by
of the urinary tract and skin), carpal tunnel




reference in its entirety. Pancreatic cells
syndrome and Dupuytren's contracture).




that may be used according to these assays
An additional highly preferred indication is




are publicly available (e.g., through the
obesity and/or complications associated with




ATCC TM) and/or may be routinely
obesity. Additional highly preferred indications




generated. Exemplary pancreatic cells that
include weight loss or alternatively, weight




may be used according to these assays
gain. Additional highly preferred indications are




include HITT15 Cells. HITT15 are an
complications associated with insulin




adherent epithelial cell line established from
resistance.




Syrian hamster islet cells transformed with





SV40. These cells express glucagon,





somatostatin, and glucocorticoid receptors.





The cells secrete insulin, which is





stimulated by glucose and glucagon and





suppressed by somatostatin or





glucocorticoids. ATTC# CRL-1777 Refs:





Lord and Ashcroft. Biochem. J. 219: 547-551;





Santerre et al. Proc. Natl. Acad. Sci.





USA 78: 4339-4343, 1981.



82
MCP-1 in Eol-1




83
MCP-1 in HUVEC




84
MIP-1a in HMC




85
Myoblast cell
Assays for muscle cell proliferation are well
Highly preferred indications include diabetes,



proliferation
known in the art and may be used or
myopathy, muscle cell atrophy, cancers of




routinely modified to assess the ability of
muscle (such as, rhabdomyoma, and




polypeptides of the invention (including
rhabdosarcoma), cardiovascular disorders (such




antibodies and agonists or antagonists of the
as congestive heart failure, cachexia, myxomas,




invention) to stimulate or inhibit myoblast
fibromas, congenital cardiovascular




cell proliferation. Exemplary assays for
abnormalities, heart disease, cardiac arrest,




myoblast cell proliferation that may be used
heart valve disease, vascular disease, and also




or routinely modified to test activity of
as described below under “Cardiovascular




polypeptides and antibodies of the invention
Disorders”), stimulating myoblast proliferation,




(including agonists or antagonists of the
and inhibiting myoblast proliferation.




invention) include, for example, assays





disclosed in: Soeta, C., et al. “Possible role





for the c-ski gene in the proliferation of





myogenic cells in regenerating skeletal





muscles of rats” Dev Growth Differ





April; 43(2): 155-64 (2001); Ewton D Z, et al.,





“IGF binding proteins-4, -5 and -6 may play





specialized roles during L6 myoblast





proliferation and differentiation” J





Endocrinol March; 144(3): 539-53 (1995); and,





Pampusch M S, et al., “Effect of transforming





growth factor beta on proliferation of L6





and embryonic porcine myogenic cells” J





Cell Physiol June; 143(3): 524-8 (1990); the





contents of each of which are herein





incorporated by reference in their entirety.





Exemplary myoblast cells that may be used





according to these assays include the rat





myoblast L6 cell line. Rat myoblast L6





cells are an adherent rat myoblast cell line,





isolated from primary cultures of rat thigh





muscle, that fuse to form multinucleated





myotubes and striated fibers after culture in





differentiation media.



86
Production of GM-
GM-CSF FMAT. GM-CSF is expressed by
A highly preferred embodiment of the invention



CSF
activated T cells, macrophages, endothelial
includes a method for stimulating the




cells, and fibroblasts. GM-CSF regulates
production of GM-CSF. An alternative highly




differentiation and proliferation of
preferred embodiment of the invention includes




granulocytes-macrophage progenitors and
a method for inhibiting the production of GM-




enhances antimicrobial activity in
CSF. Highly preferred indications include




neutrophils, monocytes and macrophage.
inflammation and inflammatory disorders. An




Additionally, GM-CSF plays an important
additional highly preferred indication is




role in the differentiation of dendritic cells
infection (e.g., as described below under




and monocytes, and increases antigen
“Infectious Disease”. Highly preferred




presentation. GM-CSF is considered to be a
indications include blood disorders (e.g.,




proinflammatory cytokine. Assays for
neutropenia (and the prevention of neutropenia




immunomodulatory proteins that promote
(e.g., in HIV infected patients), and/or as




the production of GM-CSF are well known
described below under “Immune Activity”,




in the art and may be used or routinely
“Blood-Related Disorders”, and/or




modified to assess the ability of
“Cardiovascular Disorders”). Highly preferred




polypeptides of the invention (including
indications also include autoimmune diseases




antibodies and agonists or antagonists of the
(e.g., rheumatoid arthritis, systemic lupus




invention) to mediate immunomodulation
erythematosis, multiple sclerosis and/or as




and modulate the growth and differentiation
described below) and immunodeficiencies (e.g.,




of leukocytes. Exemplary assays that test
as described below). Additional highly




for immunomodulatory proteins evaluate
preferred indications include asthma. Highly




the production of cytokines, such as GM-
preferred indications include neoplastic diseases




CSF, and the activation of T cells. Such
(e.g., leukemia (e.g., acute lymphoblastic




assays that may be used or routinely
leukemia, and acute myelogenous leukemia),




modified to test immunomodulatory activity
lymphoma (e.g., non-Hodgkin''s lymphoma and




of polypeptides of the invention (including
Hodgkin''s disease), and/or as described below




antibodies and agonists or antagonists of the
under “Hyperproliferative Disorders”). Highly




invention) include the assays disclosed in
preferred indications include neoplasms and




Miraglia et al., J Biomolecular Screening
cancers, such as, leukemia, lymphoma,




4: 193-204 (1999); Rowland et al.,
melanoma, and prostate, breast, lung, colon,




“Lymphocytes: a practical approach”
pancreatic, esophageal, stomach, brain, liver




Chapter 6: 138-160 (2000); and Ye et al., J
and urinary cancer. Other preferred indications




Leukoc Biol (58(2): 225-233, the contents of
include benign dysproliferative disorders and




each of which are herein incorporated by
pre-neoplastic conditions, such as, for example,




reference in its entirety. Natural killer cells
hyperplasia, metaplasia, and/or dysplasia.




that may be used according to these assays
Highly preferred indications include:




are publicly available (e.g., through the
suppression of immune reactions to




ATCC TM) or may be isolated using
transplanted organs and tissues (e.g., bone




techniques disclosed herein or otherwise
marrow transplant); accelerating myeloid




known in the art. Natural killer (NK) cells
recovery; and mobilizing hematopoietic




are large granular lymphocytes that have
progenitor cells. Preferred indications include




cytotoxic activity but do bind antigen. NK
boosting a T cell-mediated immune response,




cells show antibody-independent killing of
and alternatively, suppressing a T cell-mediated




tumor cells and also recognize antibody
immune response. Preferred indications




bound on target cells, via NK Fc receptors,
include anemia, pancytopenia, leukopenia,




leading to cell-mediated cytotoxicity.
thrombocytopenia, acute lymphocytic anemia





(ALL), plasmacytomas, multiple myeloma,





Burkitt's lymphoma, arthritis, AIDS,





granulomatous disease, inflammatory bowel





disease, sepsis, neutrophilia, psoriasis,





hemophilia, hypercoagulation, diabetes





mellitus, endocarditis, meningitis, Lyme





Disease, and allergy.


87
Production of ICAM
Endothelial cells, which are cells that line
Highly preferred indications include



in endothelial cells
blood vessels, and are involved in functions
inflammation (acute and chronic), restnosis,



(such as human
that include, but are not limited to,
atherosclerosis, asthma and allergy. Highly



umbilical vein
angiogenesis, vascular permeability,
preferred indications include inflammation and



endothelial cells
vascular tone, and immune cell
inflammatory disorders, immunological



(HUVEC))
extravasation. Exemplary endothelial cells
disorders, neoplastic disorders (e.g.




that may be used in ICAM production
cancer/tumorigenesis), and cardiovascular




assays include human umbilical vein
disorders (such as described below under




endothelial cells (HUVEC), and are
“Immune Activity”, “Blood-Related




available from commercial sources. The
Disorders”, “Hyperproliferative Disorders”




expression of ICAM (CD54), a intergral
and/or “Cardiovascular Disorders”). Highly




membrane protein, can be upregulated by
preferred indications include neoplasms and




cytokines or other factors, and ICAM
cancers such as, for example, leukemia,




expression is important in mediating
lymphoma, melanoma, renal cell carcinoma,




immune and endothelial cell interactions
and prostate, breast, lung, colon, pancreatic,




leading to immune and inflammatory
esophageal, stomach, brain, liver and urinary




responses. Assays for measuring expression
cancer. Other preferred indications include




of ICAM-1 are well-known in the art and
benign dysproliferative disorders and pre-




may be used or routinely modified to assess
neoplastic conditions, such as, for example,




the ability of polypeptides of the invention
hyperplasia, metaplasia, and/or dysplasia.




(including antibodies and agonists or





antagonists of the invention) to regulate





ICAM-1 expression. Exemplary assays that





may be used or routinely modified to





measure ICAM-1 expression include assays





disclosed in: Rolfe B E, et al.,





Atherosclerosis, 149(1): 99-110 (2000);





Panettieri R A Jr, et al., J Immunol,





154(5): 2358-2365 (1995); and, Grunstein M M,





et al., Am J Physiol Lung Cell Mol





Physiol, 278(6): L1154-L1163 (2000), the





contents of each of which is herein





incorporated by reference in its entirety.



88
Production of ICAM-1
Assays for measuring expression of ICAM-
Preferred embodiments of the invention include




1 are well-known in the art and may be used
using polypeptides of the invention (or




or routinely modified to assess the ability of
antibodies, agonists, or antagonists thereof) in




polypeptides of the invention (including
detection, diagnosis, prevention, and/or




antibodies and agonists or antagonists of the
treatment of Vascular Disease, Atherosclerosis,




invention) to regulate ICAM-1 expression.
Restenosis, Stroke, and Asthma.




Exemplary assays that may be used or





routinely modified to measure ICAM-1





expression include assays disclosed in:





Rolfe B E, et al., Atherosclerosis, 149(1): 99-110





(2000); Panettieri R A Jr, et al., J





Immunol, 154(5): 2358-2365 (1995); and,





Grunstein M M, et al., Am J Physiol Lung





Cell Mol Physiol, 278(6): L1154-L1163





(2000), the contents of each of which is





herein incorporated by reference in its





entirety. Cells that may be used according





to these assays are publicly available (e.g.,





through the ATCC TM) and/or may be





routinely generated. Exemplary cells that





may be used according to these assays





include Aortic Smooth Muscle Cells





(AOSMC); such as bovine AOSMC.





Exemplary cells that may be used according





to these assays include microvascular





endothelial cells (MVEC).



89
Production of
IFNgamma FMAT. IFNg plays a central
A highly preferred embodiment of the invention



IFNgamma using a T
role in the immune system and is considered
includes a method for stimulating the



cells
to be a proinflammatory cytokine. IFNg
production of IFNg. An alternative highly




promotes TH1 and inhibits TH2
preferred embodiment of the invention includes




differentiation; promotes IgG2a and inhibits
a method for inhibiting the production of IFNg.




IgE secretion; induces macrophage
Highly preferred indications include blood




activation; and increases MHC expression.
disorders (e.g., as described below under




Assays for immunomodulatory proteins
“Immune Activity”, “Blood-Related




produced by T cells and NK cells that
Disorders”, and/or ““Cardiovascular




regulate a variety of inflammatory activities
Disorders””), and infection (e.g., viral




and inhibit TH2 helper cell functions are
infections, tuberculosis, infections associated




well known in the art and may be used or
with chronic granulomatosus disease and




routinely modified to assess the ability of
malignant osteoporosis, and/or as described




polypeptides of the invention (including
below under “Infectious Disease”). Highly




antibodies and agonists or antagonists of the
preferred indications include autoimmune




invention) to mediate immunomodulation,
disease (e.g., rheumatoid arthritis, systemic




regulate inflammatory activities, modulate
lupus erythematosis, multiple sclerosis and/or




TH2 helper cell function, and/or mediate
as described below), immunodeficiency (e.g., as




humoral or cell-mediated immunity.
described below), boosting a T cell-mediated




Exemplary assays that test for
immune response, and suppressing a T cell-




immunomodulatory proteins evaluate the
mediated immune response. Additional highly




production of cytokines, such as Interferon
preferred indications include inflammation and




gamma (IFNg), and the activation of T
inflammatory disorders. Additional preferred




cells. Such assays that may be used or
indications include idiopathic pulmonary




routinely modified to test
fibrosis. Highly preferred indications include




immunomodulatory activity of polypeptides
neoplastic diseases (e.g., leukemia, lymphoma,




of the invention (including antibodies and
melanoma, and/or as described below under




agonists or antagonists of the invention)
“Hyperproliferative Disorders”). Highly




include the assays disclosed in Miraglia et
preferred indications include neoplasms and




al., J Biomolecular Screening 4: 193-204
cancers, such as, for example, leukemia,




(1999); Rowland et al., ““Lymphocytes: a
lymphoma, melanoma, and prostate, breast,




practical approach”” Chapter 6: 138-160
lung, colon, pancreatic, esophageal, stomach,




(2000); Gonzalez et al., J Clin Lab Anal
brain, liver and urinary cancer. Other preferred




8(5): 225-233 (1995); Billiau et al., Ann NY
indications include benign dysproliferative




Acad Sci 856: 22-32 (1998); Boehm et al.,
disorders and pre-neoplastic conditions, such




Annu Rev Immunol 15: 749-795 (1997), and
as, for example, hyperplasia, metaplasia, and/or




Rheumatology (Oxford) 38(3): 214-20
dysplasia. Preferred indications include




(1999), the contents of each of which are
anemia, pancytopenia, leukopenia,




herein incorporated by reference in its
thrombocytopenia, Hodgkin's disease, acute




entirety. Human T cells that may be used
lymphocytic anemia (ALL), plasmacytomas,




according to these assays may be isolated
multiple myeloma, Burkitt's lymphoma,




using techniques disclosed herein or
arthritis, AIDS, granulomatous disease,




otherwise known in the art. Human T cells
inflammatory bowel disease, sepsis,




are primary human lymphocytes that mature
neutropenia, neutrophilia, psoriasis, suppression




in the thymus and express a T Cell receptor
of immune reactions to transplanted organs and




and CD3, CD4, or CD8. These cells
tissues, hemophilia, hypercoagulation, diabetes




mediate humoral or cell-mediated immunity
mellitus, endocarditis, meningitis, Lyme




and may be preactivated to enhance
Disease, asthma and allergy.




responsiveness to immunomodulatory





factors.



90
Production of
IFNgamma FMAT. IFNg plays a central




IFNgamma using
role in the immune system and is considered




Natural Killer cells
to be a proinflammatory cytokine. IFNg





promotes TH1 and inhibits TH2; promotes





IgG2a and inhibits IgE; induces





macrophage activation; and increases MHC





expression. Assays for immunomodulatory





proteins produced by T cells and NK cells





that regulate a variety of inflammatory





activities and inhibit TH2 helper cell





functions are well known in the art and may





be used or routinely modified to assess the





ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to mediate





immunomodulation, regulate inflammatory





activities, modulate TH2 helper cell





function, and/or mediate humoral or cell-





mediated immunity. Exemplary assays that





test for immunomodulatory proteins





evaluate the production of cytokines, such





as Interferon gamma (IFNg), and the





activation of T cells. Such assays that may





be used or routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include the assays disclosed in Miraglia et





al., J Biomolecular Screening 4: 193-204





(1999); Rowland et al., “Lymphocytes: a





practical approach” Chapter 6: 138-160





(2000); Gonzalez et al., J Clin Lab Anal





8(5): 225-233 (1995); Billiau et al., Ann NY





Acad Sci 856: 22-32 (1998); Boehm et al.,





Annu Rev Immunol 15: 749-795 (1997), and





Rheumatology (Oxford) 38(3): 214-20





(1999), the contents of each of which are





herein incorporated by reference in its





entirety. Natural Killer (NK) cells that may





be used according to these assays are





publicly available (e.g., through the





ATCC TM) or may be isolated using





techniques disclosed herein or otherwise





known in the art. Natural killer (NK) cells





are large granular lymphocytes that have





cytotoxic activity but do bind antigen. NK





cells show antibody-independent killing of





tumor cells and also recognize antibody





bound on target cells, via NK Fc receptors,





leading to cell-mediated cytotoxicity.



91
Production of IL-10
Assays for production of IL-10 and
Highly preferred indications include allergy and



and activation of T-
activation of T-cells are well known in the
asthma. Additional highly preferred indications



cells.
art and may be used or routinely modified to
include immune and hematopoietic disorders




assess the ability of polypeptides of the
(e.g., as described below under “Immune




invention (including antibodies and agonists
Activity”, and “Blood-Related Disorders”),




or antagonists of the invention) to stimulate
autoimmune diseases (e.g., rheumatoid arthritis,




or inhibit production of IL-10 and/or
systemic lupus erythematosis, Crohn''s disease,




activation of T-cells. Exemplary assays that
multiple sclerosis and/or as described below),




may be used or routinely modified to assess
immunodeficiencies (e.g., as described below),




the ability of polypeptides and antibodies of
boosting a T cell-mediated immune response,




the invention (including agonists or
and suppressing a T cell-mediated immune




antagonists of the invention) to modulate
response.




IL-10 production and/or T-cell proliferation





include, for example, assays such as





disclosed and/or cited in: Robinson, D S, et





al., “Th-2 cytokines in allergic disease” Br





Med Bull; 56 (4): 956-968 (2000), and





Cohn, et al., “T-helper type 2 cell-directed





therapy for asthma” Pharmacology &





Therapeutics; 88: 187-196 (2000); the





contents of each of which are herein





incorporated by reference in their entirety.





Exemplary cells that may be used according





to these assays include Th2 cells. IL10





secreted from Th2 cells may be measured as





a marker of Th2 cell activation. Th2 cells





are a class of T cells that secrete IL4, IL10,





IL13, IL5 and IL6. Factors that induce





differentiation and activation of Th2 cells





play a major role in the initiation and





pathogenesis of allergy and asthma.





Primary T helper 2 cells are generated via in





vitro culture under Th2 polarizing





conditions using peripheral blood





lymphocytes isolated from cord blood.



92
Production of IL-10
IL-10 FMAT. Assays for




and downregulation
immunomodulatory proteins produced by




of immune responses
activated T cells, B cells, and monocytes





that exhibit anti-inflammatory activity and





downregulate monocyte/macrophage





function and expression of cytokines are





well known in the art and may be used or





routinely modified to assess the ability of





the polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to mediate immunomodulation,





regulate inflammatory activities, and





modulate immune cell function and





cytokine production. Exemplary assays that





test for immunomodulatory proteins





evaluate the production of cytokines, such





as IL-10, and the downmodulation of





immune responses. Such assays that may





be used or routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include the assays disclosed in Miraglia et





al., J Biomolecular Screening 4: 193-204





(1999); Rowland et al., “Lymphocytes: a





practical approach” Chapter 6: 138-160





(2000); and Koning et al., Cytokine





9(6): 427-436 (1997), the contents of each of





which are herein incorporated by reference





in its entirety. Human T cells that may be





used according to these assays may be





isolated using techniques disclosed herein





or otherwise known in the art. Human T





cells are primary human lymphocytes that





mature in the thymus and express a T cell





receptor and CD3, CD4, or CD8. These





cells mediate humoral or cell-mediated





immunity and may be preactivated to





enhance responsiveness to





immunomodulatory factors.



93
Production of IL-13
IL-13 FMAT. IL-13 enhances IgM, IgG,





and IgE production and induces FcER1. IL-





13 has anti-inflammatory activity on





monocytes and macrophages. Assays for





immunomodulatory proteins produced by T





cells that inhibit activation and release of





cytokines by macrophages are well known





in the art and may be used or routinely





modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to mediate immunomodulation,





regulate cytokine release, stimulate immune





cells through the binding of IL-13 and IL-4





receptors, and/or mediate humoral or cell-





mediated immunity. Exemplary assays that





test for immunomodulatory proteins





evaluate the production of cytokines, such





as IL-13, the inhibition of cytokines





released by macrophages. Such assays that





may be used or routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include the assays disclosed in Miraglia et





al., J Biomolecular Screening 4: 193-204





(1999); Rowland et al., “Lymphocytes: a





practical approach” Chapter 6: 138-160





(2000); and Ohshima et al., Blood





92(9): 3338-3345 (1998), the contents of





each of which are herein incorporated by





reference in its entirety. Human T cells that





may be used according to these assays may





be isolated using techniques disclosed





herein or otherwise known in the art.





Human T cells are primary human





lymphocytes that mature in the thymus and





express a T cell receptor and CD3, CD4, or





CD8. These cells mediate humoral or cell-





mediated immunity and may be preactivated





to enhance responsiveness to





immunomodulatory factors.



94
Production of IL-13
Assays for production of IL-13 and




and activation of T-
activation of T-cells are well known in the




cells.
art and may be used or routinely modified to





assess the ability of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) to stimulate





or inhibit production of IL-13 and/or





activation of T-cells. Exemplary assays for





IL-13 production that may be used or





routinely modified to test activity of





polypeptides and antibodies of the invention





(including agonists or antagonists of the





invention) include, for example, assays such





as disclosed and/or cited in: Grunig, G, et





al., “Requirement for IL-13 independently





of IL-4 in Experimental asthma”





Science; 282: 2261-2263 (1998), and Wills-





Karp M, et al., “Interleukin-13: central





mediator of allergic asthma” Science; 282:





2258-2261 (1998); the contents of each of





which are herein incorporated by reference





in their entirety. Exemplary cells that may





be used according to these assays include





Th2 cells. IL13, a Th2 type cytokine, is a





potent stimulus for mucus production,





airway hyper-responsiveness and allergic





asthma. Th2 cells are a class of T cells that





secrete IL4, IL10, IL13, IL5 and IL6.





Factors that induce differentiation and





activation of Th2 cells play a major role in





the initiation and pathogenesis of allergy





and asthma. Primary T helper 2 cells are





generated in in vitro culture under Th2





polarizing conditions using peripheral blood





lymphocytes isolated from cord blood.



95
Production of IL-2
IL-2 FMAT. IL-2 is the principal T cell




and activation of T
factor that allows T cell expansion and




cells
differentiation into effector cells. Assays





for immunomodulatory proteins secreted by





TH1 cells that promote T cell and NK cell





growth and differentiation are well known





in the art and may be used or routinely





modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to mediate immunomodulation,





promote immune cell growth and





differentiation, and/or mediate humoral or





cell-mediated immunity. Exemplary assays





that test for immunomodulatory proteins





evaluate the production of cytokines, such





as IL-2, and the activation of T cells. Such





assays that may be used or routinely





modified to test immunomodulatory activity





of polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) include the assays disclosed in





Miraglia et al., J Biomolecular Screening





4: 193-204 (1999); Rowland et al.,





“Lymphocytes: a practical approach”





Chapter 6: 138-160 (2000); Laduda et al.,





Immunology 94(4): 496-502 (1998); and





Powell et al., Immunol Rev 165: 287-300





(1998), the contents of each of which are





herein incorporated by reference in its





entirety. Human T cells that may be used





according to these assays may be isolated





using techniques disclosed herein or





otherwise known in the art. Human T cells





are primary human lymphocytes that mature





in the thymus and express a T cell receptor





and CD3, CD4, or CD8. These cells





mediate humoral or cell-mediated immunity





and may be preactivated to enhance





responsiveness to immunomodulatory





factors.



96
Production of IL-4
IL-4 FMAT. Assays for
A highly preferred embodiment of the invention




immunomodulatory proteins secreted by
includes a method for stimulating (e.g.,




TH2 cells that stimulate B cells, T cells,
increasing) IL-4 production. An alternative




macrophages and mast cells and promote
highly preferred embodiment of the invention




polarization of CD4+ cells into TH2 cells
includes a method for inhibiting (e.g., reducing)




are well known in the art and may be used
IL-4 production. A highly preferred




or routinely modified to assess the ability of
indication includes asthma. A highly




polypeptides of the invention (including
preferred indication includes allergy. A




antibodies and agonists or antagonists of the
highly preferred indication includes rhinitis.




invention) to mediate immunomodulation,
Additional highly preferred indications include




stimulate immune cells, modulate immune
inflammation and inflammatory disorders.




cell polarization, and/or mediate humoral or
Highly preferred indications include neoplastic




cell-mediated immunity. Exemplary assays
diseases (e.g., leukemia, lymphoma,




that test for immunomodulatory proteins
melanoma, and/or as described below under




evaluate the production of cytokines, such
“Hyperproliferative Disorders”). Preferred




as IL-4, and the stimulation of immune
indications include neoplasms and cancers, such




cells, such as B cells, T cells, macrophages
as, for example, leukemia, lymphoma,




and mast cells. Such assays that may be
melanoma, and prostate, breast, lung, colon,




used or routinely modified to test
pancreatic, esophageal, stomach, brain, liver




immunomodulatory activity of polypeptides
and urinary cancer. Other preferred indications




of the invention (including antibodies and
include benign dysproliferative disorders and




agonists or antagonists of the invention)
pre-neoplastic conditions, such as, for example,




include the assays disclosed in Miraglia et
hyperplasia, metaplasia, and/or dysplasia.




al., J Biomolecular Screening 4: 193-204
Preferred indications include blood disorders




(1999); Rowland et al., “Lymphocytes: a
(e.g., as described below under “Immune




practical approach” Chapter 6: 138-160
Activity”, “Blood-Related Disorders”, and/or




(2000); Gonzalez et al., J Clin Lab Anal
“Cardiovascular Disorders”). Preferred




8(5): 277-283 (1194); Yssel et al., Res
indications include autoimmune diseases (e.g.,




Immunol 144(8): 610-616 (1993); Bagley et
rheumatoid arthritis, systemic lupus




al., Nat Immunol 1(3): 257-261 (2000); and
erythematosis, multiple sclerosis and/or as




van der Graaff et al., Rheumatology
described below) and immunodeficiencies (e.g.,




(Oxford) 38(3): 214-220 (1999), the contents
as described below). Preferred indications




of each of which are herein incorporated by
include anemia, pancytopenia, leukopenia,




reference in its entirety. Human T cells that
thrombocytopenia, Hodgkin's disease, acute




may be used according to these assays may
lymphocytic anemia (ALL), plasmacytomas,




be isolated using techniques disclosed
multiple myeloma, Burkitt's lymphoma,




herein or otherwise known in the art.
arthritis, AIDS, granulomatous disease,




Human T cells are primary human
inflammatory bowel disease, sepsis,




lymphocytes that mature in the thymus and
neutropenia, neutrophilia, psoriasis, suppression




express a T cell receptor and CD3, CD4, or
of immune reactions to transplanted organs and




CD8. These cells mediate humoral or cell-
tissues, hemophilia, hypercoagulation, diabetes




mediated immunity and may be preactivated
mellitus, endocarditis, meningitis, and Lyme




to enhance responsiveness to
Disease. An additonal preferred indication




immunomodulatory factors.
is infection (e.g., an infectious disease as





described below under “Infectious Disease”).


97
Production of IL-5
IL-5 FMAT. Assays for
A highly preferred embodiment of the invention




immunomodulatory proteins secreted by
includes a method for inhibiting (e.g., reducing)




TH2 cells, mast cells, basophils, and
IL-5 production. An alternative highly preferred




eosinophils that stimulate eosinophil
embodiment of the invention includes a method




function and B cell Ig production and
for stimulating (e.g., increasing) IL-5




promote polarization of CD4+ cells into
production. A highly preferred embodiment




TH2 cells are well known in the art and may
of the invention includes a method for




be used or routinely modified to assess the
stimulating (e.g., increasing) immunoglobulin




ability of polypeptides of the invention
production. An alternative highly preferred




(including antibodies and agonists or
embodiment of the invention includes a method




antagonists of the invention) to mediate
for inhibiting (e.g., decreasing)




immunomodulation, stimulate immune cell
immunoglobulin production. A highly




function, modulate B cell Ig production,
preferred indication includes allergy. A




modulate immune cell polarization, and/or
highly preferred indication includes asthma.




mediate humoral or cell-mediated
A highly preferred indication includes rhinitis.




immunity. Exemplary assays that test for
An additional highly preferred indication is




immunomodulatory proteins evaluate the
infection (e.g., an infectious disease as




production of cytokines, such as IL-5, and
described below under ““Infectious Disease””),




the stimulation of eosinophil function and B
and inflammation and inflammatory disorders.




cell Ig production. Such assays that may be
Preferred indications include blood disorders




used or routinely modified to test
(e.g., as described below under “Immune




immunomodulatory activity of polypeptides
Activity”, “Blood-Related Disorders”, and/or




of the invention (including antibodies and
““Cardiovascular Disorders””). Preferred




agonists or antagonists of the invention)
indications include autoimmune diseases (e.g.,




include the assays disclosed in Miraglia et
rheumatoid arthritis, systemic lupus




al., J Biomolecular Screening 4: 193-204
erythematosis, multiple sclerosis and/or as




(1999); Rowland et al., ““Lymphocytes: a
described below) and immunodeficiencies (e.g.,




practical approach”” Chapter 6: 138-160
as described below). Preferred indications




(2000); Ohshima et al., Blood 92(9): 3338-3345
include neoplastic diseases (e.g., leukemia,




(1998); Jung et al., Eur J Immunol
lymphoma, melanoma, and/or as described




25(8): 2413-2416 (1995); Mori et al., J
below under “Hyperproliferative Disorders”).




Allergy Clin Immunol 106(1 Pt 2): 558-564
Preferred indications include neoplasms and




(2000); and Koning et al., Cytokine
cancers, such as, leukemia, lymphoma,




9(6): 427-436 (1997), the contents of each of
melanoma, and prostate, breast, lung, colon,




which are herein incorporated by reference
pancreatic, esophageal, stomach, brain, liver




in its entirety. Human T cells that may be
and urinary cancer. Other preferred indications




used according to these assays may be
include benign dysproliferative disorders and




isolated using techniques disclosed herein
pre-neoplastic conditions, such as, for example,




or otherwise known in the art. Human T
hyperplasia, metaplasia, and/or dysplasia.




cells are primary human lymphocytes that
Preferred indications include anemia,




mature in the thymus and express a T cell
pancytopenia, leukopenia, thrombocytopenia,




receptor and CD3, CD4, or CD8. These
leukemias, Hodgkin's disease, acute




cells mediate humoral or cell-mediated
lymphocytic anemia (ALL), plasmacytomas,




immunity and may be preactivated to
multiple myeloma, Burkitt's lymphoma,




enhance responsiveness to
arthritis, AIDS, granulomatous disease,




immunomodulatory factors.
inflammatory bowel disease, sepsis,





neutropenia, neutrophilia, psoriasis, immune





reactions to transplanted organs and tissues,





hemophilia, hypercoagulation, diabetes





mellitus, endocarditis, meningitis, and Lyme





Disease.


98
Production of IL-6
IL-6 FMAT. IL-6 is produced by T cells
A highly preferred embodiment of the invention




and has strong effects on B cells. IL-6
includes a method for stimulating (e.g.,




participates in IL-4 induced IgE production
increasing) IL-6 production. An alternative




and increases IgA production (IgA plays a
highly preferred embodiment of the invention




role in mucosal immunity). IL-6 induces
includes a method for inhibiting (e.g., reducing)




cytotoxic T cells. Deregulated expression
IL-6 production. A highly preferrred indication




of IL-6 has been linked to autoimmune
is the stimulation or enhancement of mucosal




disease, plasmacytomas, myelomas, and
immunity. Highly preferred indications




chronic hyperproliferative diseases. Assays
include blood disorders (e.g., as described




for immunomodulatory and differentiation
below under “Immune Activity”, “Blood-




factor proteins produced by a large variety
Related Disorders”, and/or ““Cardiovascular




of cells where the expression level is
Disorders””), and infection (e.g., as described




strongly regulated by cytokines, growth
below under “Infectious Disease”). Highly




factors, and hormones are well known in the
preferred indications include autoimmune




art and may be used or routinely modified to
diseases (e.g., rheumatoid arthritis, systemic




assess the ability of polypeptides of the
lupus erythematosis, multiple sclerosis and/or




invention (including antibodies and agonists
as described below) and immunodeficiencies




or antagonists of the invention) to mediate
(e.g., as described below). Highly preferred




immunomodulation and differentiation and
indications also include boosting a B cell-




modulate T cell proliferation and function.
mediated immune response and alternatively




Exemplary assays that test for
suppressing a B cell-mediated immune




immunomodulatory proteins evaluate the
response. Highly preferred indications include




production of cytokines, such as IL-6, and
inflammation and inflammatory




the stimulation and upregulation of T cell
disorders. Additional highly preferred




proliferation and functional activities. Such
indications include asthma and allergy.




assays that may be used or routinely
Highly preferred indications include neoplastic




modified to test immunomodulatory and
diseases (e.g., myeloma, plasmacytoma,




differentiation activity of polypeptides of
leukemia, lymphoma, melanoma, and/or as




the invention (including antibodies and
described below under “Hyperproliferative




agonists or antagonists of the invention)
Disorders”). Highly preferred indications




include assays disclosed in Miraglia et al., J
include neoplasms and cancers, such as,




Biomolecular Screening 4: 193-204(1999);
myeloma, plasmacytoma, leukemia, lymphoma,




Rowland et al., ““Lymphocytes: a practical
melanoma, and prostate, breast, lung, colon,




approach”” Chapter 6: 138-160 (2000); and
pancreatic, esophageal, stomach, brain, liver




Verhasselt et al., J Immunol 158: 2919-2925
and urinary cancer. Other preferred indications




(1997), the contents of each of which are
include benign dysproliferative disorders and




herein incorporated by reference in its
pre-neoplastic conditions, such as, for example,




entirety. Human dendritic cells that may be
hyperplasia, metaplasia, and/or dysplasia.




used according to these assays may be
Preferred indications include anemia,




isolated using techniques disclosed herein
pancytopenia, leukopenia, thrombocytopenia,




or otherwise known in the art. Human
Hodgkin's disease, acute lymphocytic anemia




dendritic cells are antigen presenting cells in
(ALL), multiple myeloma, Burkitt's lymphoma,




suspension culture, which, when activated
arthritis, AIDS, granulomatous disease,




by antigen and/or cytokines, initiate and
inflammatory bowel disease, sepsis,




upregulate T cell proliferation and
neutropenia, neutrophilia, psoriasis, suppression




functional activities.
of immune reactions to transplanted organs and





tissues, hemophilia, hypercoagulation, diabetes





mellitus, endocarditis, meningitis, and Lyme





Disease. An additonal preferred indication





is infection (e.g., an infectious disease as





described below under ““Infectious Disease””).


99
Production of IL-8 by
Assays measuring production of IL-8 are
Highly preferred indications include



by endothelial cells
well known in the art and may be used or
immunological and inflammatory disorders



(such as Human
routinely modified to assess the ability of
(e.g., such as allergy, asthma, leukemia, etc. and



Umbilical Cord
polypeptides of the invention (including
as described below under “Immune Activity”,



Endothelial Cells).
antibodies and agonists or antagonists of the
and “Blood-Related Disorders”). Highly




invention) to regulate production and/or
preferred indications also includie autoimmune




secretion of IL-8. For example, FMAT may
disorders (e.g., rheumatoid arthritis, systemic




be used or routinely modified to assess the
lupus erythematosis, Crohn''s disease, multiple




ability of polypeptides of the invention
sclerosis and/or as described below), neoplastic




(including antibodies and agonists or
disorders (e.g., organ cancers such as lung,




antagonists of the invention) to regulate
liver, colon cancer, and/or as described below




production and/or secretion of IL-8 from
under “Hyperproliferative Disorders”), and




endothelial cells (such as human umbilical
cardiovascular disorders (e.g. such as described




vein endothelial cells (HUVEC)). HUVECs
below under “Cardiovascular Disorders”).




are endothelial cells which line venous
Preferred indications include thrombosis,




blood vessels, and are involved in functions
bacteremia and sepsis syndrome and




that include, but are not limited to,
consequent complications (such as acute




angiogenesis, vascular permeability,
respiratory distress syndrome and systemic




vascular tone, and immune cell
ischemia-reperfusion resulting from septic




extravasation. Endothelial cells play a
shock), restnosis and atherosclerosis.




pivotal role in the initiation and





perpetuation of inflammation and secretion





of IL-8 may play an important role in





recruitment and activation of immune cells





such as neutrophils, macrophages, and





lymphocytes.



100
Production of IL-8 by
Assays measuring production of IL-8 are




endothelial cells
well known in the art and may be used or




(such as Human
routinely modified to assess the ability of




Umbilical Cord
polypeptides of the invention (including




Endothelial Cells).
antibodies and agonists or antagonists of the





invention) to regulate production and/or





secretion of IL-8. For example, FMAT may





be used or routinely modified to assess the





ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to regulate





production and/or secretion of IL-8 from





endothelial cells (such as human umbilical





vein endothelial cells (HUVEC)). HUVECs





are endothelial cells which line venous





blood vessels, and are involved in functions





that include, but are not limited to,





angiogenesis, vascular permeability,





vascular tone, and immune cell





extravasation. Endothelial cells play a





pivotal role in the initiation and





perpetuation of inflammation and secretion





of IL-8 may play an important role in





recruitment and activation of immune cells





such as neutrophils, macrophages, and





lymphocytes.



101
Production of IL-8 by
Assay that measures the production of the




immune cells (such
chemokine interleukin-8 (IL-8) from




as the human EOL-1
immune cells (such as the EOL-1 human




eosinophil cells)
eosinophil cell line) are well known in the





art (for example, measurement of IL-8





production by FMAT) and may be used or





routinely modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to promote or inhibit.





Eosinophils are a type of immune cell





important in allergic responses; they are





recruited to tissues and mediate the





inflammatory response of late stage allergic





reaction. IL8 is a strong immunomodulator





and may have a potential proinflammatory





role in immunological diseases and





disorders (such as allergy and asthma).



102
Production of IL6 by
Assay to measure regulation of production




primary human aortic
of Interleukin-6 (IL-6) by either human




smooth muscle or
aortic smooth muscle cells or normal human




normal human dermal
dermal fibroblasts minus or plus




fibroblast cells
costimulation with TNFalpha (TNFa).




(without or with
Human aortic smooth muscle cells or




costimulation with
normal human dermal fibroblasts may be




TNFalpha).
obtained from commercial sources; these





cells are important structural and functional





components of blood vessels and connective





tissue, respectively. Interleukin-6 (IL-6) is





a key molecule in chronic inflammation and





has been implicated in the progression of





arteriosclerosis, stroke, arthritis and other





vascular and inflammatory diseases.





Deregulated expression of IL-6 has been





linked to autoimmune disease,





plasmacytomas, myelomas, and chronic





hyperproliferative diseases. Assays for





immunomodulatory and differentiation





factor proteins produced by a large variety





of cells where the expression level is





strongly regulated by cytokines, growth





factors, and hormones are well known in the





art and may be used or routinely modified to





assess the ability of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) to mediate





immunomodulation and production of IL-6.



103
Production of MCP-1
MCP-1 FMAT. Assays for
A highly preferred embodiment of the invention




immunomodulatory proteins that are
includes a method for stimulating (e.g.,




produced by a large variety of cells and act
increasing) MCP-1 production. An alternative




to induce chemotaxis and activation of
highly preferred embodiment of the invention




monocytes and T cells are well known in
includes a method for inhibiting (e.g., reducing)




the art and may be used or routinely
MCP-1 production. A highly preferred




modified to assess the ability of
indication is infection (e.g., an infectious




polypeptides of the invention (including
disease as described below under “Infectious




antibodies and agonists or antagonists of the
Disease”). Additional highly preferred




invention) to mediate immunomodulation,
indications include inflammation and




induce chemotaxis, and modulate immune
inflammatory disorders. Preferred




cell activation. Exemplary assays that test
indications include blood disorders (e.g., as




for immunomodulatory proteins evaluate
described below under “Immune Activity”,




the production of cell surface markers, such
“Blood-Related Disorders”, and/or




as monocyte chemoattractant protein
““Cardiovascular Disorders””). Highly preferred




(MCP), and the activation of monocytes and
indications include autoimmune diseases (e.g.,




T cells. Such assays that may be used or
rheumatoid arthritis, systemic lupus




routinely modified to test
erythematosis, multiple sclerosis and/or as




immunomodulatory and differentiation
described below) and immunodeficiencies (e.g.,




activity of polypeptides of the invention
as described below). Preferred indications




(including antibodies and agonists or
also include anemia, pancytopenia, leukopenia,




antagonists of the invention) include assays
thrombocytopenia, Hodgkin's disease, acute




disclosed in Miraglia et al., J Biomolecular
lymphocytic anemia (ALL), plasmacytomas,




Screening 4: 193-204(1999); Rowland et al.,
multiple myeloma, Burkitt's lymphoma,




““Lymphocytes: a practical approach””
arthritis, AIDS, granulomatous disease,




Chapter 6: 138-160 (2000); Satthaporn and
inflammatory bowel disease, sepsis,




Eremin, J R Coll Surg Ednb 45(1): 9-19
neutropenia, neutrophilia, psoriasis, suppression




(2001); and Verhasselt et al., J Immunol
of immune reactions to transplanted organs and




158: 2919-2925 (1997), the contents of each
tissues, hemophilia, hypercoagulation, diabetes




of which are herein incorporated by
mellitus, endocarditis, meningitis (bacterial and




reference in its entirety. Human dendritic
viral), Lyme Disease, asthma, and allergy




cells that may be used according to these
Preferred indications also include neoplastic




assays may be isolated using techniques
diseases (e.g., leukemia, lymphoma, and/or as




disclosed herein or otherwise known in the
described below under “Hyperproliferative




art. Human dendritic cells are antigen
Disorders”). Highly preferred indications




presenting cells in suspension culture,
include neoplasms and cancers, such as,




which, when activated by antigen and/or
leukemia, lymphoma, prostate, breast, lung,




cytokines, initiate and upregulate T cell
colon, pancreatic, esophageal, stomach, brain,




proliferation and functional activities.
liver, and urinary cancer. Other preferred





indications include benign dysproliferative





disorders and pre-neoplastic conditions, such





as, for example, hyperplasia, metaplasia, and/or





dysplasia.


104
Production of
MIP-1alpha FMAT. Assays for
A highly preferred embodiment of the invention



MIP1alpha
immunomodulatory proteins produced by
includes a method for stimulating MIP1a




activated dendritic cells that upregulate
production. An alternative highly preferred




monocyte/macrophage and T cell
embodiment of the invention includes a method




chemotaxis are well known in the art and
for inhibiting (e.g., reducing) MIP1a




may be used or routinely modified to assess
production. A highly preferred indication is




the ability of polypeptides of the invention
infection (e.g., an infectious disease as




(including antibodies and agonists or
described below under “Infectious Disease”).




antagonists of the invention) to mediate
Preferred indications include blood disorders




immunomodulation, modulate chemotaxis,
(e.g., as described below under “Immune




and modulate T cell differentiation.
Activity”, “Blood-Related Disorders”, and/or




Exemplary assays that test for
““Cardiovascular Disorders””). Highly preferred




immunomodulatory proteins evaluate the
indications include autoimmune diseases (e.g.,




production of chemokines, such as
rheumatoid arthritis, systemic lupus




macrophage inflammatory protein 1 alpha
erythematosis, multiple sclerosis and/or as




(MIP-1a), and the activation of
described below) and immunodeficiencies (e.g.,




monocytes/macrophages and T cells. Such
as described below). Additional highly




assays that may be used or routinely
preferred indications include inflammation and




modified to test immunomodulatory and
inflammatory disorders. Preferred indications




chemotaxis activity of polypeptides of the
also include anemia, pancytopenia, leukopenia,




invention (including antibodies and agonists
thrombocytopenia, Hodgkin's disease, acute




or antagonists of the invention) include
lymphocytic anemia (ALL), plasmacytomas,




assays disclosed in Miraglia et al., J
multiple myeloma, Burkitt's lymphoma,




Biomolecular Screening 4: 193-204(1999);
arthritis, AIDS, granulomatous disease,




Rowland et al., ““Lymphocytes: a practical
inflammatory bowel disease, sepsis,




approach”” Chapter 6: 138-160 (2000);
neutropenia, neutrophilia, psoriasis, suppression




Satthaporn and Eremin, J R Coll Surg Ednb
of immune reactions to transplanted organs and




45(1): 9-19 (2001); Drakes et al., Transp
tissues, hemophilia, hypercoagulation, diabetes




Immunol 8(1): 17-29 (2000); Verhasselt et
mellitus, endocarditis, meningitis, Lyme




al., J Immunol 158: 2919-2925 (1997); and
Disease, asthma, and allergy. Preferred




Nardelli et al., J Leukoc Biol 65: 822-828
indications also include neoplastic diseases




(1999), the contents of each of which are
(e.g., leukemia, lymphoma, and/or as described




herein incorporated by reference in its
below under “Hyperproliferative Disorders”).




entirety. Human dendritic cells that may be
Highly preferred indications include neoplasms




used according to these assays may be
and cancers, such as, leukemia, lymphoma,




isolated using techniques disclosed herein
prostate, breast, lung, colon, pancreatic,




or otherwise known in the art. Human
esophageal, stomach, brain, liver, and urinary




dendritic cells are antigen presenting cells in
cancer. Other preferred indications include




suspension culture, which, when activated
benign dysproliferative disorders and pre-




by antigen and/or cytokines, initiate and
neoplastic conditions, such as, for example,




upregulate T cell proliferation and
hyperplasia, metaplasia, and/or dysplasia.




functional activities.



105
Production of
RANTES FMAT. Assays for




RANTES
immunomodulatory proteins that induce





chemotaxis of T cells, monocytes, and





eosinophils are well known in the art and





may be used or routinely modified to assess





the ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to mediate





immunomodulation, induce chemotaxis,





and/or mediate humoral or cell-mediated





immunity. Exemplary assays that test for





immunomodulatory proteins evaluate the





production of cytokines, such as RANTES,





and the induction of chemotactic responses





in immune cells. Such assays that may be





used or routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include the assays disclosed in Miraglia et





al., J Biomolecular Screening 4: 193-204





(1999); Rowland et al., “Lymphocytes: a





practical approach” Chapter 6: 138-160





(2000): Cocchi et al., Science





270(5243): 1811-1815 (1995); and Robinson





et al., Clin Exp Immunol 101(3): 398-407





(1995), the contents of each of which are





herein incorporated by reference in its





entirety. Human immune cells that may be





used according to these assays may be





isolated using techniques disclosed herein





or otherwise known in the art.



106
Production of
RANTES FMAT. Assays for




RANTES in
immunomodulatory proteins that induce




bronchial epithelium
chemotaxis of T cells, monocytes, and




cells
eosinophils are well known in the art and





may be used or routinely modified to assess





the ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to mediate





immunomodulation, induce chemotaxis,





and/or mediate humoral or cell-mediated





immunity. Exemplary assays that test for





immunomodulatory proteins evaluate the





production of cytokines, such as RANTES,





and the induction of chemotactic responses





in immune cells. Such assays that may be





used or routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include the assays disclosed in Miraglia et





al., J Biomolecular Screening 4: 193-204





(1999); Rowland et al., “Lymphocytes: a





practical approach” Chapter 6: 138-160





(2000): Cocchi et al., Science





270(5243): 1811-1815 (1995); and Robinson





et al., Clin Exp Immunol 101(3): 398-407





(1995), the contents of each of which are





herein incorporated by reference in its





entirety. Epithelial cells were isolated from





bronchia/trachea immediately postmortem





from humans who were free of known





respiratory diseases. See Wu et al., Am Rev





Respir Dis. 132(2): 311-20 (1985), the





contents of which are herein incorporated





by reference in its entirety.



107
Production of
RANTES FMAT. Assays for




RANTES in
immunomodulatory proteins that induce




endothelial cells
chemotaxis of T cells, monocytes, and




(such as human
eosinophils are well known in the art and




umbilical vein
may be used or routinely modified to assess




endothelial cells
the ability of polypeptides of the invention




(HUVEC))
(including antibodies and agonists or





antagonists of the invention) to mediate





immunomodulation, induce chemotaxis,





and/or mediate humoral or cell-mediated





immunity. Exemplary assays that test for





immunomodulatory proteins evaluate the





production of cytokines, such as RANTES,





and the induction of chemotactic responses





in immune cells. Such assays that may be





used or routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include the assays disclosed in Miraglia et





al., J Biomolecular Screening 4: 193-204





(1999); Rowland et al., “Lymphocytes: a





practical approach” Chapter 6: 138-160





(2000): Cocchi et al., Science





270(5243): 1811-1815 (1995); and Robinson





et al., Clin Exp Immunol 101(3): 398-407





(1995), the contents of each of which are





herein incorporated by reference in its





entirety. Endothelial cells that may be used





according to these assays are publicly





available (e.g., through the ATCC TM).





Exemplary endothelial cells that may be





used according to these assays include





human umbilical vein endothelial cells





(HUVEC), which are endothelial cells





which line venous blood vessels, and are





involved in functions that include, but are





not limited to, angiogenesis, vascular





permeability, vascular tone, and immune





cell extravasation.



108
Production of TNF
TNFa FMAT. Assays for
A highly preferred embodiment of the invention



alpha by dendritic
immunomodulatory proteins produced by
includes a method for inhibiting (e.g.,



cells
activated macrophages, T cells, fibroblasts,
decreasing) TNF alpha production. An




smooth muscle, and other cell types that
alternative highly preferred embodiment of the




exert a wide variety of inflammatory and
invention includes a method for stimulating




cytotoxic effects on a variety of cells are
(e.g., increasing) TNF alpha production.




well known in the art and may be used or
Highly preferred indications include blood




routinely modified to assess the ability of
disorders (e.g., as described below under




polypeptides of the invention (including
“Immune Activity”, “Blood-Related




antibodies and agonists or antagonists of the
Disorders”, and/or ““Cardiovascular




invention) to mediate immunomodulation,
Disorders””), Highly preferred indications




modulate inflammation and cytotoxicity.
include autoimmune diseases (e.g., rheumatoid




Exemplary assays that test for
arthritis, systemic lupus erythematosis, Crohn's




immunomodulatory proteins evaluate the
disease, multiple sclerosis and/or as described




production of cytokines such as tumor
below), immunodeficiencies (e.g., as described




necrosis factor alpha (TNFa), and the
below), boosting a T cell-mediated immune




induction or inhibition of an inflammatory
response, and suppressing a T cell-mediated




or cytotoxic response. Such assays that may
immune response. Additional highly preferred




be used or routinely modified to test
indications include inflammation and




immunomodulatory activity of polypeptides
inflammatory disorders, and treating joint




of the invention (including antibodies and
damage in patients with rheumatoid arthritis.




agonists or antagonists of the invention)
An additional highly preferred indication is




include assays disclosed in Miraglia et al., J
sepsis. Highly preferred indications include




Biomolecular Screening 4: 193-204(1999);
neoplastic diseases (e.g., leukemia, lymphoma,




Rowland et al., ““Lymphocytes: a practical
and/or as described below under




approach”” Chapter 6: 138-160 (2000);
“Hyperproliferative Disorders”). Additionally,




Verhasselt et al., Eur J Immunol
highly preferred indications include neoplasms




28(11): 3886-3890 (1198); Dahlen et al., J
and cancers, such as, leukemia, lymphoma,




Immunol 160(7): 3585-3593 (1998);
melanoma, glioma (e.g., malignant glioma),




Verhasselt et al., J Immunol 158: 2919-2925
solid tumors, and prostate, breast, lung, colon,




(1997); and Nardelli et al., J Leukoc Biol
pancreatic, esophageal, stomach, brain, liver




65: 822-828 (1999), the contents of each of
and urinary cancer. Other preferred indications




which are herein incorporated by reference
include benign dysproliferative disorders and




in its entirety. Human dendritic cells that
pre-neoplastic conditions, such as, for example,




may be used according to these assays may
hyperplasia, metaplasia, and/or dysplasia.




be isolated using techniques disclosed
Preferred indications include anemia,




herein or otherwise known in the art.
pancytopenia, leukopenia, thrombocytopenia,




Human dendritic cells are antigen
Hodgkin's disease, acute lymphocytic anemia




presenting cells in suspension culture,
(ALL), plasmacytomas, multiple myeloma,




which, when activated by antigen and/or
Burkitt's lymphoma, arthritis, AIDS,




cytokines, initiate and upregulate T cell
granulomatous disease, inflammatory bowel




proliferation and functional activities.
disease, neutropenia, neutrophilia, psoriasis,





suppression of immune reactions to





transplanted organs and tissues, hemophilia,





hypercoagulation, diabetes mellitus,





endocarditis, meningitis, Lyme Disease, cardiac





reperfusion injury, and asthma and allergy.





An additional preferred indication is infection





(e.g., an infectious disease as described below





under “Infectious Disease”).


109
Production of TNF
TNFa FMAT. Assays for




alpha by T cells
immunomodulatory proteins produced by





activated macrophages, T cells, fibroblasts,





smooth muscle, and other cell types that





exert a wide variety of inflammatory and





cytotoxic effects on a variety of cells are





well known in the art and may be used or





routinely modified to assess the ability of





polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) to mediate immunomodulation,





modulate inflammation and cytotoxicity,





and mediate humoral and/or cell-mediated





immunity. Exemplary assays that test for





immunomodulatory proteins evaluate the





production of cytokines such as tumor





necrosis factor alpha (TNFa), and the





induction or inhibition of an inflammatory





or cytotoxic response. Such assays that may





be used or routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include assays disclosed in Miraglia et al., J





Biomolecular Screening 4: 193-204 (1999);





Rowland et al., “Lymphocytes: a practical





approach” Chapter 6: 138-160 (2000);





Verhasselt et al., Eur J Immunol





28(11): 3886-3890 (1198); Dahlen et al., J





Immunol 160(7): 3585-3593 (1998);





Verhasselt et al., J Immunol 158: 2919-2925





(1997); and Nardelli et al., J Leukoc Biol





65: 822-828 (1999), the contents of each of





which are herein incorporated by reference





in its entirety. Human T cells that may be





used according to these assays may be





isolated using techniques disclosed herein





or otherwise known in the art. Human T





cells are primary human lymphocytes that





mature in the thymus and express a T cell





receptor and CD3, CD4, or CD8. These





cells mediate humoral or cell-mediated





immunity and may be preactivated to





enhance responsiveness to





immunomodulatory factors.



110
Production of VCAM
Assays for measuring expression of VCAM
Highly preferred indications include



in endothelial cells
are well-known in the art and may be used
inflammation (acute and chronic), restnosis,



(such as human
or routinely modified to assess the ability of
atherosclerosis, asthma and allergy. Highly



umbilical vein
polypeptides of the invention (including
preferred indications include inflammation and



endothelial cells
antibodies and agonists or antagonists of the
inflammatory disorders, immunological



(HUVEC))
invention) to regulate VCAM expression.
disorders, neoplastic disorders (e.g.




For example, FMAT may be used to meaure
cancer/tumorigenesis), and cardiovascular




the upregulation of cell surface VCAM-1
disorders (such as described below under




expresssion in endothelial cells. Endothelial
“Immune Activity”, “Blood-Related




cells are cells that line blood vessels, and
Disorders”, “Hyperproliferative Disorders”




are involved in functions that include, but
and/or “Cardiovascular Disorders”). Highly




are not limited to, angiogenesis, vascular
preferred indications include neoplasms and




permeability, vascular tone, and immune
cancers such as, for example, leukemia,




cell extravasation. Exemplary endothelial
lymphoma, melanoma, renal cell carcinoma,




cells that may be used according to these
and prostate, breast, lung, colon, pancreatic,




assays include human umbilical vein
esophageal, stomach, brain, liver and urinary




endothelial cells (HUVEC), which are
cancer. Other preferred indications include




available from commercial sources. The
benign dysproliferative disorders and pre-




expression of VCAM (CD106), a
neoplastic conditions, such as, for example,




membrane-associated protein, can be
hyperplasia, metaplasia, and/or dysplasia.




upregulated by cytokines or other factors,





and contributes to the extravasation of





lymphocytes, leucocytes and other immune





cells from blood vessels; thus VCAM





expression plays a role in promoting





immune and inflammatory responses.



111
Proliferation of
Assays for the regulation (i.e. increases or
Highly preferred indications include asthma,



immune cells (such
decreases) of viability and proliferation of
allergy, mastocytosis (a rare, heterogeneous



as the HMC-1 human
cells in vitro are well-known in the art and
disorder characterized by excessive



mast cell line)
may be used or routinely modified to assess
accumulation of mast cells, and their




the ability of polypeptides of the invention
proliferation and action in the skin, central




(including antibodies and agonists or
nervous system, and other organs). Preferred




antagonists of the invention) to regulate
indications also include hematopoietic and




viability and proliferation of eosinophil
immunological disorders (e.g., as described




cells and cell lines. For example, the
below under “Immune Activity”, and “Blood-




CellTiter-Gloô Luminescent Cell Viability
Related Disorders”), infection (e.g., as




Assay (Promega Corp., Madison, WI, USA)
described below under “Infectious Disease”),




can be used to measure the number of
autoimmune diseases (e.g., rheumatoid arthritis,




viable cells in culture based on quantitation
systemic lupus erythematosis, multiple sclerosis




of the ATP present which signals the
and/or as described below), and




presence of metabolically active cells.
immunodeficiencies (e.g., as described below).




Mast cells are found in connective and





mucosal tissues throughout the body. Mast





cell activation (via immunoglobulin E-





antigen, promoted by T helper cell type 2





cytokines) is an important component of





allergic disease. Dysregulation of mast cell





apoptosis may play a role in allergic disease





and mast cell tumor survival. Mast cell





lines that may be used according to these





assays are publicly available and/or may be





routinely generated. Exemplary mast cells





that may be used according to these assays





include HMC-1, which is an immature





human mast cell line established from the





peripheral blood of a patient with mast cell





leukemia, and exhibits many characteristics





of immature mast cells.



112
Proliferation of pre-
Assays for the regulation (i.e. increases or




adipose cells (such as
decreases) of viability and proliferation of




3T3-L1 cells)
cells in vitro are well-known in the art and





may be used or routinely modified to assess





the ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to regulate





viability and proliferation of pre-adipose





cells and cell lines. For example, the





CellTiter-Gloô Luminescent Cell Viability





Assay (Promega Corp., Madison, WI, USA)





can be used to measure the number of viable





cells in culture based on quantitation of the





ATP present which signals the presence of





metabolically active cells. 3T3-L1 is a





mouse preadipocyte cell line. It is a





continuous substrain of 3T3 fibroblast cells





developed through clonal isolation. Cells





were differentiated to an adipose-like state





before being used in the screen. See Green H





and Meuth M., Cell 3: 127-133 (1974),





which is herein incorporated by reference in





its entirety.



113
Protection from
Caspase Apoptosis Rescue. Assays for
A highly preferred embodiment of the invention



Endothelial Cell
caspase apoptosis rescue are well known in
includes a method for stimulating endothelial



Apoptosis.
the art and may be used or routinely
cell growth. An alternative highly preferred




modified to assess the ability of the
embodiment of the invention includes a method




polypeptides of the invention (including
for inhibiting endothelial cell growth. A




antibodies and agonists or antagonists of the
highly preferred embodiment of the invention




invention) to inhibit caspase protease-
includes a method for stimulating endothelial




mediated apoptosis. Exemplary assays for
cell proliferation. An alternative highly




caspase apoptosis that may be used or
preferred embodiment of the invention includes




routinely modified to test caspase apoptosis
a method for inhibiting endothelial cell




rescue of polypeptides of the invention
proliferation. A highly preferred




(including antibodies and agonists or
embodiment of the invention includes a method




antagonists of the invention) include the
for stimulating endothelial cell growth. An




assays disclosed in Romeo et al.,
alternative highly preferred embodiment of the




Cardiovasc Res 45(3): 788-794 (2000);
invention includes a method for inhibiting




Messmer et al., Br J Pharmacol 127(7):
endothelial cell growth. A highly preferred




1633-1640 (1999); and J Atheroscler
embodiment of the invention includes a method




Thromb 3(2): 75-80 (1996); the contents of
for stimulating apoptosis of endothelial cells.




each of which are herein incorporated by
An alternative highly preferred embodiment of




reference in its entirety. Endothelial cells
the invention includes a method for inhibiting




that may be used according to these assays
(e.g., decreasing) apoptosis of endothelial cells.




are publicly available (e.g., through
A highly preferred embodiment of the invention




commercial sources). Exemplary
includes a method for stimulating angiogenisis.




endothelial cells that may be used according
An alternative highly preferred embodiment of




to these assays include bovine aortic
the invention includes a method for inhibiting




endothelial cells (bAEC), which are an
angiogenesis. A highly preferred




example of endothelial cells which line
embodiment of the invention includes a method




blood vessels and are involved in functions
for reducing cardiac hypertrophy. An




that include, but are not limited to,
alternative highly preferred embodiment of the




angiogenesis, vascular permeability,
invention includes a method for inducing




vascular tone, and immune cell
cardiac hypertrophy. Highly preferred




extravasation.
indications include neoplastic diseases (e.g., as





described below under “Hyperproliferative





Disorders”), and disorders of the cardiovascular





system (e.g., heart disease, congestive heart





failure, hypertension, aortic stenosis,





cardiomyopathy, valvular regurgitation, left





ventricular dysfunction, atherosclerosis and





atherosclerotic vascular disease, diabetic





nephropathy, intracardiac shunt, cardiac





hypertrophy, myocardial infarction, chronic





hemodynamic overload, and/or as described





below under “Cardiovascular Disorders”).





Highly preferred indications include





cardiovascular, endothelial and/or angiogenic





disorders (e.g., systemic disorders that affect





vessels such as diabetes mellitus, as well as





diseases of the vessels themselves, such as of





the arteries, capillaries, veins and/or





lymphatics). Highly preferred are indications





that stimulate angiogenesis and/or





cardiovascularization. Highly preferred are





indications that inhibit angiogenesis and/or





cardiovascularization. Highly preferred





indications include antiangiogenic activity to





treat solid tumors, leukemias, and Kaposi''s





sarcoma, and retinal disorders. Highly preferred





indications include neoplasms and cancer, such





as, Kaposi''s sarcoma, hemangioma (capillary





and cavernous), glomus tumors, telangiectasia,





bacillary angiomatosis, hemangioendothelioma,





angiosarcoma, haemangiopericytoma,





lymphangioma, lymphangiosarcoma. Highly





preferred indications also include cancers such





as, prostate, breast, lung, colon, pancreatic,





esophageal, stomach, brain, liver, and urinary





cancer. Preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia. Highly





preferred indications also include arterial





disease, such as, atherosclerosis, hypertension,





coronary artery disease, inflammatory





vasculitides, Reynaud''s disease and Reynaud''s





phenomenom, aneurysms, restenosis; venous





and lymphatic disorders such as





thrombophlebitis, lymphangitis, and





lymphedema; and other vascular disorders such





as peripheral vascular disease, and cancer.





Highly preferred indications also include





trauma such as wounds, burns, and injured





tissue (e.g., vascular injury such as, injury





resulting from balloon angioplasty, and





atheroschlerotic lesions), implant fixation,





scarring, ischemia reperfusion injury,





rheumatoid arthritis, cerebrovascular disease,





renal diseases such as acute renal failure, and





osteoporosis. Additional highly preferred





indications include stroke, graft rejection,





diabetic or other retinopathies, thrombotic and





coagulative disorders, vascularitis, lymph





angiogenesis, sexual disorders, age-related





macular degeneration, and treatment/





prevention of endometriosis and related





conditions. Additional highly preferred





indications include fibromas, heart disease,





cardiac arrest, heart valve disease, and vascular





disease. Preferred indications include blood





disorders (e.g., as described below under





“Immune Activity”, “Blood-Related





Disorders”, and/or “Cardiovascular Disorders”).





Preferred indications include autoimmune





diseases (e.g., rheumatoid arthritis, systemic





lupus erythematosis, multiple sclerosis and/or





as described below) and immunodeficiencies





(e.g., as described below). Additional preferred





indications include inflammation and





inflammatory disorders (such as acute and





chronic inflammatory diseases, e.g.,





inflammatory bowel disease and Crohn's





disease), and pain management.


114
Regulation
The mixed lymphocyte reaction assay




(inhibition or
(MLR) (see e.g., Example: “Detection of




activation) of
Inhibition of a Mixed Lymphocyte




immune cell
Reaction” below) is a complex in vitro




proliferation.
assay of T-cell responsiveness and immune





cell activation. This assay is useful, for





example, as an in vitro model of allograft





rejection and graft versus host disease. In





this assays PBMCs from human donors are





mixed, cultured, and monitored for





thymidine incorporation (a measure of cell





proliferation) to identify polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) that may





activate or inhibit immune responses.



115
Regulation of
Caspase Apoptosis. Assays for caspase
A highly preferred indication is diabetes



apoptosis in
apoptosis are well known in the art and may
mellitus. An additional highly preferred



pancreatic beta cells.
be used or routinely modified to assess the
indication is a complication associated with




ability of polypeptides of the invention
diabetes (e.g., diabetic retinopathy, diabetic




(including antibodies and agonists or
nephropathy, kidney disease (e.g., renal failure,




antagonists of the invention) to promote
nephropathy and/or other diseases and disorders




caspase protease-mediated apoptosis.
as described in the ““Renal Disorders”” section




Apoptosis in pancreatic beta is associated
below), diabetic neuropathy, nerve disease and




with induction and progression of diabetes.
nerve damage (e.g., due to diabetic neuropathy),




Exemplary assays for caspase apoptosis that
blood vessel blockage, heart disease, stroke,




may be used or routinely modified to test
impotence (e.g., due to diabetic neuropathy or




capase apoptosis activity of polypeptides of
blood vessel blockage), seizures, mental




the invention (including antibodies and
confusion, drowsiness, nonketotic




agonists or antagonists of the invention)
hyperglycemic-hyperosmolar coma,




include the assays disclosed in: Loweth, A C,
cardiovascular disease (e.g., heart disease,




et al., FEBS Lett, 400(3): 285-8 (1997);
atherosclerosis, microvascular disease,




Saini, K S, et al., Biochem Mol Biol Int,
hypertension, stroke, and other diseases and




39(6): 1229-36 (1996); Krautheim, A., et al.,
disorders as described in the ““Cardiovascular




Br J Pharmacol, 129(4): 687-94 (2000);
Disorders”” section below), dyslipidemia,




Chandra J, et al., Diabetes, 50 Suppl 1: S44-7
endocrine disorders (as described in the




(2001); Suk K, et al., J Immunol,
““Endocrine Disorders”” section below),




166(7): 4481-9 (2001); Tejedo J, et al.,
neuropathy, vision impairment (e.g., diabetic




FEBS Lett, 459(2): 238-43 (1999); Zhang, S.,
retinopathy and blindness), ulcers and impaired




et al., FEBS Lett, 455(3): 315-20 (1999);
wound healing, and infection (e.g., infectious




Lee et al., FEBS Lett 485(2-3): 122-126
diseases and disorders as described in the




(2000); Nor et al., J Vasc Res 37(3): 209-218
““Infectious Diseases”” section below,




(2000); and Karsan and Harlan, J
especially of the urinary tract and skin), carpal




Atheroscler Thromb 3(2): 75-80 (1996); the
tunnel syndrome and Dupuytren's contracture).




contents of each of which are herein
An additional highly preferred indication is




incorporated by reference in its entirety.
obesity and/or complications associated with




Pancreatic cells that may be used according
obesity. Additional highly preferred indications




to these assays are publicly available (e.g.,
include weight loss or alternatively, weight




through the ATCC TM) and/or may be
gain. Aditional highly preferred




routinely generated. Exemplary pancreatic
indications are complications associated with




cells that may be used according to these
insulin resistance.




assays include RIN-m. RIN-m is a rat





adherent pancreatic beta cell insulinoma cell





line derived from a radiation induced





transplantable rat islet cell tumor. The cells





produce and secrete islet polypeptide





hormones, and produce insulin,





somatostatin, and possibly glucagon.





ATTC: #CRL-2057 Chick et al. Proc.





Natl. Acad. Sci. 1977 74: 628; AF et al.





Proc. Natl. Acad. Sci. 1980 77: 3519.



116
Regulation of
Caspase Apoptosis. Assays for caspase
Preferred embodiments of the invention include



apoptosis of immune
apoptosis are well known in the art and may
using polypeptides of the invention (or



cells (such as mast
be used or routinely modified to assess the
antibodies, agonists, or antagonists thereof) in



cells).
ability of polypeptides of the invention
detection, diagnosis, prevention, and/or




(including antibodies and agonists or
treatment of asthma, allergy, hypersensitivity




antagonists of the invention) to regulate
and inflammation.




caspase protease-mediated apoptosis in





immune cells (such as, for example, in mast





cells). Mast cells are found in connective





and mucosal tissues throughout the body,





and their activation via immunoglobulin E-





antigen, promoted by T helper cell type 2





cytokines, is an important component of





allergic disease. Dysregulation of mast cell





apoptosis may play a role in allergic disease





and mast cell tumor survival. Exemplary





assays for caspase apoptosis that may be





used or routinely modified to test capase





apoptosis activity induced by polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include the assays disclosed in: Masuda A,





et al., J Biol Chem, 276(28): 26107-26113





(2001); Yeatman C F 2nd, et al., J Exp Med,





192(8): 1093-1103 (2000); Lee et al., FEBS





Lett 485(2-3): 122-126 (2000); Nor et al., J





Vasc Res 37(3): 209-218 (2000); and





Karsan and Harlan, J Atheroscler Thromb





3(2): 75-80 (1996); the contents of each of





which are herein incorporated by reference





in its entirety. Immune cells that may be





used according to these assays are publicly





available (e.g., through commercial





sources). Exemplary immune cells that may





be used according to these assays include





mast cells such as the HMC human mast





cell line.



117
Regulation of
Kinase assays, for example an Elk-1 kinase
Preferred embodiments of the invention include



proliferation and/or
assay for ERK signal transduction that
using polypeptides of the invention (or



differentiation in
regulates cell proliferation or
antibodies, agonists, or antagonists thereof) in



immune cells (such
differentiation, are well known in the art
detection, diagnosis, prevention, and/or



as mast cells).
and may be used or routinely modified to
treatment of asthma, allergy, hypersensitivity




assess the ability of polypeptides of the
and inflammation.




invention (including antibodies and agonists





or antagonists of the invention) to promote





or inhibit cell proliferation, activation, and





differentiation. Exemplary assays for ERK





kinase activity that may be used or routinely





modified to test ERK kinase-induced





activity of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) include the





assays disclosed in: Ali H, et al., J Immunol,





165(12): 7215-7223 (2000); Tam S Y, et al.,





Blood, 90(5): 1807-1820 (1997); Forrer et





al., Biol Chem 379(8-9): 1101-1110 (1998);





Berra et al., Biochem Pharmacol





60(8): 1171-1178 (2000); Gupta et al., Exp





Cell Res 247(2): 495-504 (1999); Chang and





Karin, Nature 410(6824): 37-40 (2001); and





Cobb M H, Prog Biophys Mol Biol 71(3-4):





479-500 (1999); the contents of each of





which are herein incorporated by reference





in its entirety. Exemplary immune cells that





may be used according to these assays





include human mast cells such as the HMC-





1 cell line.



118
Regulation of
Assays for the regulation of transcription of
A highly preferred indication is diabetes



transcription of Malic
Malic Enzyme are well-known in the art
mellitus. An additional highly preferred



Enzyme in adipocytes
and may be used or routinely modified to
indication is a complication associated with




assess the ability of polypeptides of the
diabetes (e.g., diabetic retinopathy, diabetic




invention (including antibodies and agonists
nephropathy, kidney disease (e.g., renal failure,




or antagonists of the invention) to regulate
nephropathy and/or other diseases and disorders




transcription of Malic Enzyme, a key
as described in the “Renal Disorders” section




enzyme in lipogenesis. Malic enzyme is
below), diabetic neuropathy, nerve disease and




involved in lipogenesis and its expression is
nerve damage (e.g., due to diabetic neuropathy),




stimulted by insulin. ME promoter contains
blood vessel blockage, heart disease, stroke,




two direct repeat (DR1)-like elements
impotence (e.g., due to diabetic neuropathy or




MEp and MEd identified as putative PPAR
blood vessel blockage), seizures, mental




response elements. ME promoter may also
confusion, drowsiness, nonketotic




responds to AP1 and other transcription
hyperglycemic-hyperosmolar coma,




factors. Exemplary assays that may be used
cardiovascular disease (e.g., heart disease,




or routinely modified to test for regulation
atherosclerosis, microvascular disease,




of transcription of Malic Enzyme (in
hypertension, stroke, and other diseases and




adipoocytes) by polypeptides of the
disorders as described in the “Cardiovascular




invention (including antibodies and agonists
Disorders” section below), dyslipidemia,




or antagonists of the invention) include
endocrine disorders (as described in the




assays disclosed in: Streeper, R. S., et al.,
“Endocrine Disorders” section below),




Mol Endocrinol, 12(11): 1778-91 (1998);
neuropathy, vision impairment (e.g., diabetic




Garcia-Jimenez, C., et al., Mol Endocrinol,
retinopathy and blindness), ulcers and impaired




8(10): 1361-9 (1994); Barroso, I., et al., J
wound healing, and infection (e.g., infectious




Biol Chem, 274(25): 17997-8004 (1999);
diseases and disorders as described in the




Ijpenberg, A., et al., J Biol Chem,
“Infectious Diseases” section below, especially




272(32): 20108-20117 (1997); Berger, et al.,
of the urinary tract and skin), carpal tunnel




Gene 66: 1-10 (1988); and, Cullen, B., et al.,
syndrome and Dupuytren's contracture).




Methods in Enzymol. 216: 362-368 (1992),
An additional highly preferred indication is




the contents of each of which is herein
obesity and/or complications associated with




incorporated by reference in its entirety.
obesity. Additional highly preferred indications




Hepatocytes that may be used according to
include weight loss or alternatively, weight




these assays are publicly available (e.g.,
gain. Aditional highly preferred




through the ATCC TM) and/or may be
indications are complications associated with




routinely generated. Exemplary
insulin resistance.




hepatocytes that may be used according to





these assays includes the H4IIE rat liver





hepatoma cell line.



119
Regulation of
Assays for the regulation of transcription of
A highly preferred indication is diabetes



transcription of Malic
Malic Enzyme are well-known in the art
mellitus. An additional highly preferred



Enzyme in
and may be used or routinely modified to
indication is a complication associated with



hepatocytes
assess the ability of polypeptides of the
diabetes (e.g., diabetic retinopathy, diabetic




invention (including antibodies and agonists
nephropathy, kidney disease (e.g., renal failure,




or antagonists of the invention) to regulate
nephropathy and/or other diseases and disorders




transcription of Malic Enzyme, a key
as described in the ““Renal Disorders”” section




enzyme in lipogenesis. Malic enzyme is
below), diabetic neuropathy, nerve disease and




involved in lipogenesis and its expression is
nerve damage (e.g., due to diabetic neuropathy),




stimulted by insulin. ME promoter contains
blood vessel blockage, heart disease, stroke,




two direct repeat (DR1)-like elements
impotence (e.g., due to diabetic neuropathy or




MEp and MEd identified as putative PPAR
blood vessel blockage), seizures, mental




response elements. ME promoter may also
confusion, drowsiness, nonketotic




responds to AP1 and other transcription
hyperglycemic-hyperosmolar coma,




factors. Exemplary assays that may be used
cardiovascular disease (e.g., heart disease,




or routinely modified to test for regulation
atherosclerosis, microvascular disease,




of transcription of Malic Enzyme (in
hypertension, stroke, and other diseases and




hepatocytes) by polypeptides of the
disorders as described in the ““Cardiovascular




invention (including antibodies and agonists
Disorders”” section below), dyslipidemia,




or antagonists of the invention) include
endocrine disorders (as described in the




assays disclosed in: Streeper, R. S., et al.,
““Endocrine Disorders”” section below),




Mol Endocrinol, 12(11): 1778-91 (1998);
neuropathy, vision impairment (e.g., diabetic




Garcia-Jimenez, C., et al., Mol Endocrinol,
retinopathy and blindness), ulcers and impaired




8(10): 1361-9 (1994); Barroso, I., et al., J
wound healing, and infection (e.g., infectious




Biol Chem, 274(25): 17997-8004 (1999);
diseases and disorders as described in the




Ijpenberg, A., et al., J Biol Chem,
““Infectious Diseases”” section below,




272(32): 20108-20117 (1997); Berger, et al.,
especially of the urinary tract and skin), carpal




Gene 66: 1-10 (1988); and, Cullen, B., et al.,
tunnel syndrome and Dupuytren's contracture).




Methods in Enzymol. 216: 362-368 (1992),
An additional highly preferred indication is




the contents of each of which is herein
obesity and/or complications associated with




incorporated by reference in its entirety.
obesity. Additional highly preferred indications




Hepatocytes that may be used according to
include weight loss or alternatively, weight




these assays are publicly available (e.g.,
gain. Aditional highly preferred




through the ATCC TM) and/or may be
indications are complications associated with




routinely generated. Exemplary
insulin resistance.




hepatocytes that may be used according to





these assays includes the mouse 3T3-L1 cell





line. 3T3-L1 is a mouse preadipocyte cell





line (adherent). It is a continuous substrain





of 3T3 fibroblasts developed through clonal





isolation. Cells undergo a pre-adipocyte to





adipose-like conversion under appropriate





differentiation culture conditions.



120
Regulation of
Assays for the regulation of transcription
A highly preferred indication is diabetes



transcription through
through the FAS promoter element are well-
mellitus. An additional highly preferred



the FAS promoter
known in the art and may be used or
indication is a complication associated with



element in
routinely modified to assess the ability of
diabetes (e.g., diabetic retinopathy, diabetic



hepatocytes
polypeptides of the invention (including
nephropathy, kidney disease (e.g., renal failure,




antibodies and agonists or antagonists of the
nephropathy and/or other diseases and disorders




invention) to activate the FAS promoter
as described in the “Renal Disorders” section




element in a reporter construct and to
below), diabetic neuropathy, nerve disease and




regulate transcription of FAS, a key enzyme
nerve damage (e.g., due to diabetic neuropathy),




for lipogenesis. FAS promoter is regulated
blood vessel blockage, heart disease, stroke,




by many transcription factors including
impotence (e.g., due to diabetic neuropathy or




SREBP. Insulin increases FAS gene
blood vessel blockage), seizures, mental




transcription in livers of diabetic mice. This
confusion, drowsiness, nonketotic




stimulation of transcription is also
hyperglycemic-hyperosmolar coma,




somewhat glucose dependent. Exemplary
cardiovascular disease (e.g., heart disease,




assays that may be used or routinely
atherosclerosis, microvascular disease,




modified to test for FAS promoter element
hypertension, stroke, and other diseases and




activity (in hepatocytes) by polypeptides of
disorders as described in the “Cardiovascular




the invention (including antibodies and
Disorders” section below), dyslipidemia,




agonists or antagonists of the invention)
endocrine disorders (as described in the




include assays disclosed in Xiong, S., et al.,
“Endocrine Disorders” section below),




Proc Natl Acad Sci U.S.A., 97(8): 3948-53
neuropathy, vision impairment (e.g., diabetic




(2000); Roder, K., et al., Eur J Biochem,
retinopathy and blindness), ulcers and impaired




260(3): 743-51 (1999); Oskouian B, et al.,
wound healing, and infection (e.g., infectious




Biochem J, 317 (Pt 1): 257-65 (1996);
diseases and disorders as described in the




Berger, et al., Gene 66: 1-10 (1988); and,
“Infectious Diseases” section below, especially




Cullen, B., et al., Methods in Enzymol.
of the urinary tract and skin), carpal tunnel




216: 362-368 (1992), the contents of each of
syndrome and Dupuytren's contracture).




which is herein incorporated by reference in
An additional highly preferred indication is




its entirety. Hepatocytes that may be used
obesity and/or complications associated with




according to these assays, such as H4IIE
obesity. Additional highly preferred indications




cells, are publicly available (e.g., through
include weight loss or alternatively, weight




the ATCC TM) and/or may be routinely
gain. Aditional highly preferred




generated. Exemplary hepatocytes that may
indications are complications associated with




be used according to these assays include
insulin resistance.




rat liver hepatoma cell line(s) inducible with





glucocorticoids, insulin, or cAMP





derivatives.



121
Regulation of
Assays for the regulation of transcription
A highly preferred indication is diabetes



transcription through
through the PEPCK promoter are well-
mellitus. An additional highly preferred



the PEPCK promoter
known in the art and may be used or
indication is a complication associated with



in hepatocytes
routinely modified to assess the ability of
diabetes (e.g., diabetic retinopathy, diabetic




polypeptides of the invention (including
nephropathy, kidney disease (e.g., renal failure,




antibodies and agonists or antagonists of the
nephropathy and/or other diseases and disorders




invention) to activate the PEPCK promoter
as described in the “Renal Disorders” section




in a reporter construct and regulate liver
below), diabetic neuropathy, nerve disease and




gluconeogenesis. Exemplary assays for
nerve damage (e.g., due to diabetic neuropathy),




regulation of transcription through the
blood vessel blockage, heart disease, stroke,




PEPCK promoter that may be used or
impotence (e.g., due to diabetic neuropathy or




routinely modified to test for PEPCK
blood vessel blockage), seizures, mental




promoter activity (in hepatocytes) of
confusion, drowsiness, nonketotic




polypeptides of the invention (including
hyperglycemic-hyperosmolar coma,




antibodies and agonists or antagonists of the
cardiovascular disease (e.g., heart disease,




invention) include assays disclosed in
atherosclerosis, microvascular disease,




Berger et al., Gene 66: 1-10 (1998); Cullen
hypertension, stroke, and other diseases and




and Malm, Methods in Enzymol 216: 362-368
disorders as described in the “Cardiovascular




(1992); Henthorn et al., Proc Natl Acad
Disorders” section below), dyslipidemia,




Sci USA 85: 6342-6346 (1988); Lochhead et
endocrine disorders (as described in the




al., Diabetes 49(6): 896-903 (2000); and
“Endocrine Disorders” section below),




Yeagley et al., J Biol Chem 275(23): 17814-17820
neuropathy, vision impairment (e.g., diabetic




(2000), the contents of each of which
retinopathy and blindness), ulcers and impaired




is herein incorporated by reference in its
wound healing, infection (e.g., an infectious




entirety. Hepatocyte cells that may be used
diseases or disorders as described in the




according to these assays are publicly
“Infectious Diseases” section below, especially




available (e.g., through the ATCC TM) and/or
of the urinary tract and skin), carpal tunnel




may be routinely generated. Exemplary
syndrome and Dupuytren's contracture).




liver hepatoma cells that may be used
An additional highly preferred indication is




according to these assays include H4lle
obesity and/or complications associated with




cells, which contain a tyrosine amino
obesity. Additional highly preferred indications




transferase that is inducible with
include weight loss or alternatively, weight




glucocorticoids, insulin, or cAMP
gain. Additional highly preferred




derivatives.
indications are complications associated with





insulin resistance. Additional highly





preferred indications are disorders of the





musculoskeletal systems including myopathies,





muscular dystrophy, and/or as described herein.





Additional highly preferred indications include





glycogen storage disease (e.g., glycogenoses),





hepatitis, gallstones, cirrhosis of the liver,





degenerative or necrotic liver disease, alcoholic





liver diseases, fibrosis, liver regeneration,





metabolic disease, dyslipidemia and cholesterol





metabolism, and hepatocarcinomas.





Highly preferred indications include blood





disorders (e.g., as described below under





“Immune Activity”, “Cardiovascular





Disorders”, and/or “Blood-Related Disorders”),





immune disorders (e.g., as described below





under “Immune Activity”), infection (e.g., an





infectious disease and/or disorder as described





below under “Infectious Disease”), endocrine





disorders (e.g., as described below under





“Endocrine Disorders”), and neural disorders





(e.g., as described below under “Neural Activity





and Neurological Diseases”).





Additional preferred indications include





neoplastic diseases (e.g., as described below





under “Hyperproliferative Disorders”).





Preferred indications include neoplasms and





cancers, such as, leukemia, lymphoma, prostate,





breast, lung, colon, pancreatic, esophageal,





stomach, brain, and urinary cancer. A highly





preferred indication is liver cancer. Other





preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia.


122
Regulation of
Assays for the regulation of transcription
A highly preferred indication is diabetes



transcription via
through the DMEF1 response element are
mellitus. Additional highly preferred



DMEF1 response
well-known in the art and may be used or
indications include complications associated



element in adipocytes
routinely modified to assess the ability of
with diabetes (e.g., diabetic retinopathy,



and pre-adipocytes
polypeptides of the invention (including
diabetic nephropathy, kidney disease (e.g., renal




antibodies and agonists or antagonists of the
failure, nephropathy and/or other diseases and




invention) to activate the DMEF1 response
disorders as described in the “Renal Disorders”




element in a reporter construct (such as that
section below), diabetic neuropathy, nerve




containing the GLUT4 promoter) and to
disease and nerve damage (e.g., due to diabetic




regulate insulin production. The DMEF1
neuropathy), blood vessel blockage, heart




response element is present in the GLUT4
disease, stroke, impotence (e.g., due to diabetic




promoter and binds to MEF2 transcription
neuropathy or blood vessel blockage), seizures,




factor and another transcription factor that is
mental confusion, drowsiness, nonketotic




required for insulin regulation of Glut4
hyperglycemic-hyperosmolar coma,




expression in skeletal muscle. GLUT4 is
cardiovascular disease (e.g., heart disease,




the primary insulin-responsive glucose
atherosclerosis, microvascular disease,




transporter in fat and muscle tissue.
hypertension, stroke, and other diseases and




Exemplary assays that may be used or
disorders as described in the “Cardiovascular




routinely modified to test for DMEF1
Disorders” section below), dyslipidemia,




response element activity (in adipocytes and
endocrine disorders (as described in the




pre-adipocytes) by polypeptides of the
“Endocrine Disorders” section below),




invention (including antibodies and agonists
neuropathy, vision impairment (e.g., diabetic




or antagonists of the invention) include
retinopathy and blindness), ulcers and impaired




assays disclosed in Thai, M. V., et al., J Biol
wound healing, and infection (e.g., infectious




Chem, 273(23): 14285-92 (1998); Mora, S.,
diseases and disorders as described in the




et al., J Biol Chem, 275(21): 16323-8
“Infectious Diseases” section below, especially




(2000); Liu, M. L., et al., J Biol Chem,
of the urinary tract and skin). An additional




269(45): 28514-21 (1994); “Identification of
highly preferred indication is obesity and/or




a 30-base pair regulatory element and novel
complications associated with obesity.




DNA binding protein that regulates the
Additional highly preferred indications include




human GLUT4 promoter in transgenic
weight loss or alternatively, weight gain.




mice”, J Biol Chem. 2000 August
Additional highly preferred indications are




4; 275(31): 23666-73; Berger, et al., Gene
complications associated with insulin




66: 1-10 (1988); and, Cullen, B., et al.,
resistance.




Methods in Enzymol. 216: 362-368 (1992),





the contents of each of which is herein





incorporated by reference in its entirety.





Adipocytes and pre-adipocytes that may be





used according to these assays are publicly





available (e.g., through the ATCC TM) and/or





may be routinely generated. Exemplary





cells that may be used according to these





assays include the mouse 3T3-L1 cell line





which is an adherent mouse preadipocyte





cell line. Mouse 3T3-L1 cells are a





continuous substrain of 3T3 fibroblasts





developed through clonal isolation. These





cells undergo a pre-adipocyte to adipose-





like conversion under appropriate





differentiation culture conditions.



123
Regulation of
Assays for the regulation of viability and
A highly preferred indication is diabetes



viability and
proliferation of cells in vitro are well-known
mellitus. An additional highly preferred



proliferation of
in the art and may be used or routinely
indication is a complication associated with



pancreatic beta cells.
modified to assess the ability of
diabetes (e.g., diabetic retinopathy, diabetic




polypeptides of the invention (including
nephropathy, kidney disease (e.g., renal failure,




antibodies and agonists or antagonists of the
nephropathy and/or other diseases and disorders




invention) to regulate viability and
as described in the ““Renal Disorders”” section




proliferation of pancreatic beta cells. For
below), diabetic neuropathy, nerve disease and




example, the Cell Titer-Glo luminescent cell
nerve damage (e.g., due to diabetic neuropathy),




viability assay measures the number of
blood vessel blockage, heart disease, stroke,




viable cells in culture based on quantitation
impotence (e.g., due to diabetic neuropathy or




of the ATP present which signals the
blood vessel blockage), seizures, mental




presence of metabolically active cells.
confusion, drowsiness, nonketotic




Exemplary assays that may be used or
hyperglycemic-hyperosmolar coma,




routinely modified to test regulation of
cardiovascular disease (e.g., heart disease,




viability and proliferation of pancreatic beta
atherosclerosis, microvascular disease,




cells by polypeptides of the invention
hypertension, stroke, and other diseases and




(including antibodies and agonists or
disorders as described in the ““Cardiovascular




antagonists of the invention) include assays
Disorders”” section below), dyslipidemia,




disclosed in: Friedrichsen B N, et al., Mol
endocrine disorders (as described in the




Endocrinol, 15(1): 136-48 (2001); Huotari M A,
““Endocrine Disorders”” section below),




et al., Endocrinology, 139(4): 1494-9
neuropathy, vision impairment (e.g., diabetic




(1998); Hugl S R, et al., J Biol Chem 1998
retinopathy and blindness), ulcers and impaired




July10; 273(28): 17771-9 (1998), the contents
wound healing, and infection (e.g., infectious




of each of which is herein incorporated by
diseases and disorders as described in the




reference in its entirety. Pancreatic cells
““Infectious Diseases”” section below,




that may be used according to these assays
especially of the urinary tract and skin), carpal




are publicly available (e.g., through the
tunnel syndrome and Dupuytren's contracture).




ATCC TM) and/or may be routinely
An additional highly preferred indication is




generated. Exemplary pancreatic cells that
obesity and/or complications associated with




may be used according to these assays
obesity. Additional highly preferred indications




include rat INS-1 cells. INS-1 cells are a
include weight loss or alternatively, weight




semi-adherent cell line established from
gain. Additional highly preferred




cells isolated from an X-ray induced rat
indications are complications associated with




transplantable insulinoma. These cells
insulin resistance.




retain characteristics typical of native





pancreatic beta cells including glucose





inducible insulin secretion. References:





Asfari et al. Endocrinology 1992 130: 167.





Additional exemplary pancreatic cells that





may be used according to these assays





include HITT15 Cells. HITT15 are an





adherent epithelial cell line established from





Syrian hamster islet cells transformed with





SV40. These cells express glucagon,





somatostatin, and glucocorticoid receptors.





The cells secrete insulin, which is





stimulated by glucose and glucagon and





suppressed by somatostatin or





glucocorticoids. ATTC# CRL-1777 Refs:





Lord and Ashcroft. Biochem. J. 219: 547-551;





Santerre et al. Proc. Natl. Acad. Sci.





USA 78: 4339-4343, 1981.



124
Regulation of
Assays for the regulation (i.e. increases or
Highly preferred indications include



viability or
decreases) of viability and proliferation of
eosinophilia, asthma, allergy, hypersensitivity



proliferation of
cells in vitro are well-known in the art and
reactions, inflammation, and inflammatory



immune cells (such
may be used or routinely modified to assess
disorders. Additional highly preferred



as human eosinophil
the ability of polypeptides of the invention
indications include immune and hematopoietic



EOL-1 cells).
(including antibodies and agonists or
disorders (e.g., as described below under




antagonists of the invention) to regulate
“Immune Activity”, and “Blood-Related




viability and proliferation of eosinophil
Disorders”), autoimmune diseases (e.g.,




cells and cell lines. For example, the
rheumatoid arthritis, systemic lupus




CellTiter-Gloô Luminescent Cell Viability
erythematosis, Crohn''s disease, multiple




Assay (Promega Corp., Madison, WI, USA)
sclerosis and/or as described below),




can be used to measure the number of
immunodeficiencies (e.g., as described below).




viable cells in culture based on quantitation
Highly preferred indications also include




of the ATP present which signals the
boosting or inhibiting immune cell




presence of metabolically active cells.
proliferation. Preferred indications include




Eosinophils are a type of immune cell
neoplastic diseases (e.g., leukemia, lymphoma,




important in allergic responses; they are
and/or as described below under




recruited to tissues and mediate the
“Hyperproliferative Disorders”). Highly




inflammtory response of late stage allergic
preferred indications include boosting an




reaction. Eosinophil cell lines that may be
eosinophil-mediated immune response, and




used according to these assays are publicly
suppressing an eosinophil-mediated immune




available and/or may be routinely generated.
response.




Exemplary eosinophil cells that may be





used according to these assays include





EOL-1 Cells.



125
SEAP in 293/ISRE




126
SEAP in Alk Phos





C2C12




127
SEAP in ATP-3T3-





L1




128
SEAP in





HepG2/Squale-





synthetase(stimulation)




129
SEAP in HIB/CRE




130
SEAP in Jurkat-AP1




131
SEAP in Jurkat/IL4





promoter




132
SEAP in Jurkat/IL4





promoter (antiCD3





co-stim)




133
SEAP in





Ku812/NFkB (TNF





synergy)




134
SEAP in Molt4/SRE




135
SEAP in





NK16/STAT6




136
SEAP in OE-21




137
SEAP in OE-33




138
SEAP in SW480




139
SEAP in UMR-106




140
Stimulation of
Assays for measuring calcium flux are well-
A highly preferred indication is diabetes



Calcium Flux in
known in the art and may be used or
mellitus. An additional highly preferred



pancreatic beta cells.
routinely modified to assess the ability of
indication is a complication associated with




polypeptides of the invention (including
diabetes (e.g., diabetic retinopathy, diabetic




antibodies and agonists or antagonists of the
nephropathy, kidney disease (e.g., renal failure,




invention) to mobilize calcium. For
nephropathy and/or other diseases and disorders




example, the FLPR assay may be used to
as described in the “Renal Disorders” section




measure influx of calcium. Cells normally
below), diabetic neuropathy, nerve disease and




have very low concentrations of cytosolic
nerve damage (e.g., due to diabetic neuropathy),




calcium compared to much higher
blood vessel blockage, heart disease, stroke,




extracellular calcium. Extracellular factors
impotence (e.g., due to diabetic neuropathy or




can cause an influx of calcium, leading to
blood vessel blockage), seizures, mental




activation of calcium responsive signaling
confusion, drowsiness, nonketotic




pathways and alterations in cell functions.
hyperglycemic-hyperosmolar coma,




Exemplary assays that may be used or
cardiovascular disease (e.g., heart disease,




routinely modified to measure calcium flux
atherosclerosis, microvascular disease,




by polypeptides of the invention (including
hypertension, stroke, and other diseases and




antibodies and agonists or antagonists of the
disorders as described in the “Cardiovascular




invention) include assays disclosed in: Satin
Disorders” section below), dyslipidemia,




L S, et al., Endocrinology, 136(10): 4589-601
endocrine disorders (as described in the




(1995); Mogami H, et al., Endocrinology,
“Endocrine Disorders” section below),




136(7): 2960-6 (1995); Richardson S B, et
neuropathy, vision impairment (e.g., diabetic




al., Biochem J, 288 (Pt 3): 847-51 (1992);
retinopathy and blindness), ulcers and impaired




and, Meats, J E, et al., Cell Calcium 1989
wound healing, and infection (e.g., infectious




November-December; 10(8): 535-41 (1989), 
diseases and disorders as described in the




contents the of each of which is herein incorporated 
“Infectious Diseases” section below, especially




by reference in its entirety. Pancreatic cells
of the urinary tract and skin), carpal tunnel




that may be used according to these assays
syndrome and Dupuytren's contracture).




are publicly available (e.g., through the
An additional highly preferred indication is




ATCC TM) and/or may be routinely
obesity and/or complications associated with




generated. Exemplary pancreatic cells that
obesity. Additional highly preferred indications




may be used according to these assays
include weight loss or alternatively, weight




include HITT15 Cells. HITT15 are an
gain. Aditional highly preferred




adherent epithelial cell line established from
indications are complications associated with




Syrian hamster islet cells transformed with
insulin resistance.




SV40. These cells express glucagon,





somatostatin, and glucocorticoid receptors.





The cells secrete insulin, which is





stimulated by glucose and glucagon and





suppressed by somatostatin or





glucocorticoids. ATTC# CRL-1777 Refs:





Lord and Ashcroft. Biochem. J. 219: 547-551;





Santerre et al. Proc. Natl. Acad. Sci.





USA 78: 4339-4343, 1981.



141
Stimulation of insulin
Assays for measuring secretion of insulin
A highly preferred indication is diabetes



secretion from
are well-known in the art and may be used
mellitus. An additional highly preferred



pancreatic beta cells.
or routinely modified to assess the ability of
indication is a complication associated with




polypeptides of the invention (including
diabetes (e.g., diabetic retinopathy, diabetic




antibodies and agonists or antagonists of the
nephropathy, kidney disease (e.g., renal failure,




invention) to stimulate insulin secretion.
nephropathy and/or other diseases and disorders




For example, insulin secretion is measured
as described in the ““Renal Disorders”” section




by FMAT using anti-rat insulin antibodies.
below), diabetic neuropathy, nerve disease and




Insulin secretion from pancreatic beta cells
nerve damage (e.g., due to diabetic neuropathy),




is upregulated by glucose and also by
blood vessel blockage, heart disease, stroke,




certain proteins/peptides, and disregulation
impotence (e.g., due to diabetic neuropathy or




is a key component in diabetes. Exemplary
blood vessel blockage), seizures, mental




assays that may be used or routinely
confusion, drowsiness, nonketotic




modified to test for stimulation of insulin
hyperglycemic-hyperosmolar coma,




secretion (from pancreatic cells) by
cardiovascular disease (e.g., heart disease,




polypeptides of the invention (including
atherosclerosis, microvascular disease,




antibodies and agonists or antagonists of the
hypertension, stroke, and other diseases and




invention) include assays disclosed in:
disorders as described in the ““Cardiovascular




Ahren, B., et al., Am J Physiol, 277(4 Pt
Disorders”” section below), dyslipidemia,




2): R959-66 (1999); Li, M., et al.,
endocrine disorders (as described in the




Endocrinology, 138(9): 3735-40 (1997);
““Endocrine Disorders”” section below),




Kim, K. H., et al., FEBS Lett, 377(2): 237-9
neuropathy, vision impairment (e.g., diabetic




(1995); and, Miraglia S et. al., Journal of
retinopathy and blindness), ulcers and impaired




Biomolecular Screening, 4: 193-204 (1999),
wound healing, and infection (e.g., infectious




the contents of each of which is herein
diseases and disorders as described in the




incorporated by reference in its entirety.
““Infectious Diseases”” section below,




Pancreatic cells that may be used according
especially of the urinary tract and skin), carpal




to these assays are publicly available (e.g.,
tunnel syndrome and Dupuytren's contracture).




through the ATCC TM) and/or may be
An additional highly preferred indication is




routinely generated. Exemplary pancreatic
obesity and/or complications associated with




cells that may be used according to these
obesity. Additional highly preferred indications




assays include rat INS-1 cells. INS-1 cells
include weight loss or alternatively, weight




are a semi-adherent cell line established
gain. Aditional highly preferred




from cells isolated from an X-ray induced
indications are complications associated with




rat transplantable insulinoma. These cells
insulin resistance.




retain characteristics typical of native





pancreatic beta cells including glucose





inducible insulin secretion. References:





Asfari et al. Endocrinology 1992 130: 167.



142
TNFa in Human T-





cell 293T




143
TNFa in Human T-





cell 2B9




144
Upregulation of
CD152 FMAT. CD152 (a.k.a. CTLA-4)
A highly preferred embodiment of the invention



CD152 and activation
expression is restricted to activated T cells.
includes a method for activating T cells. An



of T cells
CD152 is a negative regulator of T cell
alternative highly preferred embodiment of the




proliferation. Reduced CD152 expression
invention includes a method for inhibiting the




has been linked to hyperproliferative and
activation of and/or inactivating T cells. A




autoimmune diseases. Overexpression of
highly preferred embodiment of the invention




CD152 may lead to impaired
includes a method for inhibiting T cell




immunoresponses. Assays for
proliferation. An alternative highly preferred




immunomodulatory proteins important in
embodiment of the invention includes a method




the maintenance of T cell homeostasis and
for stimulating T cell proliferation. Highly




expressed almost exclusively on CD4+ and
preferred indications include blood disorders




CD8+ T cells are well known in the art and
(e.g., as described below under “Immune




may be used or routinely modified to assess
Activity”, “Blood-Related Disorders”, and/or




the ability of polypeptides of the invention
““Cardiovascular Disorders””), Highly




(including antibodies and agonists or
preferred indications include autoimmune




antagonists of the invention) to modulate
diseases (e.g., rheumatoid arthritis, systemic




the activation of T cells, maintain T cell
lupus erythematosis, multiple sclerosis and/or




homeostasis, and/or mediate humoral or
as described below), immunodeficiencies (e.g.,




cell-mediated immunity. Exemplary assays
as described below), boosting a T cell-mediated




that test for immunomodulatory proteins
immune response, and suppressing a T cell-




evaluate the upregulation of cell surface
mediated immune response. Highly




markers, such as CD152, and the activation
preferred indications include neoplastic diseases




of T cells. Such assays that may be used or
(e.g., leukemia, lymphoma, and/or as described




routinely modified to test
below under “Hyperproliferative Disorders”).




immunomodulatory activity of polypeptides
Additionally, highly preferred indications




of the invention (including antibodies and
include neoplasms and cancers, such as, for




agonists or antagonists of the invention)
example, leukemia, lymphoma, melanoma, and




include, for example, the assays disclosed in
prostate, breast, lung, colon, pancreatic,




Miraglia et al., J Biomolecular Screening
esophageal, stomach, brain, liver and urinary




4: 193-204 (1999); Rowland et al.,
cancer. Other preferred indications include




““Lymphocytes: a practical approach””
benign dysproliferative disorders and pre-




Chapter 6: 138-160 (2000); McCoy et al.,
neoplastic conditions, such as, for example,




Immunol Cell Biol 77(1): 1-10 (1999);
hyperplasia, metaplasia, and/or dysplasia.




Oostervegal et al., Curr Opin Immunol
Preferred indications include anemia,




11(3): 294-300 (1999); and Saito T, Curr
pancytopenia, leukopenia, thrombocytopenia,




Opin Immunol 10(3): 313-321 (1998), the
Hodgkin's disease, acute lymphocytic anemia




contents of each of which are herein
(ALL), plasmacytomas, multiple myeloma,




incorporated by reference in its entirety.
Burkitt's lymphoma, arthritis, AIDS,




Human T cells that may be used according
granulomatous disease, inflammatory bowel




to these assays may be isolated using
disease, sepsis, neutropenia, neutrophilia,




techniques disclosed herein or otherwise
psoriasis, immune reactions to transplanted




known in the art. Human T cells are
organs and tissues, hemophilia,




primary human lymphocytes that mature in
hypercoagulation, diabetes mellitus,




the thymus and express a T Cell receptor
endocarditis, meningitis, Lyme Disease,




and CD3, CD4, or CD8. These cells
inflammation and inflammatory disorders, and




mediate humoral or cell-mediated immunity
asthma and allergy. An additional preferred




and may be preactivated to enhance
indication is infection (e.g., as described below




responsiveness to immunomodulatory
under “Infectious Disease”).




factors.



145
Upregulation of
CD154 FMAT. CD154 (a.k.a., CD40L)




CD154 and activation
expression is induced following activation




of T cells
of T cells. Interaction between CD154 and





CD40 on B cells is required for correct





antibody class switching and germinal





center formation. Mutations in CD154 are





linked to immunodeficiencies and increased





susceptibility to infections. Assays for





immunomodulatory proteins important for





antibody class switching and TH1 function





and expressed on activated T helper





lymphocytes are well known in the art and





may be used or routinely modified to assess





the ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to modulate





the activation of T cells, modulate antibody





class switching, mediate TH1 function,





and/or mediate humoral or cell-mediated





immunity. Exemplary assays that test for





immunomodulatory proteins evaluate the





upregulation of cell surface markers, such





as CD154, and the activation of T cells.





Such assays that may be used or routinely





modified to test immunomodulatory activity





of polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) include, for example, the assays





disclosed in Miraglia et al., J Biomolecular





Screening 4: 193-204 (1999); Rowland et





al., “Lymphocytes: a practical approach”





Chapter 6: 138-160 (2000); Mackey et al., J





Leukoc Biol 63(4): 418: 428 (1998); and





Skov et al., 164(7): 3500-3505 (2000), the





contents of each of which are herein





incorporated by reference in its entirety.





Human T cells that may be used according





to these assays may be isolated using





techniques disclosed herein or otherwise





known in the art. Human T cells are





primary human lymphocytes that mature in





the thymus and express a T Cell receptor





and CD3, CD4, or CD8. These cells





mediate humoral or cell-mediated immunity





and may be preactivated to enhance





responsiveness to immunomodulatory





factors.



146
Upregulation of
CD69 FMAT. CD69 is an activation
A highly preferred embodiment of the invention



CD69 and activation
marker that is expressed on activated T
includes a method for activating T cells. An



of T cells
cells, B cells, and NK cells. CD69 is not
alternative highly preferred embodiment of the




expressed on resting T cells, B cells, or NK
invention includes a method for inhibiting the




cells. CD69 has been found to be associated
activation of and/or inactivating T cells. A




with inflammation. Assays for
highly preferred embodiment of the invention




immunomodulatory proteins expressed in T
includes a method for activation B cells. An




cells, B cells, and leukocytes are well
alternative highly preferred embodiment of the




known in the art and may be used or
invention includes a method for inhibiting the




routinely modified to assess the ability of
activation of and/or inactivating B cells. A




polypeptides of the invention (including
highly preferred embodiment of the invention




antibodies and agonists or antagonists of the
includes a method for activating NK cells. An




invention) to modulate the activation of T
alternative highly preferred embodiment of the




cells, and/or mediate humoral or cell-
invention includes a method for inhibiting




mediated immunity. Exemplary assays that
activation of and/or inactivation NK cells.




test for immunomodulatory proteins
Highly preferred indications include




evaluate the upregulation of cell surface
inflammation and inflammatory disorders (e.g.,




markers, such as CD69, and the activation
as described below under “Immune Activity”).




of T cells. Such assays that may be used or
Preferred indications include blood disorders




routinely modified to test
(e.g., as described below under “Immune




immunomodulatory activity of polypeptides
Activity”, “Blood-Related Disorders”, and/or




of the invention (including antibodies and
““Cardiovascular Disorders””). Highly preferred




agonists or antagonists of the invention)
indications include autoimmune diseases (e.g.,




include, for example, the assays disclosed in
rheumatoid arthritis, systemic lupus




Miraglia et al., J Biomolecular Screening
erythematosis, multiple sclerosis and/or as




4: 193-204 (1999); Rowland et al.,
described below), immunodeficiencies (e.g., as




““Lymphocytes: a practical approach””
described below), boosting a T cell-mediated




Chapter 6: 138-160 (2000); Ferenczi et al., J
immune response and alternatively suppressing




Autoimmun 14(1): 63-78 (200); Werfel et
a T cell-mediated immune response, and




al., Allergy 52(4): 465-469 (1997); Taylor-
boosting a B cell-mediated immune response




Fishwick and Siegel, Eur J Immunol
and alternatively suppressing a B cell-mediated




25(12): 3215-3221 (1995); and Afetra et al.,
immune response. An additional highly




Ann Rheum Dis 52(6): 457-460 (1993), the
preferred indication includes infection (e.g., as




contents of each of which are herein
described below under “Infectious Disease”).




incorporated by reference in its entirety.
Preferred indications also include anemia,




Human T cells that may be used according
pancytopenia, leukopenia, thrombocytopenia,




to these assays may be isolated using
Hodgkin's disease, acute lymphocytic anemia




techniques disclosed herein or otherwise
(ALL), plasmacytomas, multiple myeloma,




known in the art. Human T cells are
Burkitt's lymphoma, arthritis, AIDS,




primary human lymphocytes that mature in
granulomatous disease, inflammatory bowel




the thymus and express a T Cell receptor
disease, sepsis, neutropenia, neutrophilia,




and CD3, CD4, or CD8. These cells
psoriasis, suppression of immune reactions to




mediate humoral or cell-mediated immunity
transplanted organs and tissues, hemophilia,




and may be preactivated to enhance
hypercoagulation, diabetes mellitus,




responsiveness to immunomodulatory
endocarditis, meningitis, Lyme Disease,




factors.
inflammation and inflammatory disorders,





asthma, and allergies. Preferred indications





also include neoplastic diseases (e.g., leukemia,





lymphoma, and/or as described below under





“Hyperproliferative Disorders”). Preferred





indications include neoplasms, such as, for





example, leukemia, lymphoma, and prostate,





breast, lung, colon, pancreatic, esophageal,





stomach, brain, liver and urinary cancer. Other





preferred indications include benign





dysproliferative disorders and pre-neoplastic





conditions, such as, for example, hyperplasia,





metaplasia, and/or dysplasia.


147
Upregulation of
CD71 FMAT. CD71 is the transferrin




CD71 and activation
receptor. Transferrin is a major iron




of T cells
carrying protein that is essential for cell





proliferation. CD71 is expressed





predominantly on cells that are actively





proliferating. Assays for





immunomodulatory proteins expressed on





activated T cells, B cells, and most





proliferating cells are well known in the art





and may be used or routinely modified to





assess the ability of polypeptides of the





invention (including antibodies and agonists





or antagonists of the invention) to modulate





the activation of T cells, and/or mediate





humoral or cell-mediated immunity.





Exemplary assays that test for





immunomodulatory proteins evaluate the





upregulation of cell surface markers, such





as CD71, and the activation of T cells.





Such assays that may be used or routinely





modified to test immunomodulatory activity





of polypeptides of the invention (including





antibodies and agonists or antagonists of the





invention) include, for example, the assays





disclosed in Miraglia et al., J Biomolecular





Screening 4: 193-204 (1999); Rowland et





al., “Lymphocytes: a practical approach”





Chapter 6: 138-160 (2000); and Afetra et al.,





Ann Rheum Dis 52(6): 457-460 (1993), the





contents of each of which are herein





incorporated by reference in its entirety.





Human T cells that may be used according





to these assays may be isolated using





techniques disclosed herein or otherwise





known in the art. Human T cells are





primary human lymphocytes that mature in





the thymus and express a T Cell receptor





and CD3, CD4, or CD8. These cells





mediate humoral or cell-mediated immunity





and may be preactivated to enhance





responsiveness to immunomodulatory





factors.



148
Upregulation of
HLA-DR FMAT. MHC class II is essential




HLA-DR and
for correct presentation of antigen to CD4+




activation of T cells
T cells. Deregulation of MHC class II has





been associated with autoimmune diseases





(e.g., diabetes, rheumatoid arthritis,





systemic lupus erythematosis, and multiple





sclerosis). Assays for immunomodulatory





proteins expressed on MHC class II





expressing T cells and antigen presenting





cells are well known in the art and may be





used or routinely modified to assess the





ability of polypeptides of the invention





(including antibodies and agonists or





antagonists of the invention) to modulate





the activation of T cells, and/or mediate





humoral or cell-mediated immunity.





Exemplary assays that test for





immunomodulatory proteins evaluate the





upregulation of MHC class II products, such





as HLA-DR antigens, and the activation of





T cells. Such assays that may be used or





routinely modified to test





immunomodulatory activity of polypeptides





of the invention (including antibodies and





agonists or antagonists of the invention)





include, for example, the assays disclosed in





Miraglia et al., J Biomolecular Screening





4: 193-204 (1999); Rowland et al.,





“Lymphocytes: a practical approach”





Chapter 6: 138-160 (2000); Lamour et al.,





Clin Exp Immunol 89(2): 217-222 (1992);





Hurme and Sihvola, Immunol Lett





20(3): 217-222 (1989); Gansbacher and Zier,





Cell Immunol 117(1): 22-34 (1988); and





Itoh et al., J Histochem Cytochem





40(11): 1675-1683, the contents of each of





which are herein incorporated by reference





in its entirety. Human T cells that may be





used according to these assays may be





isolated using techniques disclosed herein





or otherwise known in the art. Human T





cells are primary human lymphocytes that





mature in the thymus and express a T Cell





receptor and CD3, CD4, or CD8. These





cells mediate humoral or cell-mediated





immunity and may be preactivated to





enhance responsiveness to





immunomodulatory factors.



149
VEGF in SW480









Table 1F: Polynucleotides encoding polypeptides of the present invention can be used in assays to test for one or more biological activities. One such biological activity which may be tested includes the ability of polynucleotides and polypeptides of the invention to stimulate up-regulation or down-regulation of expression of particular genes and proteins. Hence, if polynucleotides and polypeptides of the present invention exhibit activity in altering particular gene and protein expression patterns, it is likely that these polynucleotides and polypeptides of the present invention may be involved in, or capable of effecting changes in, diseases associated with the altered gene and protein expression profiles. Hence, polynucleotides, polypeptides, or antibodies of the present invention could be used to treat said associated diseases.
TaqMan® assays may be performed to assess the ability of polynucleotides (and polypeptides they encode) to alter the expression pattern of particular “target” genes. TaqMan® reactions are performed to evaluate the ability of a test agent to induce or repress expression of specific genes in different cell types. TaqMan® gene expression quantification assays (“TaqMan® assays”) are well known to, and routinely performed by, those of ordinary skill in the art. TaqMan® assays are performed in a two step reverse transcription/polymerase chain reaction (RT-PCR). In the first (RT) step, cDNA is reverse transcribed from total RNA samples using random hexamer primers. In the second (PCR) step, PCR products are synthesized from the cDNA using gene specific primers.
To quantify gene expression the TaqMan® PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold® DNA Polymerase to cleave a TaqMan® probe (distinct from the primers) during PCR. The TaqMan® probe contains a reporter dye at the 5′-end of the probe and a quencher dye at the 3′ end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. AmpliTaq Gold® DNA Polymerase then cleaves the probe between the reporter and quencher when the probe hybridizes to the target, resulting in increased fluorescence of the reporter (see FIG. 2). Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.
After the probe fragments are displaced from the target, polymerization of the strand continues. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected.
For test sample preparation, vector controls or constructs containing the coding sequence for the gene of interest are transfected into cells, such as for example 293T cells, and supernatants collected after 48 hours. For cell treatment and RNA isolation, multiple primary human cells or human cell lines are used; such cells may include but are not limited to, Normal Human Dermal Fibroblasts, Aortic Smooth Muscle, Human Umbilical Vein Endothelial Cells, HepG2, Daudi, Jurkat, U937, Caco, and THP-1 cell lines. Cells are plated in growth media and growth is arrested by culturing without media change for 3 days, or by switching cells to low serum media and incubating overnight. Cells are treated for 1, 6, or 24 hours with either vector control supernatant or sample supernatant (or purified/partially purified protein preparations in buffer). Total RNA is isolated; for example, by using TRIZOLTM extraction or by using the Ambion RNAqueousTM-4PCR RNA isolation system. Expression levels of multiple genes are analyzed using TaqMan®, and expression in the test sample is compared to control vector samples to identify genes induced or repressed. Each of the above described techniques are well known to, and routinely performed by, those of ordinary skill in the art.
Table 1F indicates particular disease classes and preferred indications for which polynucleotides, polypeptides, or antibodies of the present invention may be used in detecting, diagnosing, preventing, treating and/or ameliorating said diseases and disorders based on “target” gene expression patterns which may be up- or down-regulated by polynucleotides (and the encoded polypeptides) corresponding to each indicated cDNA Clone ID (shown in Table 1F, Column 2).
Thus, in preferred embodiments, the present invention encompasses a method of detecting, diagnosing, preventing, treating, and/or ameliorating a disease or disorder listed in the “Disease Class” and/or “Preferred Indication” columns of Table 1F; comprising administering to a patient in which such detection, diagnosis, prevention, or treatment is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, diagnose, prevent, treat, or ameliorate the disease or disorder. The first and second columns of Table 1F shows the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, diagnosing, preventing, treating, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
In another embodiment, the present invention also encompasses methods of detecting, diagnosing, preventing, treating, or ameliorating a disease or disorder listed in the “Disease Class” or “Preferred Indication” Columns of Table 1F; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
The “Disease Class” Column of Table 1F provides a categorized descriptive heading for diseases, disorders, and/or conditions (more fully described below) that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
The “Preferred Indication” Column of Table 1F describes diseases, disorders, and/or conditions that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
The “Cell Line” and “Exemplary Targets” Columns of Table 1F indicate particular cell lines and target genes, respectively, which may show altered gene expression patterns (i.e., up- or down-regulation of the indicated target gene) in TaqMan® assays, performed as described above, utilizing polynucleotides of the cDNA Clone ID shown in the corresponding row. Alteration of expression patterns of the indicated “Exemplary Target” genes is correlated with a particular “Disease Class” and/or “Preferred Indication” as shown in the corresponding row under the respective column headings.
The “Exemplary Accessions” Column indicates GenBank Accessions (available online through the National Center for Biotechnology Information (NCBI) at the world wide web at ncbi.nlm.nih.gov/) which correspond to the “Exemplary Targets” shown in the adjacent row.
The recitation of “Cancer” in the “Disease Class” Column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate neoplastic diseases and/or disorders (e.g., leukemias, cancers, etc., as described below under “Hyperproliferative Disorders”).
The recitation of “Immune” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
The recitation of “Angiogenesis” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), diseases and/or disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”), diseases and/or disorders involving cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), diseases and/or disorders involving angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), to promote or inhibit cell or tissue regeneration (e.g., as described below under “Regeneration”), or to promote wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”). Highly preferred indications include diagnosis, prevention, treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti-Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.”
The recitation of “Diabetes” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diabetes (including diabetes mellitus types I and II), as well as diseases and/or disorders associated with, or consequential to, diabetes (e.g. as described below under “Endocrine Disorders,” “Renal Disorders,” and “Gastrointestinal Disorders”).













TABLE 1F





Gene
cDNA



Exemplary



No.
Clone ID
Disease Class
Preferred Indications
Cell Line
Targets
Exemplary Accessions





















14
HJACG02
Angiogenesis
Highly preferred
Adipocytes-
ICAM
gb|X06990|HSICAM1





indications include
Mar. 12, 
PAI
gb|X12701|HSENDPAI





diagnosis, prevention,
2001
Vegf1
gb|AF024710|AF024710





treatment, and/or








amelioration of diseases








and disorders involving








angiogenesis, wound








healing, neoplasia








(particularly including,








but not limited to, tumor








metastases), and








cardiovascular diseases








and disorders; as








described herein under








the headings








“Hyperproliferative








Disorders,”








“Regeneration,” “Anti-








Angiogenesis Activity,”








“Diseases at the Cellular








Level,” and “Wound








Healing and Epithelial








Cell Proliferation.”





14
HJACG02
Angiogenesis
Highly preferred
AOSMC
VCAM
gb|A30922|A30922





indications include








diagnosis, prevention,








treatment, and/or








amelioration of diseases








and disorders involving








angiogenesis, wound








healing, neoplasia








(particularly including,








but not limited to, tumor








metastases), and








cardiovascular diseases








and disorders; as








described herein under








the headings








“Hyperproliferative








Disorders,”








“Regeneration,” “Anti-








Angiogenesis Activity,”








“Diseases at the Cellular








Level,” and “Wound








Healing and Epithelial








Cell








Proliferation.”(AOSMC








cells are aortic smooth








muscle cells).





14
HJACG02
Angiogenesis
Highly preferred
Daudi
ICAM
gb|X06990|HSICAM1





indications include

VCAM
gb|A30922|A30922





diagnosis, prevention,








treatment, and/or








amelioration of diseases








and disorders involving








angiogenesis, wound








healing, neoplasia








(particularly including,








but not limited to, tumor








metastases), and








cardiovascular diseases








and disorders; as








described herein under








the headings








“Hyperproliferative








Disorders,”








“Regeneration,” “Anti-








Angiogenesis Activity,”








“Diseases at the Cellular








Level,” and “Wound








Healing and Epithelial








Cell Proliferation.”(The








Daudi cell line is a human








B lymphoblast cell line








available through the








ATCC TM as cell line








number CCL-213).





14
HJACG02
Angiogenesis
Highly preferred
HUVEC
ICAM
gb|X06990|HSICAM1





indications include

TSP-1
gb|X04665|HSTHROMR





diagnosis, prevention,

Vegf1
gb|AF024710|AF024710





treatment, and/or








amelioration of diseases








and disorders involving








angiogenesis, wound








healing, neoplasia








(particularly including,








but not limited to, tumor








metastases), and








cardiovascular diseases








and disorders; as








described herein under








the headings








“Hyperproliferative








Disorders,”








“Regeneration,” “Anti-








Angiogenesis Activity,”








“Diseases at the Cellular








Level,” and “Wound








Healing and Epithelial








Cell








Proliferation.”(HUVEC








cells are human umbilical








vein endothelial cells).





14
HJACG02
Cancer
Highly preferred
Adipocytes-
Egr1






indications include
Mar. 12,







neoplastic diseases (e.g.
2001







cancer) such as described








herein under the heading








“Hyperproliferative








Disordersî (particularly








including, but not limited








to, cancer involving








adipocytes). Highly








preferred embodiments of








the invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating cancer and








hyperproliferative








disorders. (Primary








adipocytes)





14
HJACG02
Cancer
Highly preferred
AOSMC
M1 RIBO R
gb|X59543|HSRIREM1





indications include








neoplastic diseases (e.g.








cancer) such as described








herein under the heading








“Hyperproliferative








Disordersî (particularly








including, but not limited








to, cancers of muscle








tissues and the








cardiovascular system








(e.g. heart, lungs,








circulatory system)).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating cancer and








hyperproliferative








disorders. (AOSMC cells








are aortic smooth muscle








cells).





14
HJACG02
Cancer
Highly preferred
Daudi
Cyclin A1
gb|U97680|HSU97680





indications include








neoplastic diseases (e.g.








cancer) such as described








herein under the heading








“Hyperproliferative








Disordersî (particularly








including, but not limited








to, cancers of immune








cells, such as B-cells).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating cancer and








hyperproliferative








disorders involving








immune cells (such as B-








cells). (The Daudi cell line








is a human B lymphoblast








cell line available through








the ATCC TM as cell line








number CCL-213).





14
HJACG02
Cancer
Highly preferred
HEK293
E-cadherin
gb|Z35408|HSECAD9





indications include








neoplastic diseases (e.g.








cancer) such as described








herein under the heading








“Hyperproliferative








Disordersî (particularly








including, but not limited








to, cancers of epithelial








cells or cancers involving








the renal system). Highly








preferred embodiments of








the invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating cancer and








hyperproliferative








disorders involving








epithelial cells or the








renal system. (The 293








cell line human








embryonal kidney








epithelial cell line








available through the








ATCC TM as cell line








number CRL-1573).





14
HJACG02
Cancer
Highly preferred
Jurkat
Cyclin A1
gb|U97680|HSU97680





indications include








neoplastic diseases (e.g.








cancer) such as described








herein under the heading








“Hyperproliferative








Disordersî (particularly








including, but not limited








to, cancers of immune








cells, such as T-cells).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating cancer and








hyperproliferative








disorders involving








immune cells (such as T-








cells). (The Jurkat cell line








is a human T lymphocyte








cell line available through








the ATCC TM as cell line








number TIB-152).





14
HJACG02
Cancer
Highly preferred
Liver
Cyclin D2
gb|X68452|HSCYCD2





indications include








neoplastic diseases (e.g.








cancer) such as described








herein under the heading








“Hyperproliferative








Disordersî (particularly








including, but not limited








to, cancers involving cells








of the hepatic system).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating cancer and








hyperproliferative








disorders involving cells








of the hepatic system.





14
HJACG02
Cancer
Highly preferred
NHDF
Cyclin A1
gb|U97680|HSU97680





indications include








neoplastic diseases (e.g.








cancer) such as described








herein under the heading








“Hyperproliferative








Disordersî (particularly








including, but not limited








to cancers involving cells








of the skin). Highly








preferred embodiments of








the invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating cancer and








hyperproliferative








disorders involving skin








cells. (NHDF cells are








normal human dermal








fibroblasts).





14
HJACG02
Diabetes
A highly preferred
Adipocytes
CAP
gb|AF136380|AF136380





indication is diabetes.
(4D)-
PEPCK1
gb|L05144|HUMPHOCAR





Additional highly
Sep. 1, 2001







preferred indications








include complications








associated with diabetes








(e.g., diabetic retinopathy,








diabetic nephropathy,








kidney disease (e.g., renal








failure, nephropathy








and/or other diseases and








disorders as described in








the “Renal Disorders”








section below), diabetic








neuropathy, nerve disease








and nerve damage (e.g.,








due to diabetic








neuropathy), blood vessel








blockage, heart disease,








stroke, impotence (e.g.,








due to diabetic








neuropathy or blood








vessel blockage),








seizures, mental








confusion, drowsiness,








nonketotic








hyperglycemic-








hyperosmolar coma,








cardiovascular disease








(e.g., heart disease,








atherosclerosis,








microvascular disease,








hypertension, stroke, and








other diseases and








disorders as described in








the “Cardiovascular








Disorders” section








below), dyslipidemia,








endocrine disorders (as








described in the








“Endocrine Disorders”








section below),








neuropathy, vision








impairment (e.g., diabetic








retinopathy and








blindness), ulcers and








impaired wound healing,








and infection (e.g.,








infectious diseases and








disorders as described in








the “Infectious Diseases”








section below, especially








of the urinary tract and








skin). Highly preferred








indications also include








obesity, weight gain, and








weight loss, as well as








complications associated








with obesity, weight gain,








and weight loss.








Preferred embodiments of








the invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating the above








mentioned conditions,








disorders, and diseases.





14
HJACG02
Diabetes
A highly preferred
Adipocytes-
CAP
gb|AF136380|AF136380





indication is diabetes.
Mar. 12,
Hexokinase
gb|Z46354|HSHKEX1





Additional highly
2001
II






preferred indications








include complications








associated with diabetes








(e.g., diabetic retinopathy,








diabetic nephropathy,








kidney disease (e.g., renal








failure, nephropathy








and/or other diseases and








disorders as described in








the “Renal Disorders”








section below), diabetic








neuropathy, nerve disease








and nerve damage (e.g.,








due to diabetic








neuropathy), blood vessel








blockage, heart disease,








stroke, impotence (e.g.,








due to diabetic








neuropathy or blood








vessel blockage),








seizures, mental








confusion, drowsiness,








nonketotic








hyperglycemic-








hyperosmolar coma,








cardiovascular disease








(e.g., heart disease,








atherosclerosis,








microvascular disease,








hypertension, stroke, and








other diseases and








disorders as described in








the “Cardiovascular








Disorders” section








below), dyslipidemia,








endocrine disorders (as








described in the








“Endocrine Disorders”








section below),








neuropathy, vision








impairment (e.g., diabetic








retinopathy and








blindness), ulcers and








impaired wound healing,








and infection (e.g.,








infectious diseases and








disorders as described in








the “Infectious Diseases”








section below, especially








of the urinary tract and








skin). Highly preferred








indications also include








obesity, weight gain, and








weight loss, as well as








complications associated








with obesity, weight gain,








and weight loss.








Preferred embodiments of








the invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating the above








mentioned conditions,








disorders, and diseases.





14
HJACG02
Diabetes
A highly preferred
AOSMC
IRS1
gb|X90563|HSPPARGAM





indication is diabetes.

PPARg






Additional highly








preferred indications








include complications








associated with diabetes








(e.g., diabetic retinopathy,








diabetic nephropathy,








kidney disease (e.g., renal








failure, nephropathy








and/or other diseases and








disorders as described in








the “Renal Disorders”








section below), diabetic








neuropathy, nerve disease








and nerve damage (e.g.,








due to diabetic








neuropathy), blood vessel








blockage, heart disease,








stroke, impotence (e.g.,








due to diabetic








neuropathy or blood








vessel blockage),








seizures, mental








confusion, drowsiness,








nonketotic








hyperglycemic-








hyperosmolar coma,








cardiovascular disease








(e.g., heart disease,








atherosclerosis,








microvascular disease,








hypertension, stroke, and








other diseases and








disorders as described in








the “Cardiovascular








Disorders” section








below), dyslipidemia,








endocrine disorders (as








described in the








“Endocrine Disorders”








section below),








neuropathy, vision








impairment (e.g., diabetic








retinopathy and








blindness), ulcers and








impaired wound healing,








and infection (e.g.,








infectious diseases and








disorders as described in








the “Infectious Diseases”








section below, especially








of the urinary tract and








skin). Highly preferred








indications also include








obesity, weight gain, and








weight loss, as well as








complications associated








with obesity, weight gain,








and weight loss.








Preferred embodiments of








the invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating the above








mentioned conditions,








disorders, and








diseases. (AOSMC cells








are human aortic smooth








muscle cells).





14
HJACG02
Diabetes
A highly preferred
Liver
Glucose6
gb|U91844|CFU91844





indication is diabetes.

phosphatase






Additional highly








preferred indications








include complications








associated with diabetes








(e.g., diabetic retinopathy,








diabetic nephropathy,








kidney disease (e.g., renal








failure, nephropathy








and/or other diseases and








disorders as described in








the “Renal Disorders”








section below), diabetic








neuropathy, nerve disease








and nerve damage (e.g.,








due to diabetic








neuropathy), blood vessel








blockage, heart disease,








stroke, impotence (e.g.,








due to diabetic








neuropathy or blood








vessel blockage),








seizures, mental








confusion, drowsiness,








nonketotic








hyperglycemic-








hyperosmolar coma,








cardiovascular disease








(e.g., heart disease,








atherosclerosis,








microvascular disease,








hypertension, stroke, and








other diseases and








disorders as described in








the “Cardiovascular








Disorders” section








below), dyslipidemia,








endocrine disorders (as








described in the








“Endocrine Disorders”








section below),








neuropathy, vision








impairment (e.g., diabetic








retinopathy and








blindness), ulcers and








impaired wound healing,








and infection (e.g.,








infectious diseases and








disorders as described in








the “Infectious Diseases”








section below, especially








of the urinary tract and








skin). Highly preferred








indications also include








obesity, weight gain, and








weight loss, as well as








complications associated








with obesity, weight gain,








and weight loss.








Preferred embodiments of








the invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating the above








mentioned conditions,








disorders, and diseases.





14
HJACG02
Immune
Highly preferred
Adipocytes-
ICAM
gb|X06990|HSICAM1





indications include
Mar. 12,
Il6
gb|X04403|HS26KDAR





immunological disorders
2001
Rag1
gb|M29474|HUMRAG1





such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving adipocytes).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving adipocytes).





14
HJACG02
Immune
Highly preferred
AOSMC
CD30
gb|AJ300189|HSA30018





indications include

CD40
gb|X02532|HSIL1BR





immunological disorders

IL1B
gb|X12705|HSBCDFIA





such as described herein

IL5
gb|AJ270944|HSA27094





under the heading

TNF
gb|A30922|A30922





“Immune Activity” and/or

VCAM






“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving muscle tissues








and the cardiovascular








system (e.g. heart, lungs,








circulatory system)).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving muscle tissue








or the cardiovascular








system). (AOSMC cells








are human aortic smooth








muscle cells).





14
HJACG02
Immune
Highly preferred
Caco-2
Rag1
gb|M29474|HUMRAG1





indications include








immunological disorders








such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving the cells of the








gastrointestinal tract).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving cells of the








gastrointestinal tract).








(The Caco-2 cell line is a








human colorectal








adenocarcinoma cell line








available through the








ATCC TM as cell line








number HTB-37).





14
HJACG02
Immune
Highly preferred
Daudi
ICAM
gb|X06990|HSICAM1





indications include

Rag1
gb|M29474|HUMRAG1





immunological disorders

VCAM
gb|A30922|A30922





such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving the B-cells).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving B-cells). (The








Daudi cell line is a human








B lymphoblast cell line








available through the








ATCC TM as cell line








number CCL-213).





14
HJACG02
Immune
Highly preferred
HEK293
c-maf
gb|AF055377|AF055377





indications include








immunological disorders








such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving epithelial cells








or the renal system).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving epithelial cells








or the renal system). (The








293 cell line is a human








embryonal kidney








epithelial cell line








available through the








ATCC TM as cell line








number CRL-1573).





14
HJACG02
Immune
Highly preferred
HUVEC
ICAM
gb|X06990|HSICAM1





indications include








immunological disorders








such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving endothelial








cells). Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving endothelial








cells). (HUVEC cells are








human umbilical vein








endothelial cells).





14
HJACG02
Immune
Highly preferred
Jurkat
Rag2
gb|AY011962|AY011962





indications include

TNF
gb|AJ270944|HSA27094





immunological disorders








such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving T-cells).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving T-cells). (The








Jurkat cell line is a human








T lymphocyte cell line








available through the








ATCC TM as cell line








number TIB-152).





14
HJACG02
Immune
Highly preferred
NHDF
Rag1
gb|M29474|HUMRAG1





indications include








immunological disorders








such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving the skin).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving the skin).








(NHDF cells are normal








human dermal








fibroblasts).





14
HJACG02
Immune
Highly preferred
U937
GATA1
gb|X17254|HSERYF1





indications include

IL5
gb|X12705|HSBCDFIA





immunological disorders

TNF
gb|AJ270944|HSA27094





such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving monocytes).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving monocytes).








(The U937 cell line is a








human monocyte cell line








available through the








ATCC TM as cell line








number CRL-1593.2).





14
HJACG02
Immune
Highly preferred
Adipocytes-
ICAM
gb|X06990|HSICAM1





indications include
Mar. 12,
Il6
gb|X04403|HS26KDAR





immunological disorders
2001
Rag1
gb|M29474|HUMRAG1





such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving adipocytes).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving adipocytes).





14
HJACG02
Immune
Highly preferred
AOSMC
CD30






indications include

CD40
gb|AJ300189|HSA30018





immunological disorders

IL1B
gb|X02532|HSIL1BR





such as described herein

IL5
gb|X12705|HSBCDFIA





under the heading

TNF
gb|AJ270944|HSA27094





“Immune Activity” and/or

VCAM
gb|A30922|A30922





“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving muscle tissues








and the cardiovascular








system (e.g. heart, lungs,








circulatory system)).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving muscle tissue








or the cardiovascular








system). (AOSMC cells








are human aortic smooth








muscle cells).





14
HJACG02
Immune
Highly preferred
Caco-2
Rag1
gb|M29474|HUMRAG1





indications include








immunological disorders








such as described herein








under the heading








“Immune Activity” and/or








“Blood-Related








Disorders” (particularly








including, but not limited








to, immune disorders








involving the cells of the








gastrointestinal tract).








Highly preferred








embodiments of the








invention include








methods of preventing,








detecting, diagnosing,








treating and/or








ameliorating disorders of








the immune system








(particularly including,








but not limited to,








immune disorders








involving cells of the








gastrointestinal tract).








(The Caco-2 cell line is a








human colorectal








adenocarcinoma cell line








available through the








ATCC TM as cell line








number HTB-37).









Table 2 further characterizes certain encoded polypeptides of the invention, by providing the results of comparisons to protein and protein family databases. The first column provides a unique clone identifier, “Clone ID NO:”, corresponding to a cDNA clone disclosed in Tables 1A, 1B.1, and/or 1B.2. The second column provides the unique contig identifier, “Contig ID:” which allows correlation with the information in Tables 1B.1 and 1B.2. The third column provides the sequence identifier, “SEQ ID NO:”, for the contig polynucleotide sequences. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. The fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five. Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”), as described below.
The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Tables 1B.1 and 1B.2, column 3 (e.g., SEQ ID NO:X or the ‘Query’ sequence) was used to search against the NR database. The computer program BLASTX was used to compare a 6-frame translation of the Query sequence to the NR database (for information about the BLASTX algorithm please see Altshul et al., J. Mol. Biol. 215:403-410 (1990), and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring ‘Subject’) is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring ‘Subject’ is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than 1.0e-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by the number of Query amino acids in the HSP and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.
The PFAM database, PFAM version 2.1, (Sonnhammer, Nucl. Acids Res., 26:320-322, 1998)) consists of a series of multiple sequence alignments; one alignment for each protein family. Each multiple sequence alignment is converted into a probability model called a Hidden Markov Model, or HMM, that represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin, et al., Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1B.1) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2, and the database accession number of the PFAM hit is provided in column 6. Column 7 provides the score returned by HMMER version 1.8 for the alignment. Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.
As mentioned, columns 8 and 9 in Table 2, “NT From” and “NT To”, delineate the polynucleotides of “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2. Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.
The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in ATCCTM Deposit No:Z. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A and/or 1B.
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing cDNA ATCCTM Deposit No:Z (e.g., as set forth in columns 2 and 3 of Table 1A and/or as set forth, for example, in Tables 6 and 7). The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.















TABLE 2







SEQ


PFam/NR
Score/




cDNA
Contig
ID
Analysis

Accession
Percent
NT
NT


Clone ID
ID:
NO: X
Method
PFam/NR Description
Number
Identity
From
To























HTEEB42
206980
9
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
48.5
500
706


HTEEB42
206980
9
WUblastx.64
(AAG49022) Junctional adhesion
AAG49022
94%
110
952






molecule 2.






HTEEB42
206980
9
WUblastx.64
(AAG49022) Junctional adhesion
AAG49022
99%
59
952






molecule 2.






HEMCM42
407085
11
WUblastx.64
(Q9QZW3) TYPE I
Q9QZW3
91%
372
443






TRANSMEMBRANE PROTEIN FN14.

88%
133
360


HEQCC55
884824
13
WUblastx.64
(Q9NP84) TYPE I
Q9NP84
64%
62
397






TRANSMENMBRANE PROTEIN










PRECURSOR (TYPE I










TRANSMEMBRAN






HEMAE80
409495
20
WUblastx.64
(Q9NRR1) CYTOKINE-LIKE
Q9NRR1
100% 
12
419






PROTEIN C17.






HEMAE80
1310948
22
WUblastx.64
(Q9NRR1) CYTOKINE-LIKE
Q9NRR1
100% 
136
468






PROTEIN C17.






HRDFB85
411020
28
HMMER 2.1.1
PFAM: PMP-22/EMP/MP20/Claudin
PF00822
150.2 
282
−233






family






HRDFB85
411020
28
WUblastx.64
(AAH00671) Claudin 4.
AAH00671
100% 
209
835


HDTAW95
412472
38
WUblastx.64
(Q96CG8) Similar to RIKEN cDNA
Q96CG8
100% 
130
249






1110014B07 gene.

97%
230
856


HEMCV19
423219
46
HMMER 2.1.1
PFAM: ATP1G1/PLM/MAT8 family
PF02038
70.5
475
609


HEMCV19
423219
46
WUblastx.64
(Q96DB9) FXYD domain-containing
FXY5_HUMAN
88%
79
612






ion transport regulator 5 p






HETBX14
806447
61
HMMER 2.1.1
PFAM: Trypsin
PF00089
281.2 
270
935


HETBX14
806447
61
WUblastx.64
(Q9NS65) PROSTATE-TYPE
Q9NS65
94%
111
956






HIPPOSTASIN.






HETBX14
422659
63
WUblastx.64
(Q9NS65) PROSTATE-TYPE
Q9NS65
89%
4
810






HIPPOSTASIN.






HLHSK94
1307727
72
WUblastx.64
(AAH18037) Wnt inhibitory factor-1.
AAH18037
90%
112
1248


HLHSK94
422828
74
HMMER 2.1.1
PFAM: EGF-like domain
PF00008
43.9
−527
−610


HLHSK94
422828
74
WUblastx.64
(AAH18037) Wnt inhibitory factor-1.
AAH18037
99%
112
1248


HLHFP03
460467
76
blastx.2
(AX058598) unnamed protein product
emb|CAC22514.1|
95%
390
43






[Homo sapiens]






HLHFP03
460467
78
WUblastx.64
(Q9WVC2) LY-6/NEUROTOXIN
Q9WVC2
81%
224
571






HOMOLOG (ADULT MALE










HIPPOCAMPUS CDNA, RIKEN






HHTLF25
461438
83
WUblastx.64
(Q9UMT3) KILLER ACTIVATING
Q9UMT3
91%
142
474






RECEPTOR ASSOCIATED PROTEIN,










ISOFORM B.






HHTLF25
461438
83
blastx.2
KILLER ACTIVATING RECEPTOR
sp|Q9UMT3|Q9UMT3
99%
142
474






ASSOCIATED PROTEIN,










ISOFORM B.






HTADX17
457172
90
WUblastx.64
(AK009505) putative [Mus musculus]
dbj|BAB26328.1|
67%
548
778








53%
490
585








58%
165
488


HTADX17
753289
92
WUblastx.64
(Q96A28) CD84-H1
Q96A28
93%
92
412






(CD2 FAMILY 10).

79%
408
959


HTADX17
753289
92
WUblastx.64
(Q96A28) CD84-H1
Q96A28
100% 
92
412






(CD2 FAMILY 10).

99%
408
959


HTADX17
457172
94
WUblastx.64
(Q96A28) CD84-H1
Q96A28
78%
490
585






(CD2 FAMILY 10).

97%
548
952








99%
84
488


HJACG02
509948
102
WUblastx.64
(Q9HD89) CYSTEINE-RICH
Q9HD89
100% 
47
370






SECRETED PROTEIN (C/EBP-










EPSILON REGULATED MYEL






HJACG02
509948
102
WUblastx.64
(Q9HD89) CYSTEINE-RICH
Q9HD89
100% 
92
370






SECRETED PROTEIN (C/EBP-










EPSILON REGULATED MYEL






HJACG02
1307789
104
WUblastx.64
(Q9HD89) CYSTEINE-RICH
Q9HD89
100% 
111
389






SECRETED PROTEIN (C/EBP-










EPSILON REGULATED MYEL






HJACG02
1307789
104
WUblastx.64
(Q9HD89) CYSTEINE-RICH
Q9HD89
100% 
66
389






SECRETED PROTEIN (C/EBP-










EPSILON REGULATED MYEL






HKGAJ54
498303
107
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
52.8
481
657


HKGAJ54
498303
107
WUblastx.64
(AAL31867) Magic roundabout.
AAL31867
96%
846
929








98%
31
849


HKGAJ54
498303
107
WUblastx.64
(BAB55411) CDNA FLJ14946 fis,
BAB55411
96%
846
929






clone PLACE2000034, w

90%
109
849


HKGAJ54
1300770
109
WUblastx.64
(BAB55411) CDNA FLJ14946 fis,
BAB55411
91%
102
923






clone PLACE2000034, w






HSVAK93
597462
116
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
23.6
117
362


HSVAK93
597462
116
WUblastx.64
(AAH08988) Unknown (protein for
AAH08988
100% 
21
530






MGC: 17333).






HE8CH92
609866
118
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
23.6
127
372


HE8CH92
609866
118
WUblastx.64
(AAH08988) Unknown (protein for
AAH08988
100% 
31
1164






MGC: 17333).






HSDEK49
625998
123
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
18.7
225
470


HSDEK49
625998
123
WUblastx.64
(Q9Y279) Z39IG PROTEIN
Q9Y279
88%
444
1040






PRECURSOR.

99%
126
542


HSDEK49
1352253
125
WUblastx.64
(Q9Y279) Z39IG PROTEIN
Q9Y279
100% 
60
1256






PRECURSOR.






HWBAO62
838164
127
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
27.9
202
402


HWBAO62
838164
127
WUblastx.64
(Q14288) HYPOTHETICAL PROTEIN
Q14288
45%
1331
1618






(FRAGMENT).

66%
1158
1334








62%
1847
1894








55%
1594
1839


HWBAO62
625914
129
WUblastx.64
(Q14288) HYPOTHETICAL PROTEIN
Q14288
43%
1358
1645






(FRAGMENT).

62%
1874
1921








66%
1185
1361








55%
1621
1866


HWHGU54
695695
131
HMMER 2.1.1
PFAM: Serpins (serine protease
PF00079
501.1 
277
1377






inhibitors)






HWHGU54
695695
131
WUblastx.64
(Q9CQ32) 4632419J12RIK PROTEIN.
Q9CQ32
61%
145
1383


HWHGU54
695695
131
WUblastx.64
(AAL99574) OL-64 protein.
AAL99574
62%
145
1377


HWHGU54
695695
131
WUblastx.64
(Q9CQ32) 4632419J12RIK PROTEIN.
Q9CQ32
59%
145
1383


HCEJQ69
1243825
134
HMMER 2.1.1
PFAM: Leucine Rich Repeat
PF00560
116.2 
573
644


HCEJQ69
1243825
134
WUblastx.64
(Q9BZR6) NOGO RECEPTOR.
Q9BZR6
87%
39
1457


HCEJQ69
1243825
134
WUblastx.64
(AF283463) Nogo receptor [Homo
gb|AAG53612.1|AF283463_1
87%
39
1457






sapiens]






HCEJQ69
872582
136
HMMER 2.1.1
PFAM: Leucine Rich Repeat
PF00560
116.2 
573
644


HCEJQ69
872582
136
WUblastx.64
(Q9BZR6) NOGO RECEPTOR.
Q9BZR6
90%
39
1361


HCEJQ69
609999
138
HMMER 2.1.1
PFAM: Leucine Rich Repeat
PF00560
114.5 
573
644


HCEJQ69
609999
138
WUblastx.64
(Q9BZR6) NOGO RECEPTOR.
Q9BZR6
59%
1100
1462








85%
39
1460


HCEJQ69
609999
138
WUblastx.64
(Q9BZR6) NOGO RECEPTOR.
Q9BZR6
85%
39
1364








45%
1100
1462


HT5GJ57
740767
159
WUblastx.64
(Q9NZY9) HSPC046.
Q9NZY9
90%
754
1002








70%
122
799


HT5GJ57
740767
159
WUblastx.64
(Q9GZY6) CDNA FLJ11237 FIS,
Q9GZY6
84%
122
856






CLONE PLACE1008531 (WBSCR5)










(WBSCR15 PROT






HT5GJ57
740767
159
WUblastx.64
(AX083396) unnamed protein product
emb|CAC33299.1|
84%
122
856






[Homo sapiens]






HT5GJ57
1299921
161
WUblastx.64
(Q9GZY6) CDNA FLJ11237 FIS,
Q9GZY6
89%
105
833






CLONE PLACE1008531 (WBSCR5)










(WBSCR15 PROT






HPIBX03
743314
169
WUblastx.64
(Q9H5V8) CDNA: FLJ22969 FIS,
Q9H5V8
98%
81
2207






CLONE KAT10759.






HDPBO81
892018
174
WUblastx.64
(Q9ES58) OX2 RECEPTOR
Q9ES58
57%
400
1323






PRECURSOR.

38%
265
381


HWBFY57
837478
179
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
19.2
209
442


HWBFY57
837478
179
WUblastx.64
(AAH19814) Hypothetical 25.0 kDa
AAH19814
54%
134
535






protein.






HYABV21
1281466
182
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
46
166
342


HYABV21
1281466
182
blastx.2
MMAN-g protein precursor.
sp|BAB18569|BAB18569
54%
109
672


HYABV21
1213593
184
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
46
174
350


HYABV21
1213593
184
blastx.2
MMAN-g protein precursor.
sp|BAB18569|BAB18569
49%
117
686


HOHBY69
827480
186
HMMER 2.1.1
PFAM: FG-GAP repeat
PF01839
223  
1696
1875


HOHBY69
827480
186
WUblastx.64
(Q9UKX5) INTEGRIN ALPHA-11
ITAH_HUMAN
98%
82
3648






PRECURSOR.






HOHBY69
815681
188
HMMER 2.1.1
PFAM: FG-GAP repeat
PF01839
223  
1698
1877


HOHBY69
815681
188
blastx.2
(AF109681) integrin alpha-11 subunit
gb|AAF01258.1|AF109681_1
99%
84
3170






precursor [Homo sapiens]

99%
3172
3648


HDHMA45
902513
202
WUblastx.64
(Q9H3S3) TRANSMEMBRANE
TMS5_HUMAN
91%
175
1428






PROTEASE, SERINE 5 (EC 3.4.21.—)










(SP






HDHMA45
812764
204
HMMER 2.1.1
PFAM: Trypsin
PF00089
296.3 
723
1415


HDHMA45
812764
204
WUblastx.64
(Q9H3S3) TRANSMEMBRANE
TMS5_HUMAN
99%
180
1442






PROTEASE, SERINE 5 (EC 3.4.21.—)










(SP






HMADJ14
1099342
206
WUblastx.64
(Q9H295) DC-SPECIFIC
Q9H295
99%
200
1306






TRANSMEMBRANE PROTEIN.






HMADJ14
889659
208
blastx.2
(AF305068) DC-specific
gb|AAG39167.1|AF305068_1
90%
186
1019






transmembrane protein

96%
1010
1084






[Homo sapiens]






HMADJ14
843725
210
WUblastx.64
(Q9H295) DC-SPECIFIC
Q9H295
96%
871
945






TRANSMEMBRANE PROTEIN.

90%
47
880


HMADJ14
843725
212
WUblastx.64
(Q9H295) DC-SPECIFIC
Q9H295
96%
871
945






TRANSMEMBRANE PROTEIN.

90%
47
880


HMADJ14
795479
214
WUblastx.64
(Q9H295) DC-SPECIFIC
Q9H295
100% 
1010
1393






TRANSMEMBRANE PROTEIN.

93%
186
1067


HMADJ14
426068
216
blastx.2
(AF305068) DC-specific
gb|AAG39167.1|AF305068_1
91%
47
685






transmembrane protein [Homo sapiens]

78%
675
881








96%
872
946


HEMFA84
608198
222
WUblastx.64
(Q9H6H3) CDNA: FLJ22282 FIS,
Q9H6H3
100% 
42
812






CLONE HRC03861.






HDPPA04
904765
224
HMMER 2.1.1
PFAM: Immunoglobulin domain
PF00047
19.1
373
582


HDPPA04
904765
224
WUblastx.64
(Q9BQ51) BUTYROPHILIN
Q9BQ51
92%
271
1089






PRECURSOR B7-DC (PD-1-LIGAND










2 PROTEIN).






HE2OA95
637595
230
WUblastx.64
(Q9UIK5) TMEFF2 PROTEIN
Q9UIK5
99%
1647
1231






PRECURSOR (TRANSMEMBRANE










PROTEIN TENB2) (TPEF






HKABZ65
862030
232
WUblastx.64
(Q96LB9) Peptidoglycan recognition
Q96LB9
90%
77
802






protein-I-alpha precursor.

39%
137
541


HKABZ65
862030
232
WUblastx.64
(Q96LB9) Peptidoglycan recognition
Q96LB9
99%
77
802






protein-I-alpha precursor.

45%
137
541


HKABZ65
665424
234
WUblastx.64
(Q96LB9) Peptidoglycan recognition
Q96LB9
99%
69
794






protein-I-alpha precursor.

45%
129
533









RACE Protocol for Recovery of Full-Length Genes

Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). A cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+ or total RNA is reverse transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI, SalI and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (PROMEGATM), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBLUESCRIPTTM SKII (STRATAGENETM) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.
Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from CLONTECHTM which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to Obtain Full Length Genes
Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′ RACE. While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant gene.
The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCCTM (e.g., as described in columns 2 and 3 of Table 1A, and/or as set forth in Table 6 or 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Tables 1A and 7. These deposits are referred to as “the deposits” herein. The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A (ATCCTM Deposit No:Z). A clone which is isolatable from the ATCCTM Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire coding region of a human gene or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCCTM Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCCTM Deposits by use of a sequence (or portion thereof) described in, for example Tables 1A, 1B.1, 1B.2, or 2, by procedures hereinafter further described, and others apparent to those skilled in the art.
Also provided in Tables 1A and 7 is the name of the vector which contains the cDNA clone. Each vector is routinely used in the art. The following additional information is provided for convenience.
Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPTTM (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENETM Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from STRATAGENETM.
Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIESTM, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIESTM. See, for instance, Gruber, C. E., et al., Focus 15:59-(1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIESTM. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or the deposited clone (ATCCTM Deposit No:Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in ATCCTM Deposit No:Z, using information from the sequences disclosed herein or the clones deposited with the ATCCTM. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA sequence contained in ATCCTM Deposit No:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in ATCCTM Deposit No:Z.
Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, wherein sequentially delineated sequences in the table (i.e. corresponding to those exons located closest to each other) are directly contiguous in a 5′ to 3′ orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B.1, 1B.2, or 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B.1, 1B.2, or 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1C. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same Clone ID (see Table 1C, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
Table 3
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fifth column of Table 1A and/or in fourth column of Tables 1B.1 or 1B.2, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a+14. More specifically, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides of the invention do not consist of at least one, two, three, four, five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone). In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety. Table 3, from U.S. patent application Ser. No. 10/472,532, filed Sep. 20, 2003, is herein incorporated by reference. Table 3 in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 643 to 798 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.











TABLE 3








SEQ

EST Disclaimer













cDNA
ID
Contig
Range
Range



Clone ID
NO: X
ID:
of a
of b
Accession Numbers















HNFIP24
1
351534


H04128, AA099288, AA101983


HETBY74
7
361396


R23770, R26913, R32172, R32216,







R38317, R39395, R63720, R63767,







R70906, R70940, R70993, R77169,







R82584, R82585, H01942, H04495,







H21696, H21906, H97740, H99748,







N62092, N95055, W38893,







W93362, W93394, W93606,







AA016203, AA016243, AA076610,







AA079633, AA079807, AA086218,







AA158761, AA159815, AA159816,







AA159710, AA159711, AA159565,







AA160381, AA186897, AA188520


HTEEB42
9
206980
1-1008
15-1022
AL522795, AA725566, AI421450,







AL522796, AI199779, AA406389,







AA912674, AW022835, AI952846,







AI123727, BE218057, AW022646,







N90730, BF846982, BF845761,







AI652914, BF056970, AW020783,







AI312805, AW393829, AI017553,







AW393887, AW474261,







AW264246, BF848293, AI366088,







AI418268, T89217, AI052637,







AW082343, BF221504,







AW593293, AA865038, AI201753,







BF091146, AI140139, AA987434,







AA410345, BF846977, BF846980,







AW900593, BF932982, BF932991,







AW865421, AW136481, AI650503,







AI432092, T89127, AA974715,







AW261924, BE938414,







AF255910.1, AY016009.1,







AP001694.1, AP000087.1,







AP000225.1, AP000226.1,







AP000086.1, AP000223.1.


HTEEB42
9
206980
1-1008
15-1022
AL522795, AA725566, AI421450,







AL522796, AI199779, AA406389,







AA912674, AW022835, AI952846,







AI123727, BE218057, AW022646,







N90730, BF846982, BF845761,







AI652914, BF056970, AW020783,







AI312805, AW393829, AI017553,







AW393887, AW474261,







AW264246, BF848293, AI366088,







AI418268, T89217, AI052637,







AW082343, BF221504,







AW593293, AA865038, AI201753,







BF091146, AI140139, AA987434,







AA410345, BF846977, BF846980,







AW900593, BF932982, BF932991,







AW865421, AW136481, AI650503,







AI432092, T89127, AA974715,







AW261924, BE938414, AF255910,







AP001694, AP000087, AF255911,







AX036060, AP000225, AP000226,







AP000086, and AP000223.


HEQCC55
15
1352368
1-986
15-1000
BF344112, BE275042, BE275057,







BE563860, BE876310, BE880309,







BE910481, BE733268, BF526321,







BG169508, BE876530, BE878582,







BE384718, BE562629, BG170369,







BG170840, BE304867, AI492143,







AI768116, BE876850, BG164974,







BF338794, BE274902, AA149044,







AW204761, AI768403, AI221536,







BE395085, BG032898, AW172990,







BG248912, AW051452,







AW262030, BF114971, AI800959,







AW338518, AI827127, AI568941,







AI761510, F25336, AI313436,







AI086734, N41733, AA994944,







AW938942, BF063028,







AW628237, AI219327, AW859588,







AI718198, BF108787, AI004154,







AI864228, BF197631, AI767239,







AW001699, AI796303, T56712,







AA576558, AI911799, AW149867,







AI470703, AA149043, BG236779,







AI470484, N83862, BG249788,







AA631934, AI270718, AA610401,







AI813825, AI401800, AI358289,







AI799902, H95227, AW166567,







AI264959, AI611273, R48167,







BE730123, AI867518, AW815436,







AW815444, BF338447,







AW391445, AW815697, AI167239,







AA970894, AW815514, T74424,







BE076102, AW815626, BE076131,







BG168390, BG169301, AI910684,







AI189491, BE075966, AW843216,







AA386018, BE076032, AA873480,







R33355, T74049, BF338531,







BF734770, AI919408, AI858303,







AI701259, D29265, BF331419,







BF982285, BE042437, AA996249,







BG002211, AW374913,







BC002718.1, AF191148.1,







AB035480.1, AC004643.1,







AB035481.1.


HEMCM42
15
1352150
1-986
15-1000
BF344112, BE275042, BE275057,







BE563860, BE876310, BE880309,







BE910481, BE733268, BF526321,







BG169508, BE876530, BE878582,







BE384718, BE562629, BG170369,







BG170840, BE304867, AI492143,







AI768116, BE876850, BG164974,







BF338794, BE274902, AA149044,







AW204761, AI768403, AI221536,







BE395085, BG032898, AW172990,







BG248912, AW051452,







AW262030, BF114971, AI800959,







AW338518, AI827127, AI568941,







AI761510, F25336, AI313436,







AI086734, N41733, AA994944,







AW938942, BF063028,







AW628237, AI219327, AW859588,







AI718198, BF108787, AI004154,







AI864228, BF197631, AI767239,







AW001699, AI796303, T56712,







AA576558, AI911799, AW149867,







AI470703, AA149043, BG236779,







AI470484, N83862, BG249788,







AA631934, AI270718, AA610401,







AI813825, AI401800, AI358289,







AI799902, H95227, AW166567,







AI264959, AI611273, R48167,







BE730123, AI867518, AW815436,







AW815444, BF338447,







AW391445, AW815697, AI167239,







AA970894, AW815514, T74424,







BE076102, AW815626, BE076131,







BG168390, BG169301, AI910684,







AI189491, BE075966, AW843216,







AA386018, BE076032, AA873480,







R33355, T74049, BF338531,







BF734770, AI919408, AI858303,







AI701259, D29265, BF331419,







BF982285, BE042437, AA996249,







BG002211, AW374913,







BC002718.1, AF191148.1,







AB035480.1, AC004643.1,







AB035481.1.


HEMAE80
20
409495


T71556, T90634, T82005, T83161,







H57113, H61567, AA233071


HEMAE80
22
1310948
1-1078
15-1092
AI742654, AA461037, BF949730,







AA460463, AI571360, AA701618,







AI127309, AI633508, AA233071,







AA448648, AA448744, AA897786,







BF477408, AI206655, T82005,







AI589742, T71556, T83161,







T90634, H61567, H57113, H60996,







AV707024, AV725948, AV701724,







AV727990, AV706677, AV702026,







AV729131, AV703989,







AW949478, AV728436,







AV727314, AV727935,







AW949477, AV728518,







AV728523, AV725134, AV656283,







AV696791, AV698429, AV684604,







AV692972, AV685688, AV689800,







AV693005, AV656478, AV702516,







AW964421, AV702266,







AV709660, AV687909, AV656903,







AV661704, AV697196, AV655280,







AV659322, AV709314, AV708381,







AV660728, AV691080, AV703169,







AV659536, AV706721, AV729220,







AV687035, AV655096, AV706223,







AV726816, AV695545, AV708025,







AV707933, AV708980, AV692691,







AV701914, AV708992, AV726103,







AV727029, AV728733, AV727100,







AV705280, AV686064,







AW960720, AV686060,







AV703515, AV703472,







AW955900, AW951263,







AW967047, AV704553,







AV704785, AW953797,







AV692600, AV651955, AV725208,







AW952751, AV727238,







AV709880, AV696866, AV725920,







AV693523, AV706342, AV725826,







AW949353, AV654908,







AV728546, AV692345, AV726584,







AV698290, AV698609, AV725582,







AV649758, AV708438, AV696106,







AV689111, AV728157, AV708893,







AV704217, AV702280, AV725031,







AW951280, AV696931,







AV726624, AV709604, AV652001,







AV701946, AV704955, AV702117,







AW954248, AV701707,







AV707753, AV704269, AV702832,







AV701560, AV705693, AV708704,







AV726738, AV703495,







AW964095, AV702772,







AV726156, AV697288, AV652290,







AV648263, AV647789, AV727787,







AV660608, AV698545,







AW955053, AV725497,







AW952410, AV699089,







AW950443, AV654035,







AV706854, AV728997, AV656256,







AW960601, AW956199,







AW952403, AV704234,







AV729378, AW952183,







AV705159, AW959806,







AV709407, AV703494, AV725633,







AW956075, AV645936,







AV709587, AW955723,







AV658084, AW962384,







AV650315, AV659389, AV697880,







AV727613, AV726010, AV660258,







AV708109, AW956474,







AV659294, AV703146, AV725745,







AV728148, AW951239,







AW020543, AV726590,







AV653353, AV654070,







AW951281, AV706219,







AV702385, AV658275,







AW949802, AV702790,







AV703669, AV707979, AV726194,







AV727003, AV709580, AV702372,







AV708786, AV659547,







AW957517, AW959543,







AV727526, AV705416, AV651920,







AV725618, AW954439,







AV707541, AV725577, AV725033,







AV728924, AF193766.1,







AF274956.1, Z59544.1, Y08991.1,







AF217994.1.


HRDFB85
28
411020
1-1691
15-1705
AL526995, AU133713, AU122264,







AU133716, AU133898, AU133901,







AU121966, AU141449, BE792903,







BE786350, AU133817, AU136746,







AU133887, AU121817, BG254161,







AU133655, AU133578, AU121969,







AU133752, BE907881, BG260814,







AU133916, AU136516, BG179180,







AU134354, BF038418, BF036516,







BF037422, AU138430, BE734836,







BE873066, AU133630, BE875603,







AW844114, AU122380,







AU134380, BE616872, BE871117,







BE880804, BE871864, AU134369,







BE902820, BG256045, BE536853,







AU133618, BE304643, BE787818,







AU147972, AU138507, AA700004,







AI927220, AW170580, W74492,







BF446247, BE812430, AU147690,







BE870378, AI991311, AW935041,







AA523290, AI859845, BF681403,







AU136509, AI081052, AU155374,







AA535079, AU159009, AI400364,







AU147886, AI335984, AU155390,







BG254485, AW193221,







AW874638, AU155077, BE564185,







AW170345, AA622540,







AU155064, AI273767, AA522795,







AW474974, AW770097,







BF445088, AW168283, AI188508,







AI559433, AI420481, AI246782,







AU157642, BF182989, BF825787,







AW815382, BF110949,







AW662920, AI928146, AA157892,







BG000738, AU133955, AA314960,







AW815398, AI281336, AU155007,







BE740807, AU155054, BF447199,







AU154870, AA838633, AA844471,







AW589514, AI401064, AW844815,







AW815552, AI949231, AU154943,







AI911649, AI268908, AI874198,







AI186144, AI819846, BF742353,







AW193220, AI863584, AL036495,







BE930559, AA149417, AW168206,







AA565989, W79089, BE819070,







AA506616, AI564546, BF987328,







BF435486, BE903940, AA434123,







AA149738, W02467, AW194453,







AA948146, AU155067, AL526954,







C06165, AI276313, BG002911,







AI963847, BG254948, AI874344,







BF476318, BF987337, AI660464,







AI567796, BE745379, BE673504,







BF446395, AW167101, BF990584,







AA434059, AI739607, AI560666,







AI280032, AI961910, R48300,







AW009339, BE840036, AA551656,







BE840048, AW167849,







AW513272, BF001751, AI346572,







AI923100, AI005290, AI091394,







H93341, AI588982, AI819915,







AI950029, AI991855, AI347074,







AI347076, AI660868, AA295491,







BE930558, AW874110, AI682624,







AI348165, AI949885, BF001991,







AW374558, AW516096, AI347071,







AU154878, AW014104, AI343369,







AI860565, AW167111, AI222884,







AI861959, AW194388, BE840202,







BF109128, AI283186, AI347501,







AI305833, AI031766, AI346386,







AI346944, AW189088, AI032425,







H27323, AI283162, BE048493,







BF569270, AI347072, AW510456,







BE840244, BF001523, BF111772,







AI214245, AI346606, AI743195,







BE840045, AW015201, AI347060,







AI346569, AW572261, AW275383,







AI281140, AI346475, AI743978,







AI274133, AI738882, AI347930,







AI273374, AI738627, AI991114,







AI097004, AI144005, U46417,







AI304544, AA157596, AI281141,







AA569935, AI274318, AI285074,







AI346274, AI336454, AI346908,







AI339875, AK026651, AB000712,







D88492, AX014904, AB000714,







AF007189, AF095905, AJ011656,







M74067, AJ130941, AC004643,







AJ249735, R12121, T96099,







R05961, R05962, R36883, R48403,







R50075, R50076, H13937, H27324,







H27350, H44304, H93844, N72688,







W21446, AA149303, AA149402.


HDTAW95
38
412472
1-1274
15-1288
AL532456, BE896915, BE387335,







AW071610, AA584310, AI762109,







BF691507, AW994682, AI085616,







BE550475, AI422726, AW316980,







AI809642, AI743774, BF000103,







AA482398, BE551643, AI359844,







BE550264, AI379443, AA406425,







AA482544, AW518948, BE221566,







AW276370, AI656907, AA410434,







AI380885, AW236626, BE169370,







AI963298, AA857920, AW519014,







AI352209, AV649395, AI469175,







AI920963, AI631574, AI700002,







AI750892, AI218433, AA335862,







C01758, AI760411, BF432104,







AA335551, AW594188,







AW084890, AA723450, AI653051,







AA738416, AI074769, AI081084,







AA974239, AA969841,







AW137377, AI239604, AI391517,







AW594685, AW518917,







BF000821, AI370649, AW662493,







R46762, BE843536, AA507081,







BF507778, AW630561, AI750893,







AW837990, R46857, AW608087,







AW838003, AI074870, AI369474,







BE504418, D62262, BE698661,







F13673, AW627356, D79314,







AI418593, AI337269, AW627526,







AA588673, BF573143.


HEMCV19
46
423219


R39576, R39644, R55519, R55520,







H25585, H25630, H42497, H43485,







R95168, H73675, H73419, H80718,







H80719, W95391, W95348,







AA034079, AA044081, AA187305,







AA187096


HEMCV19
48
1352162
1-722
15-736
BE614883, BE873075, BG025158,







BE253565, BE875979, BE395207,







AW954217, BE734057, BE789549,







BF529722, BE614806, BE738333,







BE785505, AA044211, BF340317,







AW238972, AW406916, R95168,







BF737302, BF812968, BF738993,







AA075901, BF792671, BG012029,







AW796084, H25630, BE876237,







BE183450, BF901660, BE810793,







H43485, AA296837, H80718,







AA287470, AA298795, AA296826,







AA296696, AI342703, AA296869,







AA218811, AW802996,







AA034079, BE737872, H73675,







R55519, W95348, BF331433,







AV743953, BF082160, BF868942,







BF809994, BE775061, C00212,







AL532494, BF997411, R39644,







BE828356, BF804848, AA806231,







AL532493, BF799156, BF895320,







AA187096, AA297863, BF245896,







BF905334, N86960, T10507,







BE904822, BF931829, BF829581,







BE140100, AI819836, BE396147,







BF747982, BF056207, BF763569,







BF807379, AA465105, BF811185,







BF446055, AA583464, AA485829,







AI582284, AA983595, BF771777,







BF750444, AA523623, BF771828,







AI952620, AA044081, BF736987,







AI346295, BF978464, BF248485,







AA912088, AW512207, AI028038,







AI066489, AI424071, AI521467,







AA699982, AI358167, AA722634,







H42497, AC002390.1, AF177940.







1.


HETBX14
61
806447
1-1278
15-1292
BE867930, BE219655, BF476474,







AW511566, AI521607, AI217150,







AI183346, AI283289, AW000834,







AA436049, AA435952, AA532717,







AA403004, BE673570, AI624187,







BE898804, W60282, AI913780,







W60374, AA402971, AA477283,







BF679282, AA412318, AA477282,







AW969530, BE150851, AA482081,







BF222155, AA402028, BE646241,







BF677072, AA514646, BE150769,







AA503821, AI611257, AI659265,







BE828491, BF515767, AW578446,







AW166796, AI874153, BF573830,







AW874467, AB041036, AB012917,







AX016289, AX016287, AB013730,







AC011473, AF164623, AF243527,







AR060847, AB016226, AF135025,







AC011473, AC011473.


HETBX14
65
422659


W60282


HLHSK94
70
422828


R55809, H83295, N92239,







W37154, W38638, N90902,







AA017680, AA040604, AA040705


HLHSK94
72
1307727
1-1775
15-1789
AW956492, BF352284, BE709448,







W29010, AV751953, AI829559,







BE179448, AI571060, AI083491,







AA905071, AW118125, AI049799,







W22553, N90902, W27632,







AI273588, AI890622, W27896,







AW195777, W22119, BF844755,







AA040604, W23268, AW269932,







W38638, W37154, AA904910,







W27944, R55894, W27681,







AW607334, AA337059,







AW367713, AW607165,







BF351195, AW966017, AA298658,







R55809, C02576, BE163743,







AI376671, AA364393, AV746728,







AI393483, W23093, BE708746,







W28670, W27851, BF844715,







H83295, BF437730, N92239,







AI194027, W27371, D81988,







BE163611, BE163731, C14616,







C14877, AA040705, AA897696,







AV747434, BF850779, AA017680,







H83294, AW974810, AF122922. 1.


HLHFP03
76
460467
1-599
15-613
H46196, AI421986, H19572,







H46195, BF947135, H19490,







BF738481, BF994257, BF127477,







AW139949, BF947011, AF141377,







AF169202, AC011976, and







AC011976.


HLHFP03
78
460467
1-599
15-613
H46196, AI421986, H19572,







H46195, BF947135, H19490,







BF738481, BF994257, BF127477,







AW139949, BF947011, AF321824.







1.


HHTLF25
83
461438
1-683
15-697
AA481924, BF343628, AI276798,







BE858514, BF915546, BG058647,







BF917552, AI299346, N41026,







BF914451, AA989053, W60864,







BF915075, AI423526, BF106006,







AI289858, AA746220, BF915128,







AI306602, AW015647, AA633118,







AI207255, BF913974, AI301688,







W92376, AI139176, AA971275,







AA480109, H12338, BF912934,







AA865668, BF901361, F30553,







AW975896, AA991168, AI302882,







BF915115, AA729941, AA627378,







AA865651, AW607348, H39980,







AA729534, T55959, T57206,







AW607175, W60940, BE155729,







AI880682, AW383808, BG058709,







AW383055, AW383057,







BE154544, AW383016,







AW383047, AW383871,







AW383051, BF901355,







AW383000, AI919456, BE154555,







AW383784, BF914191, F32872,







AI017727, AA974881, BE154538,







AW383009, BF092099, AI243983,







AA991170, R49835, R49793,







AA318120, BF893642, W74783,







AW382999, AV712713,







AW579628, AW382994, H12392,







AW372144, AW372157,







AW383836, T52100, AW372154,







AW383822, AW383837,







AW579627, AW383817,







AW372166, BF881098,







AW382997, D20493, AW372161,







AW383865, AA918360, N47127,







AW579992, AA937670,







AW579601, AW579998,







AU076484, AI245273, BF831159,







AA664094, AA878598, AA865673,







AI807718, AA937805, BF350664,







AI525220, AD000833.1,







AF019563.1, AF019562.1,







AJ010098.1, AD000864.1,







X78928.1, AF072845. 1.


HHTLF25
83
461438
1-683
15-697
AA481924, BF343628, AI276798,







BE858514, BF915546, BG058647,







BF917552, AI299346, N41026,







BF914451, AA989053, W60864,







BF915075, AI423526, BF106006,







AI289858, AA746220, BF915128,







AI306602, AW015647, AA633118,







AI207255, BF913974, AI301688,







W92376, AI139176, AA971275,







AA480109, H12338, BF912934,







AA865668, BF901361, F30553,







AW975896, AA991168, AI302882,







BF915115, AA729941, AA627378,







AA865651, AW607348, H39980,







AA729534, T55959, T57206,







AW607175, W60940, BE155729,







AI880682, AW383808, BG058709,







AW383055, AW383057,







BE154544, AW383016,







AW383047, AW383871,







AW383051, BF901355,







AW383000, AI919456, BE154555,







AW383784, BF914191, F32872,







AI017727, AA974881, BE154538,







AW383009, BF092099, AI243983,







AA991170, R49835, R49793,







AA318120, BF893642, W74783,







AW382999, AV712713,







AW579628, AW382994, H12392,







AW372144, AW372157,







AW383836, T52100, AW372154,







AW383822, AW383837,







AW579627, AW383817,







AW372166, BF881098,







AW382997, D20493, AW372161,







AW383865, AA918360, N47127,







AW579992, AA937670,







AW579601, AW579998,







AU076484, AI245273, BF831159,







AA664094, AA878598, AA865673,







AI807718, AA937805, BF350664,







AI525220, AD000833, AF019563,







AF019562, AJ010098, A83704,







AF152021, AF247680, AD000864,







AF285446, 125670, X78928, and







AF072845.


HTADX17
90
457172
1-1126
15-1140
AA446344, AA612751, AA298785,







AA298780, AA298784, AA446524,







AA298781, AA381170, AL357565,







and AL357565.


HTADX17
92
753289
1-1133
15-1147
AA446344, AA612751, AA298785,







AA298780, AA298784, AA446524,







AA298781, AA381170.


HJACG02
104
1307789
1-561
15-575
AA311223, BF002026, N41594,







N30820, BF982046, AI829327,







BE047833, AI457369, AW071417,







BF968205, AI340627, R36271,







AL036980, BF061283, BG168549,







AW022682, BG034550, AV682418,







AL047042, BF343172, BG113299,







AW020693, BF751308, AI452560,







AI690748, AI349645, AW946806,







AI340511, BF924882, AW074869,







AW196299, AL038445, BE781369,







AW302992, BG110684, BE887488,







AL514193, AI310575, BG164558,







AI340533, AI349957, AI433384,







BF680133, AV715560, AI309401,







AI345005, BG163618, AI343112,







AV743962, AI826225, AI811785,







AI494201, AW054931, AW268302,







AW301300, AI349598, BF672397,







AW072719, AW075207,







BF526020, AV741327, AI345735,







BG036846, AI697243, BE536058,







AW193134, AI889147, BF904189,







BE910373, AI500077, AA225339,







BE138712, AI307210, BG033723,







AI589267, AI269862, BE885353,







AI313320, BG058150, BE886728,







AW827106, BF527014, AI313352,







BG110517, AL039086, AW079336,







AI251434, AI274728, BF868928,







AI524780, AI589947, AV682724,







AI439717, AI312146, AI312339,







AI814087, AI345745, AL036925,







AI345258, AI932638, AI470651,







AL036857, AW050578,







AW196105, AV682227, AI306705,







AW269097, AI620639, AI611348,







AW090393, AL042628,







AW152469, AA833760, BG256090,







AI866798, AW074993, AI567351,







AI431424, AI349614, AI311604,







AW105601, BE966990, AL044207,







AW167918, AI611738, AW169604,







AW268253, AI862144, AI567612,







BE886827, BF793308, AI890806,







AI349256, AL036664, AI554821,







AI312152, AI955906, AI336495,







BF970768, BF885000, AW075084,







AL120854, BE895585, AI950664,







BE897632, BE964078, BF872670,







AW022699, AI349937, AL036923,







AW089572, AI334884, AI307543,







AW151138, AW071412,







BF885081, AI307708, AI312325,







AI500659, AI868204, AI340659,







BF816037, AI280655, AI612885,







BF092710, AW302965, BF339322,







AI334930, AI309443, AV699211,







AV734185, AI307520, AI445237,







AV724373, AI590423, AV756798,







AI345739, AI889168, AI440263,







AW117743, AI312143, AW673635,







AW806761, AI343037, AV708834,







AI434256, AI312428, W33163,







BG109270, BE966829, AI349955,







AW075093, AI371228, BE548914,







AW827206, AI348897, AA427700,







AI306613, AI312357, AI335426,







AI348777, AI308032, AI569583,







AI687127, BG249582, AI783997,







BG030364, BG104820, AW161579,







AI627988, AI344785, BG113662,







BE971716, BF970449, AL079963,







AL036718, BE047852, BE785868,







AI207454, AI382670, AW020095,







AI874166, AL036901, BE047952,







AI670009, BG180996, BF970990,







BF526262, BG027280, AL036274,







BF061286, AI497733, AL041150,







AI288285, AI890507, BG026428,







AW827115, AW268964, AI343091,







AI318280, AI567582, BG165051,







AI554245, BE963035, BE138658,







BG260037, AI310582, BG032208,







BF344691, BE885490,







AF352730.1, AF205952.1,







AF323081.1, AK024538.1,







X53587.1, AL512765.1,







AL050393.1, AK025254.1,







AF090901.1, AK026542.1,







AL136787.1, AK026597.1,







BC006525.1, AF218031.1,







BC001963.1, BC007326.1,







AB055366.1, AK027213.1,







AL389939.1, AK026528.1,







AK026855.1, AL122110.1,







BC008780.1, AF090943.1,







AL133098.1, AL136799.1,







BC008070.1, BC003687.1,







AK024524.1, AF091084.1,







AL049466.1, AK025967.1,







AK026480.1, BC002839.1,







BC006807.1, AL136789.1,







AL157482.1, AL117394.1,







AK025391.1, AL137560.1,







AY034001.1, AK025349.1,







AF125948.1, AL359615.1,







AJ242859.1, AK025906.1,







AL136915.1, AL110221.1,







AL050092.1, AL512718.1,







AB056427.1, AK027146.1,







AB060825.1, AL133075.1,







AK025484.1, AK000391.1,







AF056191.1, AB051158.1,







AK026583.1, AB060908.1,







AL133557.1, AL117457.1,







AL110225.1, AK026526.1,







AL080159.1, AL137550.1,







AK026629.1, AL133640.1,







AB063046.1, AF217987.1,







AK026452.1, U42766.1,







AL133606.1, AK026534.1,







AL133067.1, AK024588.1,







AB047615.1, AB048954.1,







AK027182.1, AL049464.1,







AL133113.1, AL133560.1,







AL137521.1, BC008983.1,







AB060912.1, AF097996.1,







AK026353.1, BC008899.1,







BC001967.1, AK026959.1,







AL137459.1, AB060839.1,







BC003548.1, AF090900.1,







AL050116.1, AB052200.1,







AL133016.1, AL359620.1,







AB060214.1, AF057300.1,







AF057299.1, X72889.1,







AK000618.1, BC002643.1,







BC008417.1, AK026164.1,







AK026506.1, AK026741.1,







AL359618.1, AL442082.1,







BC007199.1, AK025465.1,







U91329.1, L19437.2, AL049314.1,







AB056421.1, BC004958.1,







AK027164.1, AB056809.1,







AL162062.1, AL050149.1,







AL389982.1, AL137463.1,







AL122123.1, AF230496.1,







AL162002.1, AB048964.1,







AL133080.1, AL390154.1,







BC006164.1, AK000137.1,







AK026762.1, AL117460.1,







AK026630.1, AL512689.1,







AL512719.1, AL050108.1,







AK026593.1, AB063100.1,







S61953.1, AL049300.1,







AL049452.1, BC005151.1,







AK000647.1, BC001045.1,







AB063079.1, BC002733.1,







AL136893.1, BC002342.1,







AK025383.1, BC004556.1,







BC009284.1, AL080086.1,







BC005678.1, AF078844.1,







AF026816.2, AL136928.1,







AL512750.1, BC003627.1,







Y16645.1, AB062942.1,







AF051325.1, AL512754.1,







BC006103.1, U58996.2,







AB060863.1, AL136844.1,







AK000212.1, AL080074.1,







AL359601.1, AF262032.1,







AK026600.1, AL136864.1,







U80742.1, AL136845.1,







AL122093.1, BC008893.1,







AL136892.1, BC006201.1,







AL117435.1, BC008365.1,







AL110280.1, AF061943.1,







AL136749.1, AK027868.1,







AB019565.1, AF162270.1,







BC008382.1, AL133104.1,







AF003737.1, AF353396.1,







AL137557.1, AK000445.1,







BC008284.1, AK000432.1,







AF218014.1, AL136786.1,







AB050510.1, AK024601.1,







AL117583.1, AL137648.1,







AB060929.1, AK025491.1,







AL117585.1, AL122098.1,







AK025573.1, AF219137.1,







AF090903.1, AF125949.1,







AF260566.1, AL050146.1,







AF111847.1, AL442072.1,







AB062978.1, AJ006417.1,







AL122121.1, AJ012755.1,







AL136784.1, AF104032.1,







BC008488.1, AL080060.1,







AB055315.1, AL137478.1,







AB047887.1, AF132676.1,







AF061836.1, AF183393.1,







AK027204.1, AL080127.1,







AK026647.1, AK027116.1,







AL096744.1, AB055361.1,







AL353940.1, BC008387.1,







AL137556.1, X82434.1,







AF090934.1, AL122049.1,







AL162008.1, AB063070.1,







AL122050.1, AL162003.1,







AL137271.1, BC005168.1,







AB048974.1, AK025798.1,







AB055368.1, S78214.1,







AK000486.1.


HKGAJ54
107
498303
1-1332
15-1346
H97115, AA130346, and







AA193462.


HKGAJ54
109
1300770
1-1318
15-1332
AA757140, BE301253, AL528730,







AI761849, AL520073, BG150431,







AI288886, BE301262, AW380873,







BE018216, AA227523, AA227644,







AI224842, AI417635, AI094181,







H97115, AA193462, AW371297,







AL039859, AA262346, AW957515,







AA577940, AI435978, AA373648,







AW381560, AW380920, H96998,







AI240420, AA130346, W88873,







W90542, AA001144, AW381554,







AF074684, AA253322, BF083570,







AA193344, AL520074, BE173261,







AA368803, AI718525, AA831401,







AA741509, AI741117,







AK027852.1, BC008993.1.


HE8CH92
118
609866
1-1268
15-1282
BF338867, AI862534, AA861640,







AI149724, AA400490, AA759080,







AA400536, AI218853, H52956,







BE550640, AI476417, AW274868,







BE552351, H53024, AA843555,







H53025, AA936598, AW516351,







T81709, AL079515, AW292593,







AI280269, AA629002, AL079514.


HSVAK93
120
1352228
1-1226
15-1240
BF338867, AI862534, AI149724,







AA400536, AA400490, AA861640,







AA759080, H52956, AI218853,







H53024, BE550640, AW274868,







BE552351, AI476417, H53025,







AA843555, AA936598,







AW516351, T81709, AL079515,







AW292593, AL079514, AI280269,







AA629002, BC008988.1,







AC008491.6, AC010315.6.


HSDEK49
125
1352253
1-1768
15-1782
AL513706, AL513705, AV700980,







BF343961, AV710516, AV716397,







AV715849, BF351156, AV717025,







AW071975, AI922669, AI129815,







BF106386, AA702864, W32947,







AV690218, AV685715, AV693576,







AV686846, AV695322, AV697709,







BF924861, AI168499, AI343825,







AA627735, AI554367, AI335089,







AV697729, AI290781, AA875852,







AA442570, AV686969, AV698914,







AA486920, AI357884, AI088635,







W79882, R39812, AV683817,







BF932594, W17367, N78991,







AA972857, R62969, R59135,







AW961380, R56601, BE857524,







R66262, W74268, AA436814,







AA813538, H05057, AA133776,







Z43556, R14044, R81029, T48889,







AA228697, R56602, AA142932,







R63023, Z39624, F02373,







AA993978, R66723, R67603,







R59136, R80928, AA133775,







AW874480, T48888, AA228698,







AA368546, BF525711, AA115592,







AA328299, AA486747, BG001652,







AJ132502.1, AL034397.1.


HWBAO62
127
838164
1-1889
15-1903
AI683471, AI792952.


HWHGU54
131
695695
1-1431
15-1445
AA458648, BE140448, AA455546,







AL132708.3, AL132990.3.


HWHGU54
131
695695
1-1431
15-1445
AA458648, BE140448, AA455546,







AL132708, and AL132990.


HCEJQ69
134
1243825
1-1763
15-1777
BE889609, BF183110, BG248693,







AI621234, AA452195, AI821464,







AI821685, AI820935, AI745288,







AI655244, AA436362, BF432211,







AW770404, BF196926, AA427404,







H08678, AA410412, H61387,







AI432244, AA428418, H08677,







AA410413, AA410253, H14737,







BF351660, BG170733, H62405,







AI968569, AW002476, AW166810,







BE350305, BF530203, H09849,







AW748526, AF283463.1,







AC058790.14, AC007663.29,







AC006549.28, AB045987.1.


HCEJQ69
134
1243825
1-1763
15-1777
BE889609, BF183110, BG248693,







AI621234, AA452195, AI821464,







AI821685, AI820935, AI745288,







AI655244, AA436362, BF432211,







AW770404, BF196926, AA427404,







H08678, AA410412, H61387,







AI432244, AA428418, H08677,







AA410413, AA410253, H14737,







BF351660, BG170733, H62405,







AI968569, AW002476, AW166810,







BE350305, BF530203, H09849,







AW748526, AX047642, AC058790,







AC007663, AC006549, and







AB045987.


HT5GJ57
159
740767
1-1783
15-1797
AW574516, BG259057, BF975647,







AW575080, BF795582, BE559713,







BE396519, AI521311, BE397179,







AW237047, AI446257, BF238156,







BF663664, AI862389, AI573063,







BE513368, AW296989, AU157608,







BF128855, AA811488, AA827120,







AW338778, AI439638, BE560794,







AI250231, BG120258, AI312540,







AA633095, AV742373, AI343438,







AA604586, BE269253, AI669176,







AI149413, AA723128, AW403042,







AA722908, W57991, AI826124,







BE246032, AU138425, BE513265,







AI865336, AI219708, AI589599,







AW732709, H23560, AA765412,







AI589912, BE560732, T61448,







AW444827, AA594614,







AA648496, AA361096, N34423,







W58075, AV742389, N48728,







AA975334, AA731435, AA166766,







BE247378, AA810638, AW298682,







AI919140, AW402333, AI341517,







BE245894, AV743440, AI962720,







BF062274, T30849, AI982795,







AW405561, T25945, AA810222,







AA807717, Z39117, AV756294,







AV724221, BG120887, AA593214,







N48658, AW083122, AA639378,







H23535, AI492348, AI656821,







AF045555, AK002099, AF257135,







AF161531, AC005081, AF086239,







AC016675, AC016675, AC005081,







and AC005081.


HT5GJ57
161
1299921
1-1759
15-1773
BF975647, AW574516, BG259057,







BE397179, BE396519, AW575080,







BF795582, BF663664, AI521311,







AW237047, AI446257, AI862389,







BE559713, BF128855, AI573063,







BF238156, AW296989, AU157608,







AA811488, AA827120,







AW338778, AI439638, AI250231,







AI312540, AA633095, AV742373,







AI343438, BE513368, AA604586,







AI669176, AI149413, AW732709,







AA723128, AW403042, BE269253,







BE560794, AA722908, W57991,







AI826124, BE246032, BG120258,







AU138425, BE513265, AI865336,







AI219708, AI589599, H23560,







AA765412, AI589912, T61448,







BE247378, BE560732, AW444827,







AA594614, AA361096, AA648496,







N34423, AV743440, W58075,







N48728, AV742389, AA975334,







AA731435, AA166766, AA810638,







AW298682, AI919140, AI341517,







BE245894, AI962720, BF062274,







T30849, AW402333, AI982795,







T25945, AA810222, AA807717,







Z39117, AV756294, N48658,







AW405561, AV724221, BG120887,







AA593214, AW083122,







AA639378, AI492348, H23535,







AI656821, AF252613.1,







BC009204.1, BC001609.1,







AF252611.1, AF252614.1,







AK002099.1, AF257135.1,







AF252612.1, AF045555.1,







BC006080.1, AC005081.3,







AF086239.1.


HPIBX03
169
743314
1-2195
15-2209
BG163962, AU140819, BF336602,







BG168220, AU147884, AW996615,







BF760456, AA922764, AA770561,







AA976914, AA045899, BF063943,







BF851894, BF815498, AA045900,







BF764764, AW769303,







AW996391, AW812414,







AA132854, AB033417, AA132760,







AK026622, AK023834, AK026187,







AC010170.


HDPBO81
174
892018
1-3784
15-3798
AI393580, BE041810, AI867153,







AI307279, BE568549, AX047952,







AX047936, AX047940.


HWBFY57
179
837478
1-1782
15-1796
AW003259, BE673705,







AW236996, AW590159,







BF478096, BE218330, AI802017,







AI707979, AW590572, AI970192,







BF062678, AW615365, AA984871,







AI796486, AI365178, AW138945,







AI310573, C01628, AA324946.


HYABV21
182
1281466
1-2724
15-2738
AW969109, AA278948,







AA677057, AA813919,







AW976932, AI572979, AW294948,







AW503289, AW198126, AI419925,







AA810016, AA278822, AA809271,







T89787, AA505047, AA804243,







R09908, AW500471, R12559,







AA281955, AW383680,







AA767265, AW503702,







AW504600, T89422, H50970,







AL356276, AC024085, U85195,







AE000658, AC009248, and







AC004671.


HOHBY69
186
827480
1-4981
15-4995
BF965934, BF358074, AW753022,







BG258877, AI621089, AW851445,







AI751442, AW069755, W45174,







H15811, AW339880, AI752628,







AI751441, AW068275, BF509527,







AW604115, BF056654, H16112,







AI825217, W45078, BF749563,







BF770980, BF770976, U53091,







AA852615, AI738528, BF770978,







BF928722, BF770979, AI750918,







BF365293, AW888738, AA665807,







BF328395, BG013602, AA852614,







BF770969, AI752385, AI752488,







BF770975, BF770962, AA316377,







BF770974, AI752355, BF769640,







BF758396, BF758424, BF830392,







AI750919, AV699874, AV700783,







AW166270, AF109681, AF137378,







AL359064, Z50167, Z50157.


HDHMA45
202
902513
1-2170
15-2184
AL538140, AI653241, AI826089,







AI967938, AW003801, BE222599,







AI056603, AI085672, AI201055,







AI367072, AI052212, AI631456,







AI278127, BF515139, AI597622,







AI825589, BF961191, N47437,







AA506257, AI197773, AI040587,







BF934870, BF934966,







AB028140.1, AP002436.3.


HMADJ14
206
1099342
1-1350
15-1364
AI268407, AW450309, AI831182,







AW295136, AF305068.


HMADJ14
212
843725
1-1430
15-1444
AI268407, BE162690, AI831182,







AW450309, AW295136,







AA380009, AF305068.


HEAAL31
218
639007


AA151656, AA151652, AA151733,







AA151736


HEAAL31
220
361221


T56046, T56062, T56080, T56096,







T90475, T90482, T90571, T90577,







T74331, T89037, R10054, R11653,







R54570, H22902, H24285, H24287,







H50611, H50610, H67882, H67927,







N27199, N36263, N39942, N40544,







N44078, N46591, N58937, N73790,







N94307, N95519, N99415,







W02128, W19438, W32393,







W57988, W58072, W73132,







W90660, N90316, AA009898,







AA022614, AA022615, AA025590,







AA081613, AA084503, AA085189


HEMFA84
222
608198
1-971
15-985
BE743873, BE382707, BF316838,







BE262654, AI344021, AW451305,







AW291138, AW301011,







BE784787, BG026465, AW957567,







BG253034, AI807860, AI970736,







N99472, AA378218, H23090,







W31086, BE185755, AK025935,







AF258545.


HDPPA04
224
904765
1-2392
15-2406
AU135908, AI990290, AW961323,







AI798762, AA044757, AW105205,







AW197379, AU156359,







AA039608, AA247117,







AW889458, AA303575,







AA036918, AA247128, AI214428,







AW449368, AA044631, AI762460,







AL162253.17, AK001872.1,







AF344424.1, AF329193.1.


HE2OA95
230
637595
1-1657
15-1671
AI620217, AL528146, AI056665,







AW964795, AI744518, AI038199,







AA236476, AA747220, AI368718,







AI888960, N47941, AA234584,







H19150, AI360711, BF445507,







BF475619, AI682413, AI468582,







H05762, AA297549, R52959,







N46864, T27100, AA736703,







AA579494, H18483, BE855666,







Z40989, AI474169, R41378,







AW973144, BF982162, N49148,







AL157430, AF179274, AR052523,







AB041565, AB017270, AR052522,







AB004064.


HKABZ65
232
862030
1-1175
15-1189
AA715814, AA503019, AV762033,







BE155099, AV734997, BF917346,







AW338860, AC011666.28,







AF242518.1, AF109907.1,







AC004867.5, AC020917.4,







AC004166.12, AL356915.19,







AC005071.2, AC004878.2,







AC005052.2, AC005081.3,







AC002549.1, AL590763.1,







AC020663.1, AC006064.9,







AC008745.6, AC004858.2,







AC022405.5, AC007666.12,







AC008750.7, AL451144.5,







AP001716.1, AC009131.6,







AC004656.1, AL109825.23,







AL355312.24, AL035086.12,







AC010605.4, AC004067.1,







AC004477.1, AC008736.6,







AL109915.10, AC006023.2,







AL033529.25, AC007637.9,







AL139317.5, AL031311.1,







AL049776.3, AC004971.3,







AC009220.10, AL080243.21,







AC005015.2, AC004686.1,







AL022318.2, AC002310.1,







AC009123.6, Z93015.9,







AC021999.4, AL355353.23,







AL050318.13, AL161756.6,







AC011464.5, AL132712.4,







AL359513.12, AC007546.5,







AP001695.1, AL035683.9,







AC018711.4, D87675.1,







AL133444.4, AL139100.9,







AF030453.1, AC006077.1,







AC008895.7, AP001713.1,







Z84487.2, AL357153.4,







AL163636.6, AL359382.23,







AC004770.1, AP001972.4,







AC004675.1, AL355392.7,







AC020906.6, AL138784.30,







AC020754.4, AL162426.20,







AC002288.1, AC009068.10,







AC008101.15, AC008623.4,







AC008891.7, Z98884.11,







AL136137.15, AC011247.10,







AL133163.2, AP001727.1,







AC005098.2, AC004659.1,







AC005670.1, AL139022.4,







AC009812.17, AF088219.1,







AL035404.20, AL139801.17,







AF228703.1, AC002492.1,







AC006084.1, AL353594.13,







AC005077.5, AL160271.19,







AP001724.1, AC008537.5,







AC024561.4, AL139353.3,







AC004491.1, AC008626.5,







AL391987.15, AC010530.7,







AP003352.2, AC009267.15,







AL122013.5, AP000008.1,







AC087071.2, AC009314.4,







AC020913.6, AL078463.11,







AL096700.14, AC002369.1,







AC010102.3, AP003357.2,







AL031123.14, Z95331.2,







AL513008.14, AL118501.22,







AP001435.2, AC005200.1,







AJ400877.1, AC011469.6,







AC016772.8, AC005089.2,







AC005088.2, AF312912.1,







AL022316.2, AL080317.11,







AP001693.1, AP000553.1,







AL390294.19, AC006345.4,







AC091394.2, AL359813.23,







AC007283.3, AL353807.18,







AL109921.21, AC074121.16,







Z98742.5, AC007383.4,







AF243527.1, AC027130.5,







AC010504.7, AL035462.21,







AC010650.8, AC005180.2,







AF334404.1, AL139187.19,







AC005037.2, AL021391.2.









Table 4

Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 of Table 4 provides the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a_” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.













TABLE 4





Code
Description
Tissue
Organ
Cell Line
Disease
Vector







AR022
a_Heart
a_Heart






AR023
a_Liver
a_Liver






AR024
a_mammary gland
a_mammary gland






AR025
a_Prostate
a_Prostate






AR026
a_small intestine
a_small intestine






AR027
a_Stomach
a_Stomach






AR028
Blood B cells
Blood B cells






AR029
Blood B cells activated
Blood B cells activated






AR030
Blood B cells resting
Blood B cells resting






AR031
Blood T cells activated
Blood T cells activated






AR032
Blood T cells resting
Blood T cells resting






AR033
brain
brain






AR034
breast
breast






AR035
breast cancer
breast cancer






AR036
Cell Line CAOV3
Cell Line CAOV3






AR037
cell line PA-1
cell line PA-1






AR038
cell line transformed
cell line transformed






AR039
colon
colon






AR040
colon (9808co65R)
colon (9808co65R)






AR041
colon (9809co15)
colon (9809co15)






AR042
colon cancer
colon cancer






AR043
colon cancer (9808co64R)
colon cancer (9808co64R)






AR044
colon cancer 9809co14
colon cancer 9809co14






AR045
corn clone 5
corn clone 5






AR046
corn clone 6
corn clone 6






AR047
corn clone2
corn clone2






AR048
corn clone3
corn clone3






AR049
Corn Clone4
Corn Clone4






AR050
Donor II B Cells 24 hrs
Donor II B Cells 24 hrs






AR051
Donor II B Cells 72 hrs
Donor II B Cells 72 hrs






AR052
Donor II B-Cells 24 hrs.
Donor II B-Cells 24 hrs.






AR053
Donor II B-Cells 72 hrs
Donor II B-Cells 72 hrs






AR054
Donor II Resting B Cells
Donor II Resting B Cells






AR055
Heart
Heart






AR056
Human Lung (CLONTECH TM)
Human Lung








(CLONTECH TM)






AR057
Human Mammary
Human Mammary







(CLONTECH TM)
(CLONTECH TM)






AR058
Human Thymus (CLONTECH TM)
Human Thymus








(CLONTECH TM)






AR059
Jurkat (unstimulated)
Jurkat (unstimulated)






AR060
Kidney
Kidney






AR061
Liver
Liver






AR062
Liver (CLONTECH TM)
Liver (CLONTECH TM)






AR063
Lymphocytes chronic
Lymphocytes chronic







lymphocytic leukaemia
lymphocytic leukaemia






AR064
Lymphocytes diffuse large B cell
Lymphocytes diffuse large







lymphoma
B cell lymphoma






AR065
Lymphocytes follicular
Lymphocytes follicular







lymphoma
lymphoma






AR066
normal breast
normal breast






AR067
Normal Ovarian (4004901)
Normal Ovarian (4004901)






AR068
Normal Ovary 9508G045
Normal Ovary 9508G045






AR069
Normal Ovary 9701G208
Normal Ovary 9701G208






AR070
Normal Ovary 9806G005
Normal Ovary 9806G005






AR071
Ovarian Cancer
Ovarian Cancer






AR072
Ovarian Cancer (9702G001)
Ovarian Cancer








(9702G001)






AR073
Ovarian Cancer (9707G029)
Ovarian Cancer








(9707G029)






AR074
Ovarian Cancer (9804G011)
Ovarian Cancer








(9804G011)






AR075
Ovarian Cancer (9806G019)
Ovarian Cancer








(9806G019)






AR076
Ovarian Cancer (9807G017)
Ovarian Cancer








(9807G017)






AR077
Ovarian Cancer (9809G001)
Ovarian Cancer








(9809G001)






AR078
ovarian cancer 15799
ovarian cancer 15799






AR079
Ovarian Cancer 17717AID
Ovarian Cancer 17717AID






AR080
Ovarian Cancer 4004664B1
Ovarian Cancer 4004664B1






AR081
Ovarian Cancer 4005315A1
Ovarian Cancer 4005315A1






AR082
ovarian cancer 94127303
ovarian cancer 94127303






AR083
Ovarian Cancer 96069304
Ovarian Cancer 96069304






AR084
Ovarian Cancer 9707G029
Ovarian Cancer 9707G029






AR085
Ovarian Cancer 9807G045
Ovarian Cancer 9807G045






AR086
ovarian cancer 9809G001
ovarian cancer 9809G001






AR087
Ovarian Cancer 9905C032RC
Ovarian Cancer








9905C032RC






AR088
Ovarian cancer 9907 C00 3rd
Ovarian cancer 9907 C00








3rd






AR089
Prostate
Prostate






AR090
Prostate (CLONTECH TM)
Prostate (CLONTECH TM)






AR091
prostate cancer
prostate cancer






AR092
prostate cancer #15176
prostate cancer #15176






AR093
prostate cancer #15509
prostate cancer #15509






AR094
prostate cancer #15673
prostate cancer #15673






AR095
Small Intestine (CLONTECH TM)
Small Intestine








(CLONTECH TM)






AR096
Spleen
Spleen






AR097
Thymus T cells activated
Thymus T cells activated






AR098
Thymus T cells resting
Thymus T cells resting






AR099
Tonsil
Tonsil






AR100
Tonsil geminal center centroblast
Tonsil geminal center








centroblast






AR101
Tonsil germinal center B cell
Tonsil germinal center B








cell






AR102
Tonsil lymph node
Tonsil lymph node






AR103
Tonsil memory B cell
Tonsil memory B cell






AR104
Whole Brain
Whole Brain






AR105
Xenograft ES-2
Xenograft ES-2






AR106
Xenograft SW626
Xenograft SW626






AR119
001: IL-2
001: IL-2






AR120
001: IL-2.1
001: IL-2.1






AR121
001: IL-2_b
001: IL-2_b






AR124
002: Monocytes untreated (1 hr)
002: Monocytes untreated








(1 hr)






AR125
002: Monocytes untreated (5 hrs)
002: Monocytes untreated








(5 hrs)






AR126
002: Control.1C
002: Control.1C






AR127
002: IL2.1C
002: IL2.1C






AR130
003: Placebo-treated Rat
003: Placebo-treated Rat







Lacrimal Gland
Lacrimal Gland






AR131
003: Placebo-treated Rat
003: Placebo-treated Rat







Submandibular Gland
Submandibular Gland






AR135
004: Monocytes untreated (5 hrs)
004: Monocytes untreated








(5 hrs)






AR136
004: Monocytes untreated 1 hr
004: Monocytes untreated








1 hr






AR139
005: Placebo (48 hrs)
005: Placebo (48 hrs)






AR140
006: pC4 (24 hrs)
006: pC4 (24 hrs)






AR141
006: pC4 (48 hrs)
006: pC4 (48 hrs)






AR152
007: PHA(1 hr)
007: PHA(1 hr)






AR153
007: PHA(6 HRS)
007: PHA(6 HRS)






AR154
007: PMA(6 hrs)
007: PMA(6 hrs)






AR155
008: 1449_#2
008: 1449_#2






AR161
01: A - max 24
01: A - max 24






AR162
01: A - max 26
01: A - max 26






AR163
01: A - max 30
01: A - max 30






AR164
01: B - max 24
01: B - max 24






AR165
01: B - max 26
01: B - max 26






AR166
01: B - max 30
01: B - max 30






AR167
1449 Sample
1449 Sample






AR168
3T3P10 1.0 uM insulin
3T3P10 1.0 uM insulin






AR169
3T3P10 10 nM Insulin
3T3P10 10 nM Insulin






AR170
3T3P10 10 uM insulin
3T3P10 10 uM insulin






AR171
3T3P10 No Insulin
3T3P10 No Insulin






AR172
3T3P4
3T3P4






AR173
Adipose (41892)
Adipose (41892)






AR174
Adipose Diabetic (41611)
Adipose Diabetic (41611)






AR175
Adipose Diabetic (41661)
Adipose Diabetic (41661)






AR176
Adipose Diabetic (41689)
Adipose Diabetic (41689)






AR177
Adipose Diabetic (41706)
Adipose Diabetic (41706)






AR178
Adipose Diabetic (42352)
Adipose Diabetic (42352)






AR179
Adipose Diabetic (42366)
Adipose Diabetic (42366)






AR180
Adipose Diabetic (42452)
Adipose Diabetic (42452)






AR181
Adipose Diabetic (42491)
Adipose Diabetic (42491)






AR182
Adipose Normal (41843)
Adipose Normal (41843)






AR183
Adipose Normal (41893)
Adipose Normal (41893)






AR184
Adipose Normal (42452)
Adipose Normal (42452)






AR185
Adrenal Gland
Adrenal Gland






AR186
Adrenal Gland + Whole Brain
Adrenal Gland + Whole








Brain






AR187
B7(1 hr) + (inverted)
B7(1 hr) + (inverted)






AR188
Breast (18275A2B)
Breast (18275A2B)






AR189
Breast (4004199)
Breast (4004199)






AR190
Breast (4004399)
Breast (4004399)






AR191
Breast (4004943B7)
Breast (4004943B7)






AR192
Breast (4005570B1)
Breast (4005570B1)






AR193
Breast Cancer (4004127A30)
Breast Cancer








(4004127A30)






AR194
Breast Cancer (400443A21)
Breast Cancer (400443A21)






AR195
Breast Cancer (4004643A2)
Breast Cancer (4004643A2)






AR196
Breast Cancer (4004710A7)
Breast Cancer (4004710A7)






AR197
Breast Cancer (4004943A21)
Breast Cancer








(4004943A21)






AR198
Breast Cancer (400553A2)
Breast Cancer (400553A2)






AR199
Breast Cancer (9805C046R)
Breast Cancer








(9805C046R)






AR200
Breast Cancer (9806C012R)
Breast Cancer








(9806C012R)






AR201
Breast Cancer (ODQ 45913)
Breast Cancer (ODQ








45913)






AR202
Breast Cancer (ODQ45913)
Breast Cancer (ODQ45913)






AR203
Breast Cancer (ODQ4591B)
Breast Cancer








(ODQ4591B)






AR204
Colon Cancer (15663)
Colon Cancer (15663)






AR205
Colon Cancer (4005144A4)
Colon Cancer (4005144A4)






AR206
Colon Cancer (4005413A4)
Colon Cancer (4005413A4)






AR207
Colon Cancer (4005570B1)
Colon Cancer (4005570B1)






AR208
Control RNA #1
Control RNA #1






AR209
Control RNA #2
Control RNA #2






AR210
Cultured Preadipocyte (blue)
Cultured Preadipocyte








(blue)






AR211
Cultured Preadipocyte (Red)
Cultured Preadipocyte








(Red)






AR212
Donor II B-Cells 24 hrs
Donor II B-Cells 24 hrs






AR213
Donor II Resting B-Cells
Donor II Resting B-Cells






AR214
H114EP12 10 nM Insulin
H114EP12 10 nM Insulin






AR215
H114EP12 (10 nM insulin)
H114EP12 (10 nM insulin)






AR216
H114EP12 (2.6 ug/ul)
H114EP12 (2.6 ug/ul)






AR217
H114EP12 (3.6 ug/ul)
H114EP12 (3.6 ug/ul)






AR218
HUVEC #1
HUVEC #1






AR219
HUVEC #2
HUVEC #2






AR221
L6 undiff.
L6 undiff.






AR222
L6 Undifferentiated
L6 Undifferentiated






AR223
L6P8 + 10 nM Insulin
L6P8 + 10 nM Insulin






AR224
L6P8 + HS
L6P8 + HS






AR225
L6P8 10 nM Insulin
L6P8 10 nM Insulin






AR226
Liver (00-06-A007B)
Liver (00-06-A007B)






AR227
Liver (96-02-A075)
Liver (96-02-A075)






AR228
Liver (96-03-A144)
Liver (96-03-A144)






AR229
Liver (96-04-A138)
Liver (96-04-A138)






AR230
Liver (97-10-A074B)
Liver (97-10-A074B)






AR231
Liver (98-09-A242A)
Liver (98-09-A242A)






AR232
Liver Diabetic (1042)
Liver Diabetic (1042)






AR233
Liver Diabetic (41616)
Liver Diabetic (41616)






AR234
Liver Diabetic (41955)
Liver Diabetic (41955)






AR235
Liver Diabetic (42352R)
Liver Diabetic (42352R)






AR236
Liver Diabetic (42366)
Liver Diabetic (42366)






AR237
Liver Diabetic (42483)
Liver Diabetic (42483)






AR238
Liver Diabetic (42491)
Liver Diabetic (42491)






AR239
Liver Diabetic (99-09-A281A)
Liver Diabetic (99-09-








A281A)






AR240
Lung
Lung






AR241
Lung (27270)
Lung (27270)






AR242
Lung (2727Q)
Lung (2727Q)






AR243
Lung Cancer (4005116A1)
Lung Cancer (4005116A1)






AR244
Lung Cancer (4005121A5)
Lung Cancer (4005121A5)






AR245
Lung Cancer (4005121A5))
Lung Cancer (4005121A5))






AR246
Lung Cancer (4005340A4)
Lung Cancer (4005340A4)






AR247
Mammary Gland
Mammary Gland






AR248
Monocyte (CT)
Monocyte (CT)






AR249
Monocyte (OCT)
Monocyte (OCT)






AR250
Monocytes (CT)
Monocytes (CT)






AR251
Monocytes (INFG 18 hr)
Monocytes (INFG 18 hr)






AR252
Monocytes (INFG 18 hr)
Monocytes (INFG 18 hr)






AR253
Monocytes (INFG 8-11)
Monocytes (INFG 8-11)






AR254
Monocytes (O CT)
Monocytes (O CT)






AR255
Muscle (91-01-A105)
Muscle (91-01-A105)






AR256
Muscle (92-04-A059)
Muscle (92-04-A059)






AR257
Muscle (97-11-A056d)
Muscle (97-11-A056d)






AR258
Muscle (99-06-A210A)
Muscle (99-06-A210A)






AR259
Muscle (99-07-A203B)
Muscle (99-07-A203B)






AR260
Muscle (99-7-A203B)
Muscle (99-7-A203B)






AR261
Muscle Diabetic (42352R)
Muscle Diabetic (42352R)






AR262
Muscle Diabetic (42366)
Muscle Diabetic (42366)






AR263
NK-19 Control
NK-19 Control






AR264
NK-19 IL Treated 72 hrs
NK-19 IL Treated 72 hrs






AR265
NK-19 UK Treated 72 hrs.
NK-19 UK Treated 72 hrs.






AR266
Omentum Normal (94-08-B009)
Omentum Normal (94-08-








B009)






AR267
Omentum Normal (97-01-
Omentum Normal (97-01-







A039A)
A039A)






AR268
Omentum Normal (97-04-
Omentum Normal (97-04-







A114C)
A114C)






AR269
Omentum Normal (97-06-
Omentum Normal (97-06-







A117C)
A117C)






AR270
Omentum Normal (97-09-B004C)
Omentum Normal (97-09-








B004C)






AR271
Ovarian Cancer (17717AID)
Ovarian Cancer








(17717AID)






AR272
Ovarian Cancer (9905C023RC)
Ovarian Cancer








(9905C023RC)






AR273
Ovarian Cancer (9905C032RC)
Ovarian Cancer








(9905C032RC)






AR274
Ovary (9508G045)
Ovary (9508G045)






AR275
Ovary (9701G208)
Ovary (9701G208)






AR276
Ovary 9806G005
Ovary 9806G005






AR277
Pancreas
Pancreas






AR278
Placebo
Placebo






AR279
rIL2 Control
rIL2 Control






AR280
RSS288L
RSS288L






AR281
RSS288LC
RSS288LC






AR282
Salivary Gland
Salivary Gland






AR283
Skeletal Muscle
Skeletal Muscle






AR284
Skeletal Muscle (91-01-A105)
Skeletal Muscle (91-01-








A105)






AR285
Skeletal Muscle (42180)
Skeletal Muscle (42180)






AR286
Skeletal Muscle (42386)
Skeletal Muscle (42386)






AR287
Skeletal Muscle (42461)
Skeletal Muscle (42461)






AR288
Skeletal Muscle (91-01-A105)
Skeletal Muscle (91-01-








A105)






AR289
Skeletal Muscle (92-04-A059)
Skeletal Muscle (92-04-








A059)






AR290
Skeletal Muscle (96-08-A171)
Skeletal Muscle (96-08-








A171)






AR291
Skeletal Muscle (97-07-A190A)
Skeletal Muscle (97-07-








A190A)






AR292
Skeletal Muscle Diabetic (42352)
Skeletal Muscle Diabetic








(42352)






AR293
Skeletal Muscle Diabetic (42366)
Skeletal Muscle Diabetic








(42366)






AR294
Skeletal Muscle Diabetic (42395)
Skeletal Muscle Diabetic








(42395)






AR295
Skeletal Muscle Diabetic (42483)
Skeletal Muscle Diabetic








(42483)






AR296
Skeletal Muscle Diabetic (42491)
Skeletal Muscle Diabetic








(42491)






AR297
Skeletal Muscle Diabetic 42352
Skeletal Muscle Diabetic








42352






AR298
Skeletal Musle (42461)
Skeletal Musle (42461)






AR299
Small Intestine
Small Intestine






AR300
Stomach
Stomach






AR301
T-Cell + HDPBQ71.fc 1449
T-Cell + HDPBQ71.fc







16 hrs
1449 16 hrs






AR302
T-Cell + HDPBQ71.fc 1449 6 hrs
T-Cell + HDPBQ71.fc








1449 6 hrs






AR303
T-Cell + IL2 16 hrs
T-Cell + IL2 16 hrs






AR304
T-Cell + IL2 6 hrs
T-Cell + IL2 6 hrs






AR306
T-Cell Untreated 16 hrs
T-Cell Untreated 16 hrs






AR307
T-Cell Untreated 6 hrs
T-Cell Untreated 6 hrs






AR308
T-Cells 24 hours
T-Cells 24 hours






AR309
T-Cells 24 hrs
T-Cells 24 hrs






AR310
T-Cells 24 hrs.
T-Cells 24 hrs.






AR311
T-Cells 24 hrs
T-Cells 24 hrs






AR312
T-Cells 4 days
T-Cells 4 days






AR313
Thymus
Thymus






AR314
TRE
TRE






AR315
TREC
TREC






AR316
Virtual Mixture
Virtual Mixture






AR317
B lymphocyte,
B lymphocyte,






AR318
(non-T; non-B)
(non-T; non-B)






AR326
001-293 RNA (Vector Control)
001-293 RNA (Vector








Control)






AR327
001: Control
001: Control






AR328
001: Control.1
001: Control.1






AR355
Acute Lymphocyte Leukemia
Acute Lymphocyte








Leukemia






AR356
AML Patient #11
AML Patient #11






AR357
AML Patient #2
AML Patient #2






AR358
AML Patient #2 SGAH
AML Patient #2 SGAH






AR359
AML Patient#2
AML Patient#2






AR360
Aorta
Aorta






AR361
B Cell
B Cell






AR362
B lymphoblast
B lymphoblast






AR363
B lymphocyte
B lymphocyte






AR364
B lymphocytes
B lymphocytes






AR365
B-cell
B-cell






AR366
B-Cells
B-Cells






AR367
B-Lymphoblast
B-Lymphoblast






AR368
B-Lymphocytes
B-Lymphocytes






AR369
Bladder
Bladder






AR370
Bone Marrow
Bone Marrow






AR371
Bronchial Epithelial Cell
Bronchial Epithelial Cell






AR372
Bronchial Epithelial Cells
Bronchial Epithelial Cells






AR373
Caco-2A
Caco-2A






AR374
Caco-2B
Caco-2B






AR375
Caco-2C
Caco-2C






AR376
Cardiac #1
Cardiac #1






AR377
Cardiac #2
Cardiac #2






AR378
Chest Muscle
Chest Muscle






AR381
Dendritic Cell
Dendritic Cell






AR382
Dendritic cells
Dendritic cells






AR383
E. coli
E. coli






AR384
Epithelial Cells
Epithelial Cells






AR385
Esophagus
Esophagus






AR386
FPPS
FPPS






AR387
FPPSC
FPPSC






AR388
HepG2 Cell Line
HepG2 Cell Line






AR389
HepG2 Cell line Buffer 1 hr.
HepG2 Cell line Buffer 1 hr.






AR390
HepG2 Cell line Buffer 06 hr
HepG2 Cell line Buffer 06 hr






AR391
HepG2 Cell line Buffer 24 hr.
HepG2 Cell line Buffer 24 hr.






AR392
HepG2 Cell line Insulin 01 hr.
HepG2 Cell line Insulin 01 hr.






AR393
HepG2 Cell line Insulin 06 hr.
HepG2 Cell line Insulin 06 hr.






AR394
HepG2 Cell line Insulin 24 hr.
HepG2 Cell line Insulin 24 hr.






AR398
HMC-1
HMC-1






AR399
HMCS
HMCS






AR400
HMSC
HMSC






AR401
HUVEC #3
HUVEC #3






AR402
HUVEC #4
HUVEC #4






AR404
KIDNEY NORMAL
KIDNEY NORMAL






AR405
KIDNEY TUMOR
KIDNEY TUMOR






AR406
KIDNEY TUMOR







AR407
Lymph Node
Lymph Node






AR408
Macrophage
Macrophage






AR409
Megakarioblast
Megakarioblast






AR410
Monocyte
Monocyte






AR411
Monocytes
Monocytes






AR412
Myocardium
Myocardium






AR413
Myocardium #3
Myocardium #3






AR414
Myocardium #4
Myocardium #4






AR415
Myocardium #5
Myocardium #5






AR416
NK
NK






AR417
NK cell
NK cell






AR418
NK cells
NK cells






AR419
NKYa
NKYa






AR420
NKYa019
NKYa019






AR421
Ovary
Ovary






AR422
Patient #11
Patient #11






AR423
Peripheral blood
Peripheral blood






AR424
Primary Adipocytes
Primary Adipocytes






AR425
Promyeloblast
Promyeloblast






AR427
RSSWT
RSSWT






AR428
RSSWTC
RSSWTC






AR429
SW 480(G1)
SW 480(G1)






AR430
SW 480(G2)
SW 480(G2)






AR431
SW 480(G3)
SW 480(G3)






AR432
SW 480(G4)
SW 480(G4)






AR433
SW 480(G5)
SW 480(G5)






AR434
T Lymphoblast
T Lymphoblast






AR435
T Lymphocyte
T Lymphocyte






AR436
T-Cell
T-Cell






AR438
T-Cell,
T-Cell,






AR439
T-Cells
T-Cells






AR440
T-lymphoblast
T-lymphoblast






AR441
Th 1
Th 1






AR442
Th 2
Th 2






AR443
Th1
Th1






AR444
Th2
Th2






H0002
Human Adult Heart
Human Adult Heart
Heart


UNI-ZAP TM








XR


H0003
Human Adult Liver
Human Adult Liver
Liver


UNI-ZAP TM








XR


H0004
Human Adult Spleen
Human Adult Spleen
Spleen


UNI-ZAP TM








XR


H0006
Human Frontal Lobe of Brain




UNI-ZAP TM








XR


H0007
Human Cerebellum
Human Cerebellum
Brain


UNI-ZAP TM








XR


H0008
Whole 6 Week Old Embryo




UNI-ZAP TM








XR


H0009
Human Fetal Brain




UNI-ZAP TM








XR


H0009
Human Fetal Brain
Human Fetal Brain
Brain


UNI-ZAP TM








XR


H0010
Human Fetal Hepatic
Human Fetal Liver
Liver


UNI-ZAP TM








XR


H0011
Human Fetal Kidney
Human Fetal Kidney
Kidney


UNI-ZAP TM








XR


H0012
Human Fetal Kidney
Human Fetal Kidney
Kidney


UNI-ZAP TM








XR


H0013
Human 8 Week Whole Embryo
Human 8 Week Old
Embryo


UNI-ZAP TM




Embryo



XR


H0014
Human Gall Bladder
Human Gall Bladder
Gall Bladder


UNI-ZAP TM








XR


H0015
Human Gall Bladder, fraction II
Human Gall Bladder
Gall Bladder


UNI-ZAP TM








XR


H0016
Human Greater Omentum
Human Greater Omentum
peritoneum


UNI-ZAP TM








XR


H0017
Human Greater Omentum
Human Greater Omentum
peritoneum


UNI-ZAP TM








XR


H0018
Human Greater Omentum, fII
Human Greater Omentum
peritoneum


UNI-ZAP TM



remake




XR


H0019
Human Fetal Heart
Human Fetal Heart
Heart


pBLUESCRIPT TM


H0020
Human Hippocampus
Human Hippocampus
Brain


UNI-ZAP TM








XR


H0021
Human Infant Adrenal Gland
Human Infant Adrenal
Adrenal


UNI-ZAP TM




Gland
gland


XR


H0022
Jurkat Cells
Jurkat T-Cell Line



LAMBDA








ZAP TM II


H0023
Human Fetal Lung




UNI-ZAP TM








XR


H0024
Human Fetal Lung III
Human Fetal Lung
Lung


UNI-ZAP TM








XR


H0025
Human Adult Lymph Node
Human Adult Lymph Node
Lymph Node


LAMBDA








ZAP TM II


H0026
Namalwa Cells
Namalwa B-Cell Line,



LAMBDA




EBV immortalized



ZAP TM II


H0028
Human Old Ovary
Human Old Ovary
Ovary


pBLUESCRIPT TM


H0029
Human Pancreas
Human Pancreas
Pancreas


UNI-ZAP TM








XR


H0030
Human Placenta




UNI-ZAP TM








XR


H0031
Human Placenta
Human Placenta
Placenta


UNI-ZAP TM








XR


H0032
Human Prostate
Human Prostate
Prostate


UNI-ZAP TM








XR


H0033
Human Pituitary
Human Pituitary



UNI-ZAP TM








XR


H0034
Human Parathyroid Tumor
Human Parathyroid Tumor
Parathyroid

disease
UNI-ZAP TM








XR


H0035
Human Salivary Gland
Human Salivary Gland
Salivary


UNI-ZAP TM





gland


XR


H0036
Human Adult Small Intestine
Human Adult Small
Small Int.


UNI-ZAP TM




Intestine



XR


H0037
Human Adult Small Intestine
Human Adult Small
Small Int.


pBLUESCRIPT TM




Intestine






H0038
Human Testes
Human Testes
Testis


UNI-ZAP TM








XR


H0039
Human Pancreas Tumor
Human Pancreas Tumor
Pancreas

disease
UNI-ZAP TM








XR


H0040
Human Testes Tumor
Human Testes Tumor
Testis

disease
UNI-ZAP TM








XR


H0041
Human Fetal Bone
Human Fetal Bone
Bone


UNI-ZAP TM








XR


H0042
Human Adult Pulmonary
Human Adult Pulmonary
Lung


UNI-ZAP TM








XR


H0044
Human Cornea
Human Cornea
eye


UNI-ZAP TM








XR


H0045
Human Esophagus, Cancer
Human Esophagus, cancer
Esophagus

disease
UNI-ZAP TM








XR


H0046
Human Endometrial Tumor
Human Endometrial Tumor
Uterus

disease
UNI-ZAP TM








XR


H0047
Human Fetal Liver
Human Fetal Liver
Liver


UNI-ZAP TM








XR


H0048
Human Pineal Gland
Human Pineal Gland



UNI-ZAP TM








XR


H0049
Human Fetal Kidney
Human Fetal Kidney
Kidney


UNI-ZAP TM








XR


H0050
Human Fetal Heart
Human Fetal Heart
Heart


UNI-ZAP TM








XR


H0051
Human Hippocampus
Human Hippocampus
Brain


UNI-ZAP TM








XR


H0052
Human Cerebellum
Human Cerebellum
Brain


UNI-ZAP TM








XR


H0053
Human Adult Kidney
Human Adult Kidney
Kidney


UNI-ZAP TM








XR


H0056
Human Umbilical Vein, Endo.
Human Umbilical Vein
Umbilical


UNI-ZAP TM



remake
Endothelial Cells
vein


XR


H0057
Human Fetal Spleen




UNI-ZAP TM








XR


H0058
Human Thymus Tumor
Human Thymus Tumor
Thymus

disease
LAMBDA








ZAP TM II


H0059
Human Uterine Cancer
Human Uterine Cancer
Uterus

disease
LAMBDA








ZAP TM II


H0060
Human Macrophage
Human Macrophage
Blood
Cell Line

pBLUESCRIPT TM


H0061
Human Macrophage
Human Macrophage
Blood
Cell Line

pBLUESCRIPT TM


H0062
Human Thymus
Human Thymus
Thymus


UNI-ZAP TM








XR


H0063
Human Thymus
Human Thymus
Thymus


UNI-ZAP TM








XR


H0064
Human Right Hemisphere of
Human Brain, right
Brain


UNI-ZAP TM



Brain
hemisphere



XR


H0065
Human Esophagus, Normal
Human Esophagus, normal
Esophagus


UNI-ZAP TM








XR


H0067
Human left hemisphere, adult
Human Left Hemisphere,
Brain


LAMBDA




Adult



ZAP TM II


H0068
Human Skin Tumor
Human Skin Tumor
Skin

disease
UNI-ZAP TM








XR


H0069
Human Activated T-Cells
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0070
Human Pancreas
Human Pancreas
Pancreas


UNI-ZAP TM








XR


H0071
Human Infant Adrenal Gland
Human Infant Adrenal
Adrenal


UNI-ZAP TM




Gland
gland


XR


H0073
Human Leiomyeloid Carcinoma
Human Leiomyeloid
Muscle

disease
UNI-ZAP TM




Carcinoma



XR


H0074
Human Platelets
Human Platelets
Blood
Cell Line

UNI-ZAP TM








XR


H0075
Human Activated T-Cells (II)
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0076
Human Membrane Bound
Human Membrane Bound
Blood
Cell Line

UNI-ZAP TM



Polysomes
Polysomes



XR


H0077
Human Thymus Tumor
Human Thymus Tumor
Thymus

disease
LAMBDA








ZAP TM II


H0078
Human Lung Cancer
Human Lung Cancer
Lung

disease
LAMBDA








ZAP TM II


H0079
Human Whole 7 Week Old
Human Whole 7 Week Old
Embryo


UNI-ZAP TM



Embryo (II)
Embryo



XR


H0080
Human Whole 6 Week Old
Human Whole Six Week
Embryo


LAMBDA



Embryo (II)
Old Embryo



ZAP TM II


H0081
Human Fetal Epithelium (Skin)
Human Fetal Skin
Skin


UNI-ZAP TM








XR


H0082
Human Fetal Muscle
Human Fetal Muscle
Sk Muscle


UNI-ZAP TM








XR


H0083
HUMAN JURKAT
Jurkat Cells



UNI-ZAP TM



MEMBRANE BOUND




XR



POLYSOMES







H0085
Human Colon
Human Colon



LAMBDA








ZAP TM II


H0086
Human epithelioid sarcoma
Epithelioid Sarcoma,
Sk Muscle

disease
UNI-ZAP TM




muscle



XR


H0087
Human Thymus
Human Thymus



pBLUESCRIPT TM


H0090
Human T-Cell Lymphoma
T-Cell Lymphoma
T-Cell

disease
UNI-ZAP TM








XR


H0092
Human Pancreas Tumor
Human Pancreas Tumor
Pancreas

disease
UNI-ZAP TM








XR


H0093
Human Greater Omentum Tumor
Human Greater Omentum
peritoneum

disease
UNI-ZAP TM








XR


H0095
Human Greater Omentum, RNA
Human Greater Omentum
peritoneum


UNI-ZAP TM



Remake




XR


H0096
Human Parotid Cancer
Human Parotid Cancer
Parotid

disease
LAMBDA








ZAP TM II


H0097
Human Adult Heart, subtracted
Human Adult Heart
Heart


pBLUESCRIPT TM


H0098
Human Adult Liver, subtracted
Human Adult Liver
Liver


UNI-ZAP TM








XR


H0099
Human Lung Cancer, subtracted
Human Lung Cancer
Lung


pBLUESCRIPT TM


H0100
Human Whole Six Week Old
Human Whole Six Week
Embryo


UNI-ZAP TM



Embryo
Old Embryo



XR


H0101
Human 7 Weeks Old Embryo,
Human Whole 7 Week Old
Embryo


LAMBDA



subtracted
Embryo



ZAP TM II


H0102
Human Whole 6 Week Old
Human Whole Six Week
Embryo


pBLUESCRIPT TM



Embryo (II), subt
Old Embryo






H0103
Human Fetal Brain, subtracted
Human Fetal Brain
Brain


UNI-ZAP TM








XR


H0105
Human Fetal Heart, subtracted
Human Fetal Heart
Heart


pBLUESCRIPT TM


H0106
Human Right Hemisphere of
Human Brain, right
Brain


UNI-ZAP TM



Brain, subtrac
hemisphere



XR


H0107
Human Infant Adrenal Gland,
Human Infant Adrenal
Adrenal


pBLUESCRIPT TM



subtracted
Gland
gland





H0108
Human Adult Lymph Node,
Human Adult Lymph Node
Lymph Node


UNI-ZAP TM



subtracted




XR


H0109
Human Macrophage, subtracted
Macrophage
Blood
Cell Line

pBLUESCRIPT TM


H0110
Human Old Ovary, subtracted
Human Old Ovary
Ovary


pBLUESCRIPT TM


H0111
Human Placenta, subtracted
Human Placenta
Placenta


pBLUESCRIPT TM


H0112
Human Parathyroid Tumor,
Human Parathyroid Tumor
Parathyroid


pBLUESCRIPT TM



subtracted







H0113
Human skin Tumor, subtracted
Human Skin Tumor
Skin


UNI-ZAP TM








XR


H0116
Human Thymus Tumor,
Human Thymus Tumor
Thymus


pBLUESCRIPT TM



subtracted







H0117
Human Uterine Cancer,
Human Uterine Cancer
Uterus


pBLUESCRIPT TM



subtracted







H0118
Human Adult Kidney
Human Adult Kidney
Kidney


UNI-ZAP TM








XR


H0119
Human Pediatric Kidney
Human Pediatric Kidney
Kidney


UNI-ZAP TM








XR


H0120
Human Adult Spleen, subtracted
Human Adult Spleen
Spleen


UNI-ZAP TM








XR


H0121
Human Cornea, subtracted
Human Cornea
eye


UNI-ZAP TM








XR


H0122
Human Adult Skeletal Muscle
Human Skeletal Muscle
Sk Muscle


UNI-ZAP TM








XR


H0123
Human Fetal Dura Mater
Human Fetal Dura Mater
Brain


UNI-ZAP TM








XR


H0124
Human Rhabdomyosarcoma
Human
Sk Muscle

disease
UNI-ZAP TM




Rhabdomyosarcoma



XR


H0125
Cem cells cyclohexamide treated
Cyclohexamide Treated
Blood
Cell Line

UNI-ZAP TM




Cem, Jurkat, Raji, and Supt



XR


H0128
Jurkat cells, thiouridine activated
Jurkat Cells



UNI-ZAP TM








XR


H0129
Jurkat cells, thiouridine activated,
Jurkat Cells



UNI-ZAP TM



fract II




XR


H0130
LNCAP untreated
LNCAP Cell Line
Prostate
Cell Line

UNI-ZAP TM








XR


H0131
LNCAP + o.3 nM R1881
LNCAP Cell Line
Prostate
Cell Line

UNI-ZAP TM








XR


H0132
LNCAP + 30 nM R1881
LNCAP Cell Line
Prostate
Cell Line

UNI-ZAP TM








XR


H0134
Raji Cells, cyclohexamide treated
Cyclohexamide Treated
Blood
Cell Line

UNI-ZAP TM




Cem, Jurkat, Raji, and Supt



XR


H0135
Human Synovial Sarcoma
Human Synovial Sarcoma
Synovium


UNI-ZAP TM








XR


H0136
Supt Cells, cyclohexamide treated
Cyclohexamide Treated
Blood
Cell Line

UNI-ZAP TM




Cem, Jurkat, Raji, and Supt



XR


H0138
Activated T-Cells, 0 hrs.
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0139
Activated T-Cells, 4 hrs.
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0140
Activated T-Cells, 8 hrs.
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0141
Activated T-Cells, 12 hrs.
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0142
MCF7 Cell Line
MCF7 Cell line
Breast
Cell Line

UNI-ZAP TM








XR


H0144
Nine Week Old Early Stage
9 Wk Old Early Stage
Embryo


UNI-ZAP TM



Human
Human



XR


H0147
Human Adult Liver
Human Adult Liver
Liver


UNI-ZAP TM








XR


H0149
7 Week Old Early Stage Human,
Human Whole 7 Week Old
Embryo


UNI-ZAP TM



subtracted
Embryo



XR


H0150
Human Epididymus
Epididymis
Testis


UNI-ZAP TM








XR


H0151
Early Stage Human Liver
Human Fetal Liver
Liver


UNI-ZAP TM








XR


H0152
Early Stage Human Liver, fract
Human Fetal Liver
Liver


UNI-ZAP TM



(II)




XR


H0154
Human Fibrosarcoma
Human Skin Fibrosarcoma
Skin

disease
UNI-ZAP TM








XR


H0155
Human Thymus, subtracted
Human Thymus Tumor
Thymus


pBLUESCRIPT TM


H0156
Human Adrenal Gland Tumor
Human Adrenal Gland
Adrenal

disease
UNI-ZAP TM




Tumor
Gland


XR


H0157
Activated T-Cells, 0 hrs, ligation 2
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0158
Activated T-Cells, 4 hrs., ligation 2
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0159
Activated T-Cells, 8 hrs., ligation 2
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0160
Activated T-Cells, 12 hrs.,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



ligation 2




XR


H0161
Activated T-Cells, 24 hrs.,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



ligation 2




XR


H0163
Human Synovium
Human Synovium
Synovium


UNI-ZAP TM








XR


H0164
Human Trachea Tumor
Human Trachea Tumor
Trachea

disease
UNI-ZAP TM








XR


H0165
Human Prostate Cancer, Stage B2
Human Prostate Cancer,
Prostate

disease
UNI-ZAP TM




stage B2



XR


H0166
Human Prostate Cancer, Stage B2
Human Prostate Cancer,
Prostate

disease
UNI-ZAP TM



fraction
stage B2



XR


H0167
Activated T-Cells, 24 hrs.
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM








XR


H0168
Human Prostate Cancer, Stage C
Human Prostate Cancer,
Prostate

disease
UNI-ZAP TM




stage C



XR


H0169
Human Prostate Cancer, Stage C
Human Prostate Cancer,
Prostate

disease
UNI-ZAP TM



fraction
stage C



XR


H0170
12 Week Old Early Stage Human
Twelve Week Old Early
Embryo


UNI-ZAP TM




Stage Human



XR


H0171
12 Week Old Early Stage Human,
Twelve Week Old Early
Embryo


UNI-ZAP TM



II
Stage Human



XR


H0172
Human Fetal Brain, random
Human Fetal Brain
Brain


LAMBDA



primed




ZAP TM II


H0173
Human Cardiomyopathy, RNA
Human Cardiomyopathy
Heart

disease
UNI-ZAP TM



remake




XR


H0175
H. Adult Spleen, ziplox




pSport1


H0176
CAMA1Ee Cell Line
CAMA1Ee Cell Line
Breast
Cell Line

UNI-ZAP TM








XR


H0177
CAMA1Ee Cell Line
CAMA1Ee Cell Line
Breast
Cell Line

UNI-ZAP TM








XR


H0178
Human Fetal Brain
Human Fetal Brain
Brain


UNI-ZAP TM








XR


H0179
Human Neutrophil
Human Neutrophil
Blood
Cell Line

UNI-ZAP TM








XR


H0180
Human Primary Breast Cancer
Human Primary Breast
Breast

disease
UNI-ZAP TM




Cancer



XR


H0181
Human Primary Breast Cancer
Human Primary Breast
Breast

disease
UNI-ZAP TM




Cancer



XR


H0182
Human Primary Breast Cancer
Human Primary Breast
Breast

disease
UNI-ZAP TM




Cancer



XR


H0183
Human Colon Cancer
Human Colon Cancer
Colon

disease
UNI-ZAP TM








XR


H0184
Human Colon Cancer,
Human Colon Cancer,
Liver

disease
LAMBDA



metasticized to live
metasticized to liver



ZAP TM II


H0185
Activated T-Cell labeled with 4-
T-Cells
Blood
Cell Line

LAMBDA



thioluri




ZAP TM II


H0186
Activated T-Cell
T-Cells
Blood
Cell Line

LAMBDA








ZAP TM II


H0187
Resting T-Cell
T-Cells
Blood
Cell Line

LAMBDA








ZAP TM II


H0188
Human Normal Breast
Human Normal Breast
Breast


UNI-ZAP TM








XR


H0189
Human Resting Macrophage
Human
Blood
Cell Line

UNI-ZAP TM




Macrophage/Monocytes



XR


H0190
Human Activated Macrophage
Human
Blood
Cell Line

UNI-ZAP TM



(LPS)
Macrophage/Monocytes



XR


H0191
Human Activated Macrophage
Human
Blood
Cell Line

UNI-ZAP TM



(LPS), thiour
Macrophage/Monocytes



XR


H0192
Cem Cells, cyclohexamide
Cyclohexamide Treated
Blood
Cell Line

UNI-ZAP TM



treated, subtra
Cem, Jurkat, Raji, and Supt



XR


H0193
Cem Cells, cyclohexamide
Cyclohexamide Treated
Blood
Cell Line

UNI-ZAP TM



treated, differ
Cem, Jurkat, Raji, and Supt



XR


H0194
Human Cerebellum, subtracted
Human Cerebellum
Brain


pBLUESCRIPT TM


H0196
Human Cardiomyopathy,
Human Cardiomyopathy
Heart


UNI-ZAP TM



subtracted




XR


H0197
Human Fetal Liver, subtracted
Human Fetal Liver
Liver


UNI-ZAP TM








XR


H0198
Human Fetal Liver, subtracted,
Human Fetal Liver
Liver


UNI-ZAP TM



pos. clon




XR


H0199
Human Fetal Liver, subtracted,
Human Fetal Liver
Liver


UNI-ZAP TM



neg clone




XR


H0200
Human Greater Omentum, fract II
Human Greater Omentum
peritoneum


UNI-ZAP TM



remake,




XR


H0201
Human Hippocampus, subtracted
Human Hippocampus
Brain


pBLUESCRIPT TM


H0202
Jurkat Cells, cyclohexamide
Cyclohexamide Treated
Blood
Cell Line

UNI-ZAP TM



treated, subtraction
Cem, Jurkat, Raji, and Supt



XR


H0203
Jurkat Cells, cyclohexamide
Cyclohexamide Treated
Blood
Cell Line

UNI-ZAP TM



treated, dif
Cem, Jurkat, Raji, and Supt



XR


H0204
Human Colon Cancer, subtracted
Human Colon Cancer
Colon


pBLUESCRIPT TM


H0205
Human Colon Cancer, differential
Human Colon Cancer
Colon


pBLUESCRIPT TM


H0207
LNCAP, differential expression
LNCAP Cell Line
Prostate
Cell Line

pBLUESCRIPT TM


H0208
Early Stage Human Lung,
Human Fetal Lung
Lung


pBLUESCRIPT TM



subtracted







H0209
Human Cerebellum, differentially
Human Cerebellum
Brain


UNI-ZAP TM



expressed




XR


H0211
Human Prostate, differential
Human Prostate
Prostate


pBLUESCRIPT TM



expression







H0212
Human Prostate, subtracted
Human Prostate
Prostate


pBLUESCRIPT TM


H0213
Human Pituitary, subtracted
Human Pituitary



UNI-ZAP TM








XR


H0214
Raji cells, cyclohexamide treated,
Cyclohexamide Treated
Blood
Cell Line

pBLUESCRIPT TM



subtracted
Cem, Jurkat, Raji, and Supt






H0215
Raji cells, cyclohexamide treated,
Cyclohexamide Treated
Blood
Cell Line

pBLUESCRIPT TM



differentially expressed
Cem, Jurkat, Raji, and Supt






H0216
Supt cells, cyclohexamide treated,
Cyclohexamide Treated
Blood
Cell Line

pBLUESCRIPT TM



subtracted
Cem, Jurkat, Raji, and Supt






H0217
Supt cells, cyclohexamide treated,
Cyclohexamide Treated
Blood
Cell Line

pBLUESCRIPT TM



differentially expressed
Cem, Jurkat, Raji, and Supt






H0218
Activated T-Cells, 0 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



subtracted




XR


H0219
Activated T-Cells, 0 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



differentially expressed




XR


H0220
Activated T-Cells, 4 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



subtracted




XR


H0221
Activated T-Cells, 4 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



differentially expressed




XR


H0222
Activated T-Cells, 8 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



subtracted




XR


H0223
Activated T-Cells, 8 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



differentially expressed




XR


H0224
Activated T-Cells, 12 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



subtracted




XR


H0225
Activated T-Cells, 12 hrs,
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



differentially expressed




XR


H0228
C7MCF7 cell line, estrogen
C7MCF7 Cell Line,
Breast
Cell Line

UNI-ZAP TM



treated
estrogen treated



XR


H0229
Early Stage Human Brain,
Early Stage Human Brain
Brain


LAMBDA



random primed




ZAP TM II


H0230
Human Cardiomyopathy, diff exp
Human Cardiomyopathy
Heart

disease
UNI-ZAP TM








XR


H0231
Human Colon, subtraction
Human Colon



pBLUESCRIPT TM


H0232
Human Colon, differential
Human Colon



pBLUESCRIPT TM



expression







H0233
Human Fetal Heart, Differential
Human Fetal Heart
Heart


pBLUESCRIPT TM



(Adult-Specific)







H0234
human colon cancer, metastatic to
Human Colon Cancer,
Liver


pBLUESCRIPT TM



liver, differentially expressed
metasticized to liver






H0235
Human colon cancer, metaticized
Human Colon Cancer,
Liver


pBLUESCRIPT TM



to liver, subtraction
metasticized to liver






H0238
Human Myometrium Leiomyoma
Human Myometrium
Uterus

disease
UNI-ZAP TM




Leiomyoma



XR


H0239
Human Kidney Tumor
Human Kidney Tumor
Kidney

disease
UNI-ZAP TM








XR


H0240
C7MCF7 cell line, estrogen
C7MCF7 Cell Line,
Breast
Cell Line

UNI-ZAP TM



treated, Differential
estrogen treated



XR


H0241
C7MCF7 cell line, estrogen
C7MCF7 Cell Line,
Breast
Cell Line

UNI-ZAP TM



treated, subtraction
estrogen treated



XR


H0242
Human Fetal Heart, Differential
Human Fetal Heart
Heart


pBLUESCRIPT TM



(Fetal-Specific)







H0244
Human 8 Week Whole Embryo,
Human 8 Week Old
Embryo


UNI-ZAP TM



subtracted
Embryo



XR


H0246
Human Fetal Liver-Enzyme
Human Fetal Liver
Liver


UNI-ZAP TM



subtraction




XR


H0247
Human Membrane Bound
Human Membrane Bound
Blood
Cell Line

UNI-ZAP TM



Polysomes-Enzyme Subtraction
Polysomes



XR


H0249
HE7, subtracted by hybridization
Human Whole 7 Week Old
Embryo


UNI-ZAP TM



with E7 cDNA
Embryo



XR


H0250
Human Activated Monocytes
Human Monocytes



UNI-ZAP TM








XR


H0251
Human Chondrosarcoma
Human Chondrosarcoma
Cartilage

disease
UNI-ZAP TM








XR


H0252
Human Osteosarcoma
Human Osteosarcoma
Bone

disease
UNI-ZAP TM








XR


H0253
Human adult testis, large inserts
Human Adult Testis
Testis


UNI-ZAP TM








XR


H0254
Breast Lymph node cDNA library
Breast Lymph Node
Lymph Node


UNI-ZAP TM








XR


H0255
breast lymph node CDNA library
Breast Lymph Node
Lymph Node


LAMBDA








ZAP TM II


H0256
HL-60, unstimulated
Human HL-60 Cells,
Blood
Cell Line

UNI-ZAP TM




unstimulated



XR


H0257
HL-60, PMA 4H
HL-60 Cells, PMA
Blood
Cell Line

UNI-ZAP TM




stimulated 4H



XR


H0261
H. cerebellum, Enzyme
Human Cerebellum
Brain


UNI-ZAP TM



subtracted




XR


H0263
human colon cancer
Human Colon Cancer
Colon

disease
LAMBDA








ZAP TM II


H0264
human tonsils
Human Tonsil
Tonsil


UNI-ZAP TM








XR


H0265
Activated T-Cell
T-Cells
Blood
Cell Line

UNI-ZAP TM



(12 hs)/Thiouridine labelledEco




XR


H0266
Human Microvascular
HMEC
Vein
Cell Line

LAMBDA



Endothelial Cells, fract. A




ZAP TM II


H0267
Human Microvascular
HMEC
Vein
Cell Line

LAMBDA



Endothelial Cells, fract. B




ZAP TM II


H0268
Human Umbilical Vein
HUVE Cells
Umbilical
Cell Line

LAMBDA



Endothelial Cells, fract. A

vein


ZAP TM II


H0269
Human Umbilical Vein
HUVE Cells
Umbilical
Cell Line

LAMBDA



Endothelial Cells, fract. B

vein


ZAP TM II


H0270
HPAS (human pancreas,
Human Pancreas
Pancreas


UNI-ZAP TM



subtracted)




XR


H0271
Human Neutrophil, Activated
Human Neutrophil-
Blood
Cell Line

UNI-ZAP TM




Activated



XR


H0272
HUMAN TONSILS, FRACTION 2
Human Tonsil
Tonsil


UNI-ZAP TM








XR


H0274
Human Adult Spleen, fractionII
Human Adult Spleen
Spleen


UNI-ZAP TM








XR


H0275
Human Infant Adrenal Gland,
Human Infant Adrenal
Adrenal


pBLUESCRIPT TM



Subtracted
Gland
gland





H0279
K562 cells
K562 Cell line
cell line
Cell Line

ZAP








EXPRESS TM


H0280
K562 + PMA (36 hrs)
K562 Cell line
cell line
Cell Line

ZAP








EXPRESS TM


H0281
Lymph node, abnorm. cell line
Lymph Node, abnormal cell
Lymph Node
Cell Line

ZAP



(ATCC TM #7225)
line



EXPRESS TM


H0282
HBGB''s differential
Human Primary Breast
Breast


UNI-ZAP TM



consolidation
Cancer



XR


H0284
Human OB MG63 control
Human Osteoblastoma
Bone
Cell Line

UNI-ZAP TM



fraction I
MG63 cell line



XR


H0286
Human OB MG63 treated (10 nM
Human Osteoblastoma
Bone
Cell Line

UNI-ZAP TM



E2) fraction I
MG63 cell line



XR


H0288
Human OB HOS control fraction I
Human Osteoblastoma
Bone
Cell Line

UNI-ZAP TM




HOS cell line



XR


H0290
Human OB HOS treated (1 nM
Human Osteoblastoma
Bone
Cell Line

UNI-ZAP TM



E2) fraction I
HOS cell line



XR


H0292
Human OB HOS treated (10 nM
Human Osteoblastoma
Bone
Cell Line

UNI-ZAP TM



E2) fraction I
HOS cell line



XR


H0293
WI 38 cells




UNI-ZAP TM








XR


H0294
Amniotic Cells - TNF induced
Amniotic Cells - TNF
Placenta
Cell Line

UNI-ZAP TM




induced



XR


H0295
Amniotic Cells - Primary Culture
Amniotic Cells - Primary
Placenta
Cell Line

UNI-ZAP TM




Culture



XR


H0298
HCBB''s differential
CAMA1Ee Cell Line
Breast
Cell Line

UNI-ZAP TM



consolidation




XR


H0299
HCBA''s differential
CAMA1Ee Cell Line
Breast
Cell Line

UNI-ZAP TM



consolidation




XR


H0300
CD34 positive cells (Cord Blood)
CD34 Positive Cells
Cord Blood


ZAP








EXPRESS TM


H0305
CD34 positive cells (Cord Blood)
CD34 Positive Cells
Cord Blood


ZAP








EXPRESS TM


H0306
CD34 depleted Buffy Coat (Cord
CD34 Depleted Buffy Coat
Cord Blood


ZAP



Blood)
(Cord Blood)



EXPRESS TM


H0309
Human Chronic Synovitis
Synovium, Chronic
Synovium

disease
UNI-ZAP TM




Synovitis/Osteoarthritis



XR


H0310
human caudate nucleus
Brain
Brain


UNI-ZAP TM








XR


H0313
human pleural cancer
pleural cancer


disease
pBLUESCRIPT TM


H0316
HUMAN STOMACH
Human Stomach
Stomach


UNI-ZAP TM








XR


H0318
HUMAN B CELL LYMPHOMA
Human B Cell Lymphoma
Lymph Node

disease
UNI-ZAP TM








XR


H0320
Human frontal cortex
Human Frontal Cortex
Brain


UNI-ZAP TM








XR


H0321
HUMAN SCHWANOMA
Schwanoma
Nerve

disease
UNI-ZAP TM








XR


H0327
human corpus colosum
Human Corpus Callosum
Brain


UNI-ZAP TM








XR


H0328
human ovarian cancer
Ovarian Cancer
Ovary

disease
UNI-ZAP TM








XR


H0329
Dermatofibrosarcoma
Dermatofibrosarcoma
Skin

disease
UNI-ZAP TM



Protuberance
Protuberans



XR


H0330
HCBB''s Subtractive (-mito
CAMA1Ee Cell Line
Breast
Cell Line

UNI-ZAP TM



genes)




XR


H0331
Hepatocellular Tumor
Hepatocellular Tumor
Liver

disease
LAMBDA








ZAP TM II


H0333
Hemangiopericytoma
Hemangiopericytoma
Blood vessel

disease
LAMBDA








ZAP TM II


H0334
Kidney cancer
Kidney Cancer
Kidney

disease
UNI-ZAP TM








XR


H0339
Duodenum
Duodenum



UNI-ZAP TM








XR


H0340
Corpus Callosum
Corpus Collosum-93052



UNI-ZAP TM








XR


H0341
Bone Marrow Cell Line (RS4; 11)
Bone Marrow Cell Line
Bone
Cell Line

UNI-ZAP TM




RS4; 11
Marrow


XR


H0342
Lingual Gyrus
Lingual Gyrus
Brain


UNI-ZAP TM








XR


H0343
stomach cancer (human)
Stomach Cancer - 5383A


disease
UNI-ZAP TM




(human)



XR


H0344
Adipose tissue (human)
Adipose - 6825A (human)



UNI-ZAP TM








XR


H0345
SKIN
Skin - 4000868H
Skin


UNI-ZAP TM








XR


H0346
Brain-medulloblastoma
Brain (Medulloblastoma)-
Brain

disease
UNI-ZAP TM




9405C006R



XR


H0349
human adult liver cDNA library
Human Adult Liver
Liver


pCMVSport 1


H0350
Human Fetal Liver, mixed 10 &
Human Fetal Liver, mixed
Liver


UNI-ZAP TM



14 week
10&14 Week



XR


H0351
Glioblastoma
Glioblastoma
Brain

disease
UNI-ZAP TM








XR


H0352
wilm''s tumor
Wilm''s Tumor


disease
UNI-ZAP TM








XR


H0354
Human Leukocytes
Human Leukocytes
Blood
Cell Line

pCMVSport 1


H0355
Human Liver
Human Liver, normal Adult



pCMVSport 1


H0356
Human Kidney
Human Kidney
Kidney


pCMVSport 1


H0357
H. Normalized Fetal Liver, II
Human Fetal Liver
Liver


UNI-ZAP TM








XR


H0359
KMH2 cell line
KMH2



ZAP








EXPRESS TM


H0360
Hemangiopericytoma
Hemangiopericytoma


disease



H0361
Human rejected kidney
Human Rejected Kidney


disease
pBLUESCRIPT TM


H0362
HeLa cell line
HELA CELL LINE



pSport1


H0363
Human Brain Medulla, subtracted
Human Brain Medulla



pBLUESCRIPT TM


H0364
Human Osteoclastoma, excised
Human Osteoclastoma


disease
pBLUESCRIPT TM


H0365
Osteoclastoma-normalized B
Human Osteoclastoma


disease
UNI-ZAP TM








XR


H0366
L428 cell line
L428



ZAP








EXPRESS TM


H0369
H. Atrophic Endometrium
Atrophic Endometrium and



UNI-ZAP TM




myometrium



XR


H0370
H. Lymph node breast Cancer
Lymph node with Met.


disease
UNI-ZAP TM




Breast Cancer



XR


H0371
Eosinophils-Hypereosinophilia
Eosinophils-


disease
UNI-ZAP TM



patient
Hypereosinophilia patient



XR


H0372
Human Testes
Human Testes
Testis


pCMVSport 1


H0373
Human Heart
Human Adult Heart
Heart


pCMVSport 1


H0374
Human Brain
Human Brain



pCMVSport 1


H0375
Human Lung
Human Lung



pCMVSport 1


H0376
Human Spleen
Human Adult Spleen
Spleen


pCMVSport 1


H0379
Human Tongue, frac 1
Human Tongue



pSport1


H0380
Human Tongue, frac 2
Human Tongue



pSport1


H0381
Bone Cancer
Bone Cancer


disease
UNI-ZAP TM








XR


H0383
Human Prostate BPH, re-excision
Human Prostate BPH



UNI-ZAP TM








XR


H0384
Brain, Kozak
Human Brain



pCMVSport 1


H0385
H. Leukocytes, Kozak
Human Leukocytes
Blood
Cell Line

pCMVSport 1


H0386
Leukocyte and Lung; 4 screens
Human Leukocytes
Blood
Cell Line

pCMVSport 1


H0388
Human Rejected Kidney, 704 re-
Human Rejected Kidney


disease
pBLUESCRIPT TM



excision







H0389
H. Brain, X-Chromosome
Human Brain



pCMVSport 1



hybridization







H0390
Human Amygdala Depression, re-
Human Amygdala


disease
pBLUESCRIPT TM



excision
Depression






H0391
H. Meniingima, M6
Human Meningima
brain


pSport1


H0392
H. Meningima, M1
Human Meningima
brain


pSport1


H0393
Fetal Liver, subtraction II
Human Fetal Liver
Liver


pBLUESCRIPT TM


H0394
A-14 cell line
Redd-Sternberg cell



ZAP








EXPRESS TM


H0395
A1-CELL LINE
Redd-Sternberg cell



ZAP








EXPRESS TM


H0396
L1 Cell line
Redd-Sternberg cell



ZAP








EXPRESS TM


H0398
Human Newborn Bladder
Human Newborn Bladder



pBLUESCRIPT TM


H0399
Human Kidney Cortex, re-rescue
Human Kidney Cortex



LAMBDA








ZAP TM II


H0400
Human Striatum Depression, re-
Human Brain, Striatum
Brain


LAMBDA



rescue
Depression



ZAP TM II


H0401
Human Pituitary, subtracted V
Human Pituitary



pBLUESCRIPT TM


H0402
CD34 depleted Buffy Coat (Cord
CD34 Depleted Buffy Coat
Cord Blood


ZAP



Blood), re-excision
(Cord Blood)



EXPRESS TM


H0403
H. Umbilical Vein Endothelial
HUVE Cells
Umbilical
Cell Line

UNI-ZAP TM



Cells, IL4 induced

vein


XR


H0404
H. Umbilical Vein endothelial
HUVE Cells
Umbilical
Cell Line

UNI-ZAP TM



cells, uninduced

vein


XR


H0405
Human Pituitary, subtracted VI
Human Pituitary



pBLUESCRIPT TM


H0406
H Amygdala Depression,
Human Amygdala



UNI-ZAP TM



subtracted
Depression



XR


H0408
Human kidney Cortex, subtracted
Human Kidney Cortex



pBLUESCRIPT TM


H0409
H. Striatum Depression,
Human Brain, Striatum
Brain


pBLUESCRIPT TM



subtracted
Depression






H0410
H. Male bladder, adult
H Male Bladder, Adult
Bladder


pSport1


H0411
H Female Bladder, Adult
Human Female Adult
Bladder


pSport1




Bladder






H0412
Human umbilical vein endothelial
HUVE Cells
Umbilical
Cell Line

pSport1



cells, IL-4 induced

vein





H0413
Human Umbilical Vein
HUVE Cells
Umbilical
Cell Line

pSport1



Endothelial Cells, uninduced

vein





H0414
Ovarian Tumor I, OV5232
Ovarian Tumor, OV5232
Ovary

disease
pSport1


H0415
H. Ovarian Tumor, II, OV5232
Ovarian Tumor, OV5232
Ovary

disease
pCMVSport 2.0


H0416
Human Neutrophils, Activated,
Human Neutrophil -
Blood
Cell Line

pBLUESCRIPT TM



re-excision
Activated






H0417
Human Pituitary, subtracted VIII
Human Pituitary



pBLUESCRIPT TM


H0418
Human Pituitary, subtracted VII
Human Pituitary



pBLUESCRIPT TM


H0419
Bone Cancer, re-excision
Bone Cancer



UNI-ZAP TM








XR


H0421
Human Bone Marrow, re-excision
Bone Marrow



pBLUESCRIPT TM


H0422
T-Cell PHA 16 hrs
T-Cells
Blood
Cell Line

pSport1


H0423
T-Cell PHA 24 hrs
T-Cells
Blood
Cell Line

pSport1


H0424
Human Pituitary, subt IX
Human Pituitary



pBLUESCRIPT TM


H0427
Human Adipose
Human Adipose, left



pSport1




hiplipoma






H0428
Human Ovary
Human Ovary Tumor
Ovary


pSport1


H0429
K562 + PMA (36 hrs), re-excision
K562 Cell line
cell line
Cell Line

ZAP








EXPRESS TM


H0431
H. Kidney Medulla, re-excision
Kidney medulla
Kidney


pBLUESCRIPT TM


H0432
H. Kidney Pyramid
Kidney pyramids
Kidney


pBLUESCRIPT TM


H0433
Human Umbilical Vein
HUVE Cells
Umbilical
Cell Line

pBLUESCRIPT TM



Endothelial cells, frac B, re-

vein






excision







H0434
Human Brain, striatum, re-
Human Brain, Striatum



pBLUESCRIPT TM



excision







H0435
Ovarian Tumor Oct. 3, 1995
Ovarian Tumor, OV350721
Ovary


pCMVSport 2.0


H0436
Resting T-Cell Library, II
T-Cells
Blood
Cell Line

pSport1


H0437
H Umbilical Vein Endothelial
HUVE Cells
Umbilical
Cell Line

LAMBDA



Cells, frac A, re-excision

vein


ZAP TM II


H0438
H. Whole Brain #2, re-excision
Human Whole Brain #2



ZAP








EXPRESS TM


H0439
Human Eosinophils
Eosinophils



pBLUESCRIPT TM


H0440
FGF enriched mixed library
Mixed libraries



pCMVSport 1


H0441
H. Kidney Cortex, subtracted
Kidney cortex
Kidney


pBLUESCRIPT TM


H0442
H. Striatum Depression, subt II
Human Brain, Striatum
Brain


pBLUESCRIPT TM




Depression






H0443
H. Adipose, subtracted
Human Adipose, left



pSport1




hiplipoma






H0444
Spleen metastic melanoma
Spleen, Metastic malignant
Spleen

disease
pSport1




melanoma






H0445
Spleen, Chronic lymphocytic
Human Spleen, CLL
Spleen

disease
pSport1



leukemia







H0447
Salivary gland, re-excision
Human Salivary Gland
Salivary


UNI-ZAP TM





gland


XR


H0448
Salivary gland, subtracted
Human Salivary Gland
Salivary


LAMBDA





gland


ZAP TM II


H0449
CD34+ cell, I
CD34 positive cells



pSport1


H0450
CD34+ cells, II
CD34 positive cells



pCMVSport 2.0


H0453
H. Kidney Pyramid, subtracted
Kidney pyramids
Kidney


pBLUESCRIPT TM


H0455
H. Striatum Depression, subt
Human Brain, Striatum
Brain


pBLUESCRIPT TM




Depression






H0456
H Kidney Cortex, subtracted III
Human Kidney Cortex



pBLUESCRIPT TM


H0457
Human Eosinophils
Human Eosinophils



pSport1


H0458
CD34+ cell, I, frac II
CD34 positive cells



pSport1


H0459
CD34+ cells, II, FRACTION 2
CD34 positive cells



pCMVSport 2.0


H0461
H. Kidney Medulla, subtracted
Kidney medulla
Kidney


pBLUESCRIPT TM


H0462
H. Amygdala Depression,

Brain


pBLUESCRIPT TM



subtracted







H0477
Human Tonsil, Lib 3
Human Tonsil
Tonsil


pSport1


H0478
Salivary Gland, Lib 2
Human Salivary Gland
Salivary


pSport1





gland





H0479
Salivary Gland, Lib 3
Human Salivary Gland
Salivary


pSport1





gland





H0480
L8 cell line
L8 cell line



ZAP








EXPRESS TM


H0483
Breast Cancer cell line, MDA 36
Breast Cancer Cell line,



pSport1




MDA 36






H0484
Breast Cancer Cell line,
Breast Cancer Cell line,



pSport1



angiogenic
Angiogenic, 36T3






H0485
Hodgkin''s Lymphoma I
Hodgkin''s Lymphoma I


disease
pCMVSport 2.0


H0486
Hodgkin''s Lymphoma II
Hodgkin''s Lymphoma II


disease
pCMVSport 2.0


H0487
Human Tonsils, lib I
Human Tonsils



pCMVSport 2.0


H0488
Human Tonsils, Lib 2
Human Tonsils



pCMVSport 2.0


H0489
Crohn''s Disease
Ileum
Intestine

disease
pSport1


H0490
H1-60, untreated, subtracted
Human HL-60 Cells,
Blood
Cell Line

UNI-ZAP TM




unstimulated



XR


H0491
HL-60, PMA 4 H, subtracted
HL-60 Cells, PMA
Blood
Cell Line

UNI-ZAP TM




stimulated 4 H



XR


H0492
HL-60, RA 4 h, Subtracted
HL-60 Cells, RA stimulated
Blood
Cell Line

UNI-ZAP TM




for 4 H



XR


H0493
HL-60, PMA 1 d, subtracted
HL-60 Cells, PMA
Blood
Cell Line

UNI-ZAP TM




stimulated for 1 day



XR


H0494
Keratinocyte
Keratinocyte



pCMVSport 2.0


H0497
HEL cell line
HEL cell line

HEL 92.1.7

pSport1


H0505
Human Astrocyte
Human Astrocyte



pSport1


H0506
Ulcerative Colitis
Colon
Colon


pSport1


H0509
Liver, Hepatoma
Human Liver, Hepatoma,
Liver

disease
pCMVSport 3.0




patient 8






H0510
Human Liver, normal
Human Liver, normal,
Liver


pCMVSport 3.0




Patient # 8






H0512
Keratinocyte, lib 3
Keratinocyte



pCMVSport 2.0


H0517
Nasal polyps
Nasal polyps



pCMVSport 2.0


H0518
pBMC stimulated w/poly I/C
pBMC stimulated with poly



pCMVSport 3.0




I/C






H0519
NTERA2, control
NTERA2, Teratocarcinoma



pCMVSport 3.0




cell line






H0520
NTERA2 + retinoic acid, 14 days
NTERA2, Teratocarcinoma



pSport1




cell line






H0521
Primary Dendritic Cells, lib 1
Primary Dendritic cells



pCMVSport 3.0


H0522
Primary Dendritic cells, frac 2
Primary Dendritic cells



pCMVSport 3.0


H0523
Primary Dendritic
Primary Dendritic cells



pSport1



cells, CapFinder2, frac 1







H0524
Primary Dendritic Cells,
Primary Dendritic cells



pSport1



CapFinder, frac 2







H0525
PCR, pBMC I/C treated
pBMC stimulated with poly



pCR II




I/C



[Invitrogen]


H0528
Poly[I]/Poly[C] Normal Lung
Poly[I]/Poly[C] Normal



pCMVSport 3.0



Fibroblasts
Lung Fibroblasts






H0529
Myoloid Progenitor Cell Line
TF-1 Cell Line; Myoloid



pCMVSport 3.0




progenitor cell line






H0530
Human Dermal Endothelial
Human Dermal Endothelial



pSport1



Cells, untreated
Cells; untreated






H0533
Human Stromal endometrial
Human Stromal



pSport1



fibroblasts, treated w/estradiol
endometrial fibroblasts,








treated wit






H0535
Human ovary tumor cell
Ovarian Tumor, OV350721
Ovary

disease
pSport1



OV350721







H0537
H. Primary Dendritic Cells, lib 3
Primary Dendritic cells



pCMVSport 2.0


H0538
Merkel Cells
Merkel cells
Lymph node


pSport1


H0539
Pancreas Islet Cell Tumor
Pancreas Islet Cell Tumour
Pancreas

disease
pSport1


H0540
Skin, burned
Skin, leg burned
Skin


pSport1


H0542
T Cell helper I
Helper T cell



pCMVSport 3.0


H0543
T cell helper II
Helper T cell



pCMVSport 3.0


H0544
Human endometrial stromal cells
Human endometrial stromal



pCMVSport 3.0




cells






H0545
Human endometrial stromal cells-
Human endometrial stromal



pCMVSport 3.0



treated with progesterone
cells-treated with proge






H0546
Human endometrial stromal cells-
Human endometrial stromal



pCMVSport 3.0



treated with estradiol
cells-treated with estra






H0547
NTERA2 teratocarcinoma cell
NTERA2, Teratocarcinoma



pSport1



line + retinoic acid (14 days)
cell line






H0548
Human Skin Fibroblasts, normal
Human Skin Fibroblasts



pBLUESCRIPT TM


H0549
H. Epididiymus, caput & corpus
Human Epididiymus, caput



UNI-ZAP TM




and corpus



XR


H0550
H. Epididiymus, cauda
Human Epididiymus, cauda



UNI-ZAP TM








XR


H0551
Human Thymus Stromal Cells
Human Thymus Stromal



pCMVSport 3.0




Cells






H0552
Signal trap, Femur Bone
Femur Bone marrow,



Other



Marrow, pooled
pooled from 8 male/female






H0553
Human Placenta
Human Placenta



pCMVSport 3.0


H0555
Rejected Kidney, lib 4
Human Rejected Kidney
Kidney

disease
pCMVSport 3.0


H0556
Activated T-
T-Cells
Blood
Cell Line

UNI-ZAP TM



cell(12 h)/Thiouridine-re-excision




XR


H0559
HL-60, PMA 4 H, re-excision
HL-60 Cells, PMA
Blood
Cell Line

UNI-ZAP TM




stimulated 4 H



XR


H0560
KMH2
KMH2



pCMVSport 3.0


H0561
L428
L428



pCMVSport 3.0


H0562
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



c5-11-26







H0563
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



50021F







H0564
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



C5001F







H0565
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



100024F







H0566
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



c50F







H0567
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



A5002F







H0569
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



CO







H0570
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



C500H







H0571
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



C500HE







H0572
Human Fetal Brain, normalized
Human Fetal Brain



pCMVSport 2.0



AC5002







H0574
Hepatocellular Tumor; re-
Hepatocellular Tumor
Liver

disease
LAMBDA



excision




ZAP TM II


H0575
Human Adult Pulmonary; re-
Human Adult Pulmonary
Lung


UNI-ZAP TM



excision




XR


H0576
Resting T-Cell; re-excision
T-Cells
Blood
Cell Line

LAMBDA








ZAP TM II


H0578
Human Fetal Thymus
Fetal Thymus
Thymus


pSport1


H0579
Pericardium
Pericardium
Heart


pSport1


H0580
Dendritic cells, pooled
Pooled dendritic cells



pCMVSport 3.0


H0581
Human Bone Marrow, treated
Human Bone Marrow
Bone


pCMVSport 3.0





Marrow





H0583
B Cell lymphoma
B Cell Lymphoma
B Cell

disease
pCMVSport 3.0


H0584
Activated T-cells, 24 hrs, re-
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



excision




XR


H0585
Activated T-Cells, 12 hrs, re-
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



excision




XR


H0586
Healing groin wound, 6.5 hours
healing groin wound, 6.5
groin

disease
pCMVSport 3.0



post incision
hours post incision-2/






H0587
Healing groin wound; 7.5 hours
Groin-Feb. 19, 1997
groin

disease
pCMVSport 3.0



post incision







H0589
CD34 positive cells (cord
CD34 Positive Cells
Cord Blood


ZAP



blood), re-ex




EXPRESS TM


H0590
Human adult small intestine, re-
Human Adult Small
Small Int.


UNI-ZAP TM



excision
Intestine



XR


H0591
Human T-cell lymphoma; re-
T-Cell Lymphoma
T-Cell

disease
UNI-ZAP TM



excision




XR


H0592
Healing groin wound - zero hr
HGS wound healing


disease
pCMVSport 3.0



post-incision (control)
project; abdomen






H0593
Olfactory epithelium; nasalcavity
Olfactory epithelium from



pCMVSport 3.0




roof of left nasal cacit






H0594
Human Lung Cancer; re-excision
Human Lung Cancer
Lung

disease
LAMBDA








ZAP TM II


H0595
Stomach cancer (human); re-
Stomach Cancer-5383A


disease
UNI-ZAP TM



excision
(human)



XR


H0596
Human Colon Cancer; re-excision
Human Colon Cancer
Colon


LAMBDA








ZAP TM II


H0597
Human Colon; re-excision
Human Colon



LAMBDA








ZAP TM II


H0598
Human Stomach; re-excision
Human Stomach
Stomach


UNI-ZAP TM








XR


H0599
Human Adult Heart; re-excision
Human Adult Heart
Heart


UNI-ZAP TM








XR


H0600
Healing Abdomen wound;
Abdomen


disease
pCMVSport 3.0



70&90 min








post incision







H0601
Healing Abdomen Wound; 15
Abdomen


disease
pCMVSport 3.0



days post incision







H0602
Healing Abdomen Wound; 21&29
Abdomen


disease
pCMVSport 3.0



days post incision







H0604
Human Pituitary, re-excision
Human Pituitary



pBLUESCRIPT TM


H0606
Human Primary Breast Cancer; re-
Human Primary Breast
Breast

disease
UNI-ZAP TM



excision
Cancer



XR


H0607
H. Leukocytes, normalized cot
H. Leukocytes



pCMVSport 1



50A3







H0608
H. Leukocytes, control
H. Leukocytes



pCMVSport 1


H0609
H. Leukocytes, normalized
H. Leukocytes



pCMVSport 1



cot >500A







H0610
H. Leukocytes, normalized cot
H. Leukocytes



pCMVSport 1



5A







H0611
H. Leukocytes, normalized cot
H. Leukocytes



pCMVSport 1



500 B







H0612
H. Leukocytes, normalized cot 50 B
H. Leukocytes



pCMVSport 1


H0613
H. Leukocytes, normalized cot 5B
H. Leukocytes



pCMVSport 1


H0614
H. Leukocytes, normalized cot
H. Leukocytes



pCMVSport 1



500 A







H0615
Human Ovarian Cancer
Ovarian Cancer
Ovary

disease
UNI-ZAP TM



Reexcision




XR


H0616
Human Testes, Reexcision
Human Testes
Testis


UNI-ZAP TM








XR


H0617
Human Primary Breast Cancer
Human Primary Breast
Breast

disease
UNI-ZAP TM



Reexcision
Cancer



XR


H0618
Human Adult Testes, Large
Human Adult Testis
Testis


UNI-ZAP TM



Inserts, Reexcision




XR


H0619
Fetal Heart
Human Fetal Heart
Heart


UNI-ZAP TM








XR


H0620
Human Fetal Kidney; Reexcision
Human Fetal Kidney
Kidney


UNI-ZAP TM








XR


H0622
Human Pancreas Tumor;
Human Pancreas Tumor
Pancreas

disease
UNI-ZAP TM



Reexcision




XR


H0623
Human Umbilical Vein;
Human Umbilical Vein
Umbilical


UNI-ZAP TM



Reexcision
Endothelial Cells
vein


XR


H0624
12 Week Early Stage Human II;
Twelve Week Old Early
Embryo


UNI-ZAP TM



Reexcision
Stage Human



XR


H0625
Ku 812F Basophils Line
Ku 812F Basophils



pSport1


H0626
Saos2 Cells; Untreated
Saos2 Cell Line; Untreated



pSport1


H0627
Saos2 Cells; Vitamin D3 Treated
Saos2 Cell Line; Vitamin



pSport1




D3 Treated






H0628
Human Pre-Differentiated
Human Pre-Differentiated



UNI-ZAP TM



Adipocytes
Adipocytes



XR


H0629
Human Leukocyte, control #2
Human Normalized



pCMVSport 1




leukocyte






H0630
Human Leukocytes, normalized
Human Normalized



pCMVSport 1



control #4
leukocyte






H0631
Saos2, Dexamethosome Treated
Saos2 Cell Line;



pSport1




Dexamethosome Treated






H0632
Hepatocellular Tumor; re-excision
Hepatocellular Tumor
Liver


LAMBDA








ZAP TM II


H0633
Lung Carcinoma A549 TNFalpha
TNFalpha activated A549-


disease
pSport1



activated
Lung Carcinoma






H0634
Human Testes Tumor, re-excision
Human Testes Tumor
Testis

disease
UNI-ZAP TM








XR


H0635
Human Activated T-Cells, re-
Activated T-Cells
Blood
Cell Line

UNI-ZAP TM



excision




XR


H0637
Dendritic Cells From CD34 Cells
Dentritic cells from CD34



pSport1




cells






H0638
CD40 activated monocyte
CD40 activated monocyte



pSport1



dendridic cells
dendridic cells






H0639
Ficolled Human Stromal Cells,
Ficolled Human Stromal



Other



5Fu treated
Cells, 5Fu treated






H0640
FICOLL TMed Human Stromal
FICOLL TMed Human



Other



Cells, Untreated
Stromal Cells, Untreated






H0641
LPS activated derived dendritic
LPS activated monocyte



pSport1



cells
derived dendritic cells






H0642
Hep G2 Cells, lambda library
Hep G2 Cells



Other


H0643
Hep G2 Cells, PCR library
Hep G2 Cells



Other


H0644
Human Placenta (re-excision)
Human Placenta
Placenta


UNI-ZAP TM








XR


H0645
Fetal Heart, re-excision
Human Fetal Heart
Heart


UNI-ZAP TM








XR


H0646
Lung, Cancer (4005313 A3):
Metastatic squamous cell



pSport1



Invasive Poorly Differentiated
lung carcinoma, poorly di







Lung Adenocarcinoma,







H0647
Lung, Cancer (4005163 B7):
Invasive poorly


disease
pSport1



Invasive, Poorly Diff.
differentiated lung







Adenocarcinoma, Metastatic
adenocarcinoma






H0648
Ovary, Cancer: (4004562 B6)
Papillary Cstic neoplasm of


disease
pSport1



Papillary Serous Cystic
low malignant potentia







Neoplasm, Low Malignant Pot







H0649
Lung, Normal: (4005313 B1)
Normal Lung



pSport1


H0650
B-Cells
B-Cells



pCMVSport 3.0


H0651
Ovary, Normal: (9805C040R)
Normal Ovary



pSport1


H0652
Lung, Normal: (4005313 B1)
Normal Lung



pSport1


H0653
Stromal Cells
Stromal Cells



pSport1


H0654
Lung, Cancer: (4005313 A3)
Metastatic Squamous cell



Other



Invasive Poorly-differentiated
lung Carcinoma poorly dif







Metastatic lung adenoc







H0656
B-cells (unstimulated)
B-cells (unstimulated)



pSport1


H0657
B-cells (stimulated)
B-cells (stimulated)



pSport1


H0658
Ovary, Cancer (9809C332):
9809C332-Poorly
Ovary &

disease
pSport1



Poorly differentiated
differentiate
Fallopian






adenocarcinoma

Tubes





H0659
Ovary, Cancer (15395A1F):
Grade II Papillary
Ovary

disease
pSport1



Grade II Papillary Carcinoma
Carcinoma, Ovary






H0660
Ovary, Cancer: (15799A1F)
Poorly differentiated


disease
pSport1



Poorly differentiated carcinoma
carcinoma, ovary






H0661
Breast, Cancer: (4004943 A5)
Breast cancer


disease
pSport1


H0662
Breast, Normal: (4005522B2)
Normal Breast-
Breast


pSport1




#4005522(B2)






H0663
Breast, Cancer: (4005522 A2)
Breast Cancer-
Breast

disease
pSport1




#4005522(A2)






H0664
Breast, Cancer: (9806C012R)
Breast Cancer
Breast

disease
pSport1


H0665
Stromal cells 3.88
Stromal cells 3.88



pSport1


H0666
Ovary, Cancer: (4004332 A2)
Ovarian Cancer, Sample


disease
pSport1




#4004332A2






H0667
Stromal cells(HBM3.18)
Stromal cell(HBM 3.18)



pSport1


H0668
stromal cell clone 2.5
stromal cell clone 2.5



pSport1


H0669
Breast, Cancer: (4005385 A2)
Breast Cancer (4005385A2)
Breast


pSport1


H0670
Ovary, Cancer(4004650 A3):
Ovarian Cancer-



pSport1



Well-Differentiated
4004650A3







Micropapillary Serous Carcinoma







H0671
Breast, Cancer: (9802C02OE)
Breast Cancer-Sample #



pSport1




9802C02OE






H0672
Ovary, Cancer: (4004576 A8)
Ovarian
Ovary


pSport1




Cancer(4004576A8)






H0673
Human Prostate Cancer, Stage
Human Prostate Cancer,
Prostate


UNI-ZAP TM



B2; re-excision
stage B2



XR


H0674
Human Prostate Cancer, Stage C;
Human Prostate Cancer,
Prostate


UNI-ZAP TM



re-excission
stage C



XR


H0675
Colon, Cancer: (9808C064R)
Colon Cancer 9808C064R



pCMVSport 3.0


H0676
Colon, Cancer: (9808C064R)-
Colon Cancer 9808C064R



pCMVSport 3.0



total RNA







H0677
TNFR degenerate oligo
B-Cells



pCR II








[Invitrogen]


H0678
screened clones from placental
Placenta
Placenta


Other



library







H0679
screened clones from Tonsil
Human Tonsils



Other



library







H0682
Serous Papillary Adenocarcinoma
serous papillary



pCMVSport 3.0




adenocarcinoma








(9606G304SPA3B)






H0683
Ovarian Serous Papillary
Serous papillary



pCMVSport 3.0



Adenocarcinoma
adenocarcinoma, stage 3C








(9804G01






H0684
Serous Papillary Adenocarcinoma
Ovarian Cancer-9810G606
Ovaries


pCMVSport 3.0


H0685
Adenocarcinoma of Ovary,
Adenocarcinoma of Ovary,



pCMVSport 3.0



Human Cell Line, # OVCAR-3
Human Cell Line, #








OVCAR-






H0686
Adenocarcinoma of Ovary,
Adenocarcinoma of Ovary,



pCMVSport 3.0



Human Cell Line
Human Cell Line, # SW-








626






H0687
Human normal
Human normal
Ovary


pCMVSport 3.0



ovary(#9610G215)
ovary(#9610G215)






H0688
Human Ovarian
Human Ovarian



pCMVSport 3.0



Cancer(#9807G017)
cancer(#9807G017), mRNA








from Maura Ru






H0689
Ovarian Cancer
Ovarian Cancer,



pCMVSport 3.0




#9806G019






H0690
Ovarian Cancer, # 9702G001
Ovarian Cancer,



pCMVSport 3.0




#9702G001






H0691
Normal Ovary, #9710G208
normal ovary, #9710G208



pCMVSport 3.0


H0692
BLyS Receptor from Expression
B Cell Lymphoma
B Cell


pCMVSport 3.0



Cloning







H0693
Normal Prostate #ODQ3958EN
Normal Prostate Tissue #



pCMVSport 3.0




ODQ3958EN






H0694
Prostate gland adenocarcinoma
Prostate gland,
prostate


pCMVSport 3.0




adenocarcinoma, mod/diff,
gland







gleason






H0695
mononucleocytes from patient
mononucleocytes from



pCMVSport 3.0




patient at Shady Grove








Hospit






L0002
Atrium cDNA library Human








heart







L0004
CLONTECH TM HL 1065a







L0005
CLONTECH TM human aorta








polyA+ mRNA (#6572)







L0009
EST from 8p21.3-p22







L0012
HDMEC cDNA library







L0015
Human







L0017
Human (J. Swensen)







L0018
Human (M. Lovett)







L0021
Human adult (K. Okubo)







L0022
Human adult lung 3′′ directed








MboI cDNA







L0023
human adult testis







L0024
Human brain A RSanders







L0025
Human brain striatum







L0032
Human chromosome 12p cDNAs







L0033
Human chromosome 13q14








cDNA







L0034
Human chromosome 14







L0038
Human chromosome 6







L0040
Human colon mucosa







L0041
Human epidermal keratinocyte







L0045
Human keratinocyte differential








display (B. Lin)







L0051
Human mRNA (Tripodis and








Ragoussis)







L0052
Human normalized K562-cDNA







L0053
Human pancreatic tumor







L0055
Human promyelocyte







L0060
Human thymus NSTH II







L0065
Liver HepG2 cell line.







L0070
Selected chromosome 21 cDNA








library







L0096
Subtracted human retina







L0097
Subtracted human retinal pigment








epithelium (RPE)







L0103
DKFZphamy1
amygdala






L0105
Human aorta polyA+ (TFujiwara)
aorta






L0109
Human brain cDNA
brain






L0114
Human fetal brain (R. L. Margolis)
brain






L0117
Human fetal brain cDNA
brain







(T. M. Gress)







L0118
Human fetal brain S. Meier-Ewert
brain






L0121
Stratagene catalog #936206
brain






L0126
Human fibroblast cDNA
fibroblast






L0130
Human hippocampus, Stratagene
hippocampus







catalog #936205







L0136
Human neuroepithelium
neuroepithelium







(N. Jiang)







L0138
Human normal gingiva
normal gingiva






L0140
Human pancreatic cancer
pancreatic cancer







(C Wallrapp)







L0141
Human pancreatic islet cell
pancreatic islet






L0142
Human placenta cDNA
placenta







(T Fujiwara)







L0143
Human placenta polyA+
placenta







(T Fujiwara)







L0145
Human retina (D. Swanson)
retina






L0146
Human fovea cDNA
retinal fovea






L0149
DKFZphsnu1
subthalamic nucleus






L0151
Human testis (C. De Smet)
testis






L0157
Human fetal brain (T Fujiwara)

brain





L0158
Human fetal brain Q Boqin

brain





L0162
Human brain frontal cortex
frontal cortex
brain





L0163
Human heart cDNA

heart






(Y Nakamura)







L0171
Human lung adenocarcinoma
lung adenocarcinoma

A549





A549







L0175
Human retina cell line ARPE-19
retina

ARPE-19




L0177
Human newborn melanocytes


Clonetics





(T. Vogt)


Corp. (San








Diego, CA)








strain #68 and








2486




L0182
Human HeLa (Y. Wang)


HeLa




L0183
Human HeLa cells (M. Lovett)


HeLa




L0185
Human immortalized fibroblasts


HS74 and its





(H. L. Ozer)


SV40-








transformed








sublines




L0186
Human salivary gland cell line
salivary gland

HSG





HSG







L0187
Human fibrosarcoma cell line
fibrosarcoma

HT1080





HT1080







L0194
Human pancreatic cancer cell line
pancreatic cancer

Patu 8988t





Patu 8988t







L0295
Human liver EST (Y. L. Yu)

liver





L0307
Human C3-A11N


C3-A11N;








clonally related








variant of OCI








LY8-C3P




L0309
Human E8CASS
breast adenocarcinoma

E8CASS;








variant of








MCF7




L0351
Infant brain, Bento Soares




BA, M13-








derived


L0352
Normalized infant brain, Bento




BA, M13-



Soares




derived


L0353
21q Placenta, F. Tassone and




pBLUESCRIPT TM



K. Gardiner







L0354
JG, Human foetal Kidney tissue




pBLUESCRIPT TM


L0355
P, Human foetal Brain Whole




pBLUESCRIPT TM



tissue







L0356
S, Human foetal Adrenals tissue




pBLUESCRIPT TM


L0357
V, Human Placenta tissue




Bluescript KS








II+


L0361
STRATAGENE TM ovary

ovary


pBLUESCRIPT TM



(#937217)




SK


L0362
STRATAGENE TM ovarian cancer




pBLUESCRIPT TM



(#937219)




SK−


L0363
NCI_CGAP_GC2
germ cell tumor



pBLUESCRIPT TM








SK−


L0364
NCI_CGAP_GC5
germ cell tumor



pBLUESCRIPT TM








SK−


L0365
NCI_CGAP_Phe1
pheochromocytoma



pBLUESCRIPT TM








SK−


L0366
STRATAGENE TM schizo brain
schizophrenic brain S-11



pBLUESCRIPT TM



S11
frontal lobe



SK−


L0367
NCI_CGAP_Sch1
Schwannoma tumor



pBLUESCRIPT TM








SK−


L0368
NCI_CGAP_SS1
synovial sarcoma



pBLUESCRIPT TM








SK−


L0369
NCI_CGAP_AA1
adrenal adenoma
adrenal gland


pBLUESCRIPT TM








SK−


L0370
Johnston frontal cortex
pooled frontal lobe
brain


pBLUESCRIPT TM








SK−


L0371
NCI_CGAP_Br3
breast tumor
breast


pBLUESCRIPT TM








SK−


L0372
NCI_CGAP_Co12
colon tumor
colon


pBLUESCRIPT TM








SK−


L0373
NCI_CGAP_Co11
tumor
colon


pBLUESCRIPT TM








SK−


L0374
NCI_CGAP_Co2
tumor
colon


pBLUESCRIPT TM








SK−


L0375
NCI_CGAP_Kid6
kidney tumor
kidney


pBLUESCRIPT TM








SK−


L0376
NCI_CGAP_Lar1
larynx
larynx


pBLUESCRIPT TM








SK−


L0377
NCI_CGAP_HN2
squamous cell carcinoma
larynx


pBLUESCRIPT TM




from vocal cord



SK−


L0378
NCI_CGAP_Lu1
lung tumor
lung


pBLUESCRIPT TM








SK−


L0379
NCI_CGAP_Lym3
lymphoma
lymph node


pBLUESCRIPT TM








SK−


L0380
NCI_CGAP_HN1
squamous cell carcinoma
lymph node


pBLUESCRIPT TM








SK−


L0381
NCI_CGAP_HN4
squamous cell carcinoma
pharynx


pBLUESCRIPT TM








SK−


L0382
NCI_CGAP_Pr25
epithelium (cell line)
prostate


pBLUESCRIPT TM








SK−


L0383
NCI_CGAP_Pr24
invasive tumor (cell line)
prostate


pBLUESCRIPT TM








SK−


L0384
NCI_CGAP_Pr23
prostate tumor
prostate


pBLUESCRIPT TM








SK−


L0385
NCI_CGAP_Gas1
gastric tumor
stomach


pBLUESCRIPT TM








SK−


L0386
NCI_CGAP_HN3
squamous cell carcinoma
tongue


pBLUESCRIPT TM




from base of tongue



SK−


L0387
NCI_CGAP_GCB0
germinal center B-cells
tonsil


pBLUESCRIPT TM








SK−


L0388
NCI_CGAP_HN6
normal gingiva (cell line



pBLUESCRIPT TM




from immortalized kerati



SK−


L0389
NCI_CGAP_HN5
normal gingiva (cell line



pBLUESCRIPT TM




from primary keratinocyt



SK−


L0393
B, Human Liver tissue




gt11


L0394
H, Human adult Brain Cortex




gt11



tissue







L0404
b4HB3MA Cot109 + 103 + 85-Bio




Lafmid A


L0405
b4HB3MA Cot109 + 103-Bio




Lafmid A


L0411
1-NIB




Lafmid BA


L0414
b4HB3MA




Lafmid BA


L0415
b4HB3MA Cot8-HAP-Ft




Lafmid BA


L0416
b4HB3MA-Cot0.38-HAP-B




Lafmid BA


L0417
b4HB3MA-Cot0.38-HAP-Ft-6




Lafmid BA


L0418
b4HB3MA-Cot109 + 10-Bio




Lafmid BA


L0419
b4HB3MA-Cot109 + 103 + 85-Bio




Lafmid BA


L0422
b4HB3MA-Cot12-HAP-B




Lafmid BA


L0424
b4HB3MA-Cot14.5




Lafmid BA


L0425
b4HB3MA-Cot18-Bio




Lafmid BA


L0426
b4HB3MA-Cot51.5-HAP-Ft




Lafmid BA


L0427
b4HB3MA-FT20%-Biotin




Lafmid BA


L0428
Cot1374Ft-4HB3MA




Lafmid BA


L0430
Cot250Ft-b4HB3MA




Lafmid BA


L0433
HWM42YA




Lafmid BA


L0434
Infant brain library of Dr. M. Soares




Lafmid BA


L0435
Infant brain, LLNL array of Dr.




Lafmid BA



M. Soares 1NIB







L0437
N-b4HB3MA-Cot109




Lafmid BA


L0438
normalized infant brain cDNA
total brain
brain


Lafmid BA


L0439
Soares infant brain 1NIB

whole brain


Lafmid BA


L0441
2HB3MK




Lafmid BK


L0442
4HB3MK




Lafmid BK


L0443
b4HB3MK




Lafmid BK


L0446
N4HB3MK




Lafmid BK


L0447
NHB3MK




Lafmid BK


L0448
3HFLSK20




Lafmid K


L0451
N3HFLSK20




Lafmid K


L0453
BATM1




lambda gt10


L0454
CLONTECH TM adult human fat




lambda gt10



cell library HL1108A







L0455
Human retina cDNA randomly
retina
eye


lambda gt10



primed sublibrary







L0456
Human retina cDNA Tsp509I-
retina
eye


lambda gt10



cleaved sublibrary







L0457
multi-tissue normalized short-
multi-tissue
pooled


lambda gt10



fragment







L0459
Adult heart, CLONTECH TM




Lambda gt11


L0460
Adult heart, Lambda gt11




Lambda gt11


L0462
WATM1




lambda gt11


L0463
fetal brain cDNA
brain
brain


lambda gt11


L0465
TEST1, Human adult Testis




lambda nm1149



tissue







L0467
Fetal heart, Lambda ZAP Express




Lambda ZAP


L0468
HE6W




lambda zap


L0469
T, Human adult




Lambda Zap



Rhabdomyosarcoma cell-line







L0470
BL29 Burkitt''s lymphoma,




LAMBDA



Pascalis Sideras




ZAP TM II


L0471
Human fetal heart, LAMBDA




LAMBDA



ZAP TM Express




ZAP TM Express








(STRATAGENE TM)


L0475
KG1-a Lambda Zap Express


KG1-a

Lambda Zap



cDNA library




Express








(STRATAGENE TM)


L0476
Fetal brain, STRATAGENE TM




LAMBDA








ZAP TM II


L0477
HPLA CCLee
placenta



LAMBDA








ZAP TM II


L0480
STRATAGENE TM cat#937212




Lambda ZAP,



(1992)




pBLUESCRIPT TM








SK−


L0481
CD34 + DIRECTIONAL




LAMBDA








ZAP TM II


L0482
HT29M6




LAMBDA








ZAP TM II


L0483
Human pancreatic islet




LAMBDA








ZAP TM II


L0485
STRATAGENE TM Human
skeletal muscle
leg muscle


LAMBDA



skeletal muscle cDNA library,




ZAP TM II



cat. #936215.







L0486
Human promyelocytic HL60 cell


promyelocytic

LAMBDA



line (S. Herblot)


HL60 cell line

ZAP TM II


L0487
Human peripheral blood (Steve
whole peripheral blood



Lambda-Yes



Elledge)







L0492
Human Genomic




pAMP


L0493
NCI_CGAP_Ov26
papillary serous carcinoma
ovary


pAMP1


L0497
NCI_CGAP_HSC4
CD34+, CD38− from
bone marrow


pAMP1




normal bone marrow donor






L0498
NCI_CGAP_HSC3
CD34+, T negative, patient
bone marrow


pAMP1




with chronic myelogenou






L0499
NCI_CGAP_HSC2
stem cell 34+/38+
bone marrow


pAMP1


L0500
NCI_CGAP_Brn20
oligodendroglioma
brain


pAMP1


L0501
NCI_CGAP_Brn21
oligodendroglioma
brain


pAMP1


L0502
NCI_CGAP_Br15
adenocarcinoma
breast


pAMP1


L0503
NCI_CGAP_Br17
adenocarcinoma
breast


pAMP1


L0504
NCI_CGAP_Br13
breast carcinoma in situ
breast


pAMP1


L0505
NCI_CGAP_Br12
invasive carcinoma
breast


pAMP1


L0506
NCI_CGAP_Br16
lobullar carcinoma in situ
breast


pAMP1


L0507
NCI_CGAP_Br14
normal epithelium
breast


pAMP1


L0508
NCI_CGAP_Lu25
bronchioalveolar carcinoma
lung


pAMP1


L0509
NCI_CGAP_Lu26
invasive adenocarcinoma
lung


pAMP1


L0510
NCI_CGAP_Ov33
borderline ovarian
ovary


pAMP1




carcinoma






L0511
NCI_CGAP_Ov34
borderline ovarian
ovary


pAMP1




carcinoma






L0512
NCI_CGAP_Ov36
borderline ovarian
ovary


pAMP1




carcinoma






L0513
NCI_CGAP_Ov37
early stage papillary serous
ovary


pAMP1




carcinoma






L0514
NCI_CGAP_Ov31
papillary serous carcinoma
ovary


pAMP1


L0515
NCI_CGAP_Ov32
papillary serous carcinoma
ovary


pAMP1


L0516
Chromosome 19p12-p13.1 exon




pAMP10


L0517
NCI_CGAP_Pr1




pAMP10


L0518
NCI_CGAP_Pr2




pAMP10


L0519
NCI_CGAP_Pr3




pAMP10


L0520
NCI_CGAP_Alv1
alveolar



pAMP10




rhabdomyosarcoma






L0521
NCI_CGAP_Ew1
Ewing''s sarcoma



pAMP10


L0522
NCI_CGAP_Kid1
kidney



pAMP10


L0523
NCI_CGAP_Lip2
liposarcoma



pAMP10


L0524
NCI_CGAP_Li1
liver



pAMP10


L0525
NCI_CGAP_Li2
liver



pAMP10


L0526
NCI_CGAP_Pr12
metastatic prostate bone



pAMP10




lesion






L0527
NCI_CGAP_Ov2
ovary



pAMP10


L0528
NCI_CGAP_Pr5
prostate



pAMP10


L0529
NCI_CGAP_Pr6
prostate



pAMP10


L0530
NCI_CGAP_Pr8
prostate



pAMP10


L0531
NCI_CGAP_Pr20
prostate metastasis, liver



pAMP10


L0532
NCI_CGAP_Thy1
thyroid



pAMP10


L0533
NCI_CGAP_HSC1
stem cells
bone marrow


pAMP10


L0534
Chromosome 7 Fetal Brain cDNA
brain
brain


pAMP10



Library







L0535
NCI_CGAP_Br5
infiltrating ductal
breast


pAMP10




carcinoma






L0536
NCI_CGAP_Br4
normal ductal tissue
breast


pAMP10


L0537
NCI_CGAP_Ov6
normal cortical stroma
ovary


pAMP10


L0538
NCI_CGAP_Ov5
normal surface epithelium
ovary


pAMP10


L0539
Chromosome 7 Placental cDNA

placenta


pAMP10



Library







L0540
NCI_CGAP_Pr10
invasive prostate tumor
prostate


pAMP10


L0541
NCI_CGAP_Pr7
low-grade prostatic
prostate


pAMP10




neoplasia






L0542
NCI_CGAP_Pr11
normal prostatic epithelial
prostate


pAMP10




cells






L0543
NCI_CGAP_Pr9
normal prostatic epithelial
prostate


pAMP10




cells






L0544
NCI_CGAP_Pr4
prostatic intraepithelial
prostate


pAMP10




neoplasia - high grade






L0545
NCI_CGAP_Pr4.1
prostatic intraepithelial
prostate


pAMP10




neoplasia - high grade






L0546
NCI_CGAP_Pr18
stroma
prostate


pAMP10


L0547
NCI_CGAP_Pr16
tumor
prostate


pAMP10


L0548
Chromosome 7 Thymus cDNA
thymus
thymus


pAMP10



Library







L0549
NCI_CGAP_HN10
carcinoma in situ from



pAMP10




retromolar trigone






L0550
NCI_CGAP_HN9
normal squamous



pAMP10




epithelium from retromolar








trigone






L0551
NCI_CGAP_HN7
normal squamous



pAMP10




epithelium, floor of mouth






L0552
NCI_CGAP_HN8
well-differentiated invasive



pAMP10




carcinoma, floor of m






L0553
NCI_CGAP_Co22
colonic adenocarcinoma
colon


pAMP10


L0554
NCI_CGAP_Li8

liver


pAMP10


L0555
NCI_CGAP_Lu34
large cell carcinoma
lung


pAMP10


L0556
NCI_CGAP_Lu34.1
large cell carcinoma
lung


pAMP10


L0557
NCI_CGAP_Lu21
small cell carcinoma
lung


pAMP10


L0558
NCI_CGAP_Ov40
endometrioid ovarian
ovary


pAMP10




metastasis






L0559
NCI_CGAP_Ov39
papillary serous ovarian
ovary


pAMP10




metastasis






L0560
NCI_CGAP_HN12
moderate to poorly
tongue


pAMP10




differentiated invasive








carcino






L0561
NCI_CGAP_HN11
normal squamous
tongue


pAMP10




epithelium






L0562
Chromosome 7 HeLa cDNA


HeLa cell line;

pAMP10



Library


ATCC TM




L0563
Human Bone Marrow Stromal
bone marrow



pBLUESCRIPT TM



Fibroblast







L0564
Jia bone marrow stroma
bone marrow stroma



pBLUESCRIPT TM


L0565
Normal Human Trabecular Bone
Bone
Hip


pBLUESCRIPT TM



Cells







L0579
Human fetal brain QBoqin2
cerebrum and cerebellum



pBLUESCRIPT TM








SK


L0581
STRATAGENE TM liver

liver


pBLUESCRIPT TM



(#937224)




SK


L0583
STRATAGENE TM cDNA library




pBLUESCRIPT TM



Human fibroblast, cat#937212




SK


L0584
STRATAGENE TM cDNA library




pBLUESCRIPT TM



Human heart, cat#936208




SK


L0586
HTCDL1




pBLUESCRIPT TM








SK−


L0587
STRATAGENE TM colon HT29




pBLUESCRIPT TM



(#937221)




SK−


L0588
STRATAGENE TM endothelial




pBLUESCRIPT TM



cell 937223




SK−


L0589
STRATAGENE TM fetal retina




pBLUESCRIPT TM



937202




SK−


L0590
STRATAGENE TM fibroblast




pBLUESCRIPT TM



(#937212)




SK−


L0591
STRATAGENE TM HeLa cell s3




pBLUESCRIPT TM



937216




SK−


L0592
STRATAGENE TM hNT neuron




pBLUESCRIPT TM



(#937233)




SK−


L0593
STRATAGENE TM




pBLUESCRIPT TM



neuroepithelium (#937231)




SK−


L0594
STRATAGENE TM




pBLUESCRIPT TM



neuroepithelium NT2RAMI




SK−



937234







L0595
STRATAGENE TM NT2 neuronal
neuroepithelial cells
brain


pBLUESCRIPT TM



precursor 937230




SK−


L0596
STRATAGENE TM colon

colon


pBLUESCRIPT TM



(#937204)




SK−


L0597
STRATAGENE TM corneal stroma

cornea


pBLUESCRIPT TM



(#937222)




SK−


L0598
Morton Fetal Cochlea
cochlea
ear


pBLUESCRIPT TM








SK−


L0599
STRATAGENE TM lung

lung


pBLUESCRIPT TM



(#937210)




SK−


L0600
Weizmann Olfactory Epithelium
olfactory epithelium
nose


pBLUESCRIPT TM








SK−


L0601
STRATAGENE TM pancreas

pancreas


pBLUESCRIPT TM



(#937208)




SK−


L0602
Pancreatic Islet
pancreatic islet
pancreas


pBLUESCRIPT TM








SK−


L0603
STRATAGENE TM placenta

placenta


pBLUESCRIPT TM



(#937225)




SK−


L0604
STRATAGENE TM muscle
muscle
skeletal


pBLUESCRIPT TM



937209

muscle


SK−


L0605
STRATAGENE TM fetal spleen
fetal spleen
spleen


pBLUESCRIPT TM



(#937205)




SK−


L0606
NCI_CGAP_Lym5
follicular lymphoma
lymph node


pBLUESCRIPT TM








SK−


L0607
NCI_CGAP_Lym6
mantle cell lymphoma
lymph node


pBLUESCRIPT TM








SK−


L0608
STRATAGENE TM lung
lung carcinoma
lung
NCI-H69

pBLUESCRIPT TM



carcinoma 937218




SK−


L0609
Schiller astrocytoma
astrocytoma
brain


pBLUESCRIPT TM








SK−








(STRATAGENE TM)


L0610
Schiller glioblastoma multiforme
glioblastoma multiforme
brain


pBLUESCRIPT TM








SK−








(STRATAGENE TM)


L0611
Schiller meningioma
meningioma
brain


pBLUESCRIPT TM








SK−








(STRATAGENE TM)


L0612
Schiller oligodendroglioma
oligodendroglioma
brain


pBLUESCRIPT TM








SK−








(STRATAGENE TM)


L0615
22 week old human fetal liver




pBLUESCRIPT TM



cDNA library




II SK−


L0616
Chromosome 21 exon




pBLUESCRIPT TM








IIKS+


L0617
Chromosome 22 exon




pBLUESCRIPT TM








IIKS+


L0618
Chromosome 9 exon




pBLUESCRIPT TM








IIKS+


L0619
Chromosome 9 exon II




pBLUESCRIPT TM








IIKS+


L0622
HM1




pcDNAII








(Invitrogen)


L0623
HM3
pectoral muscle (after



pcDNAII




mastectomy)



(Invitrogen)


L0625
NCI_CGAP_AR1
bulk alveolar tumor



pCMV-








SPORT2


L0626
NCI_CGAP_GC1
bulk germ cell seminoma



pCMV-








SPORT2


L0627
NCI_CGAP_Co1
bulk tumor
colon


pCMV-








SPORT2


L0628
NCI_CGAP_Ov1
ovary bulk tumor
ovary


pCMV-








SPORT2


L0629
NCI_CGAP_Mel3
metastatic melanoma to
bowel (skin


pCMV-




bowel
primary)


SPORT4


L0630
NCI_CGAP_CNS1
substantia nigra
brain


pCMV-








SPORT4


L0631
NCI_CGAP_Br7

breast


pCMV-








SPORT4


L0632
NCI_CGAP_Li5
hepatic adenoma
liver


pCMV-








SPORT4


L0633
NCI_CGAP_Lu6
small cell carcinoma
lung


pCMV-








SPORT4


L0634
NCI_CGAP_Ov8
serous adenocarcinoma
ovary


pCMV-








SPORT4


L0635
NCI_CGAP_PNS1
dorsal root ganglion
peripheral


pCMV-





nervous


SPORT4





system





L0636
NCI_CGAP_Pit1
four pooled pituitary
brain


pCMV-




adenomas



SPORT6 (LIFE








TECHNOLOGIES TM)


L0637
NCI_CGAP_Brn53
three pooled meningiomas
brain


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L0638
NCI_CGAP_Brn35
tumor, 5 pooled (see
brain


pCMV-




description)



SPORT6 (LIFE








TECHNOLOGIES TM)


L0639
NCI_CGAP_Brn52
tumor, 5 pooled (see
brain


pCMV-




description)



SPORT6 (LIFE








TECHNOLOGIES TM)


L0640
NCI_CGAP_Br18
four pooled high-grade
breast


pCMV-




tumors, including two



SPORT6 (LIFE




prima



TECHNOLOGIES TM)


L0641
NCI_CGAP_Co17
juvenile granulosa tumor
colon


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L0642
NCI_CGAP_Co18
moderately differentiated
colon


pCMV-




adenocarcinoma



SPORT6 (LIFE








TECHNOLOGIES TM)


L0643
NCI_CGAP_Co19
moderately differentiated
colon


pCMV-




adenocarcinoma



SPORT6 (LIFE








TECHNOLOGIES TM)


L0644
NCI_CGAP_Co20
moderately differentiated
colon


pCMV-




adenocarcinoma



SPORT6 (LIFE








TECHNOLOGIES TM)


L0645
NCI_CGAP_Co21
moderately differentiated
colon


pCMV-




adenocarcinoma



SPORT6 (LIFE








TECHNOLOGIES TM)


L0646
NCI_CGAP_Co14
moderately-differentiated
colon


pCMV-




adenocarcinoma



SPORT6 (LIFE








TECHNOLOGIES TM)


L0647
NCI_CGAP_Sar4
five pooled sarcomas,
connective


pCMV-




including myxoid
tissue


SPORT6 (LIFE




liposarcoma



TECHNOLOGIES TM)


L0648
NCI_CGAP_Eso2
squamous cell carcinoma
esophagus


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L0649
NCI_CGAP_GU1
2 pooled high-grade
genitourinary


pCMV-




transitional cell tumors
tract


SPORT6 (LIFE








TECHNOLOGIES TM)


L0650
NCI_CGAP_Kid13
2 pooled Wilms'' tumors,
kidney


pCMV-




one primary and one metast



SPORT6 (LIFE








TECHNOLOGIES TM)


L0651
NCI_CGAP_Kid8
renal cell tumor
kidney


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L0652
NCI_CGAP_Lu27
four pooled poorly-
lung


pCMV-




differentiated



SPORT6 (LIFE




adenocarcinomas



TECHNOLOGIES TM)


L0653
NCI_CGAP_Lu28
two pooled squamous cell
lung


pCMV-




carcinomas



SPORT6 (LIFE








TECHNOLOGIES TM)


L0654
NCI_CGAP_Lu31

lung, cell line


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L0655
NCI_CGAP_Lym12
lymphoma, follicular mixed
lymph node


pCMV-




small and large cell



SPORT6 (LIFE








TECHNOLOGIES TM)


L0656
NCI_CGAP_Ov38
normal epithelium
ovary


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L0657
NCI_CGAP_Ov23
tumor, 5 pooled (see
ovary


pCMV-




description)



SPORT6 (LIFE








TECHNOLOGIES TM)


L0658
NCI_CGAP_Ov35
tumor, 5 pooled (see
ovary


pCMV-




description)



SPORT6 (LIFE








TECHNOLOGIES TM)


L0659
NCI_CGAP_Pan1
adenocarcinoma
pancreas


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L0661
NCI_CGAP_Mel15
malignant melanoma,
skin


pCMV-




metastatic to lymph node



SPORT6 (LIFE








TECHNOLOGIES TM)


L0662
NCI_CGAP_Gas4
poorly differentiated
stomach


pCMV-




adenocarcinoma with signet r



SPORT6 (LIFE








TECHNOLOGIES TM)


L0663
NCI_CGAP_Ut2
moderately-differentiated
uterus


pCMV-




endometrial adenocarcino



SPORT6 (LIFE








TECHNOLOGIES TM)


L0664
NCI_CGAP_Ut3
poorly-differentiated
uterus


pCMV-




endometrial



SPORT6 (LIFE




adenocarcinoma,



TECHNOLOGIES TM)


L0665
NCI_CGAP_Ut4
serous papillary carcinoma,
uterus


pCMV-




high grade, 2 pooled t



SPORT6 (LIFE








TECHNOLOGIES TM)


L0666
NCI_CGAP_Ut1
well-differentiated
uterus


pCMV-




endometrial



SPORT6 (LIFE




adenocarcinoma, 7



TECHNOLOGIES TM)


L0667
NCI_CGAP_CML1
myeloid cells, 18 pooled
whole blood


pCMV-




CML cases, BCR/ABL



SPORT6 (LIFE




rearra



TECHNOLOGIES TM)


L0669
Human MCF7 cDNA subtracted
breast adenocarcinoma
breast
MCF7

pCR II



with MDA-MB-231 cDNA




[Invitrogen]


L0681
Stanley Frontal SN individual
frontal lobe (see
brain


pCR2.1




description)



(Invitrogen)


L0682
Stanley Frontal NB pool 2
frontal lobe (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0683
Stanley Frontal NS pool 2
frontal lobe (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0684
Stanley Frontal SB pool 1
frontal lobe (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0685
Stanley Frontal SN pool 1
frontal lobe (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0686
Stanley Frontal SN pool 2
frontal lobe (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0687
Stanley Hippocampus NB pool 1
hippocampus (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0688
Stanley Hippocampus SB pool 1
hippocampus (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0689
Stanley Hippocampus SN pool 1
hippocampus (see
brain


pCR2.1-TOPO




description)



(Invitrogen)


L0690
Testis, Subtracted




pCR II








[Invitrogen]


L0695
Human Glialblastoma Cell

Brain
BT-325

pCR II








[Invitrogen]


L0697
Testis 1




PGEM 5zf(+)


L0698
Testis 2




PGEM 5zf(+)


L0700
Outward Alu-primed hncDNA




pGEM-3Z



library







L0708
NIH_MGC_17
rhabdomyosarcoma
muscle


pOTB7


L0709
NIH_MGC_21
choriocarcinoma
placenta


pOTB7


L0710
NIH_MGC_7
small cell carcinoma
lung
MGC3

pOTB7


L0716
PMA-induced HL60 cell


PMA-induced

pSport1



subtraction library


HL60 human








leukemic cell








line




L0717
Gessler Wilms tumor




pSport1


L0718
Testis 5




pSport1


L0719
human embryo cDNA library
Whole embryo



pSport1


L0720
PN001-Normal Human Prostate

prostate


pSport1


L0731
Soares_pregnant_uterus_NbHPU

uterus


pT7T3-Pac


L0738
Human colorectal cancer




pT7T3D


L0740
Soares melanocyte 2NbHM
melanocyte



pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0741
Soares adult brain N2b4HB55Y

brain


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0742
Soares adult brain N2b5HB55Y

brain


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0743
Soares breast 2NbHBst

breast


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0744
Soares breast 3NbHBst

breast


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0745
Soares retina N2b4HR
retina
eye


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0746
Soares retina N2b5HR
retina
eye


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0747
Soares_fetal_heart_NbHH19W

heart


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0748
Soares fetal liver spleen 1NFLS

Liver and


pT7T3D





Spleen


(PHARMACIA TM)








with a








modified








polylinker


L0749
Soares_fetal_liver—

Liver and


pT7T3D



spleen_1NFLS_S1

Spleen


(PHARMACIA TM)








with a








modified








polylinker


L0750
Soares_fetal_lung_NbHL19W

lung


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0751
Soares ovary tumor NbHOT
ovarian tumor
ovary


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0752
Soares_parathyroid_tumor_NbHPA
parathyroid tumor
parathyroid


pT7T3D





gland


(PHARMACIA TM)








with a








modified








polylinker


L0753
Soares_pineal_gland_N3HPG

pineal gland


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0754
Soares placenta Nb2HP

placenta


pT7T3D








(PHARMACIA TM)








with a








modified








polylinker


L0755
Soares_placenta_8to9weeks—

placenta


pT7T3D



2NbHP8to9W




(PHARMACIA TM)








with a








modified








polylinker


L0756
Soares_multiple_sclerosis—
multiple sclerosis lesions



pT7T3D



2NbHMSP




(PHARMACIA TM)








with a








modified








polylinker








V_TYPE


L0757
Soares_senescent_fibroblasts_Nb
senescent fibroblast



pT7T3D



HSF




(PHARMACIA TM)








with a








modified








polylinker








V_TYPE


L0758
Soares_testis_NHT




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0759
Soares_total_fetus_Nb2HF8_9w




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0760
Barstead aorta HPLRB3
aorta



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0761
NCI_CGAP_CLL1
B-cell, chronic lymphotic



pT7T3D-Pac




leukemia



(PHARMACIA TM)








with a








modified








polylinker


L0762
NCI_CGAP_Br1.1
breast



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0763
NCI_CGAP_Br2
breast



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0764
NCI_CGAP_Co3
colon



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0765
NCI_CGAP_Co4
colon



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0766
NCI_CGAP_GCB1
germinal center B cell



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0767
NCI_CGAP_GC3
pooled germ cell tumors



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0768
NCI_CGAP_GC4
pooled germ cell tumors



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0769
NCI_CGAP_Brn25
anaplastic
brain


pT7T3D-Pac




oligodendroglioma



(PHARMACIA TM)








with a








modified








polylinker


L0770
NCI_CGAP_Brn23
glioblastoma (pooled)
brain


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0771
NCI_CGAP_Co8
adenocarcinoma
colon


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0772
NCI_CGAP_Co10
colon tumor RER+
colon


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0773
NCI_CGAP_Co9
colon tumor RER+
colon


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0774
NCI_CGAP_Kid3

kidney


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0775
NCI_CGAP_Kid5
2 pooled tumors (clear cell
kidney


pT7T3D-Pac




type)



(PHARMACIA TM)








with a








modified








polylinker


L0776
NCI_CGAP_Lu5
carcinoid
lung


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0777
Soares_NhHMPu_S1
Pooled human melanocyte,
mixed (see


pT7T3D-Pac




fetal heart, and pregnant
below)


(PHARMACIA TM)








with a








modified








polylinker


L0778
Barstead pancreas HPLRB1

pancreas


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0779
Soares_NFL_T_GBC_S1

pooled


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0780
Soares_NSF_F8—

pooled


pT7T3D-Pac



9W_OT_PA_P_S1




(PHARMACIA TM)








with a








modified








polylinker


L0782
NCI_CGAP_Pr21
normal prostate
prostate


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0783
NCI_CGAP_Pr22
normal prostate
prostate


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0784
NCI_CGAP_Lei2
leiomyosarcoma
soft tissue


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0785
Barstead spleen HPLRB2

spleen


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0786
Soares_NbHFB

whole brain


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0787
NCI_CGAP_Sub1




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0788
NCI_CGAP_Sub2




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0789
NCI_CGAP_Sub3




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0790
NCI_CGAP_Sub4




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0791
NCI_CGAP_Sub5




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0792
NCI_CGAP_Sub6




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0793
NCI_CGAP_Sub7




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0794
NCI_CGAP_GC6
pooled germ cell tumors



pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0796
NCI_CGAP_Brn50
medulloblastoma
brain


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0800
NCI_CGAP_Co16
colon tumor, RER+
colon


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0803
NCI_CGAP_Kid11

kidney


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0804
NCI_CGAP_Kid12
2 pooled tumors (clear cell
kidney


pT7T3D-Pac




type)



(PHARMACIA TM)








with a








modified








polylinker


L0805
NCI_CGAP_Lu24
carcinoid
lung


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0806
NCI_CGAP_Lu19
squamous cell carcinoma,
lung


pT7T3D-Pac




poorly differentiated (4



(PHARMACIA TM)








with a








modified








polylinker


L0807
NCI_CGAP_Ov18
fibrotheoma
ovary


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0808
Barstead prostate BPH HPLRB41

prostate


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0809
NCI_CGAP_Pr28

prostate


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L0811
BATM2




PTZ18


L0879
BT0254

breast


puc18


L0930
BT0314

breast


puc18


L0946
BT0333

breast


puc18


L0988
BT0387

breast


puc18


L1057
BT0559

breast


puc18


L1278
BN0005

breast_normal


puc18


L1430
CT0225

colon


puc18


L1441
CT0249

colon


puc18


L1446
CT0254

colon


puc18


L1477
CT0297

colon


puc18


L1499
CT0322

colon


puc18


L1548
CN0007

colon_normal


puc18


L1561
CN0026

colon_normal


puc18


L1562
CN0027

colon_normal


puc18


L1607
DT0041

denis_drash


puc18


L1651
HT0059

head_neck


puc18


L1727
HT0158

head_neck


puc18


L1788
HT0229

head_neck


puc18


L1819
HT0268

head_neck


puc18


L1872
HT0335

head_neck


puc18


L1877
HT0340

head_neck


puc18


L1878
HT0342

head_neck


puc18


L1886
HT0350

head_neck


puc18


L1894
HT0366

head_neck


puc18


L1942
HT0452

head_neck


puc18


L1948
HT0470

head_neck


puc18


L2094
ST0125

stomach


puc18


L2138
ST0186

stomach


puc18


L2174
ST0240

stomach


puc18


L2197
ST0278

stomach


puc18


L2210
ST0293

stomach


puc18


L2242
subtracted 3′′ EST library

pancreas
AsPC-

pUC18






1(ATCC TM:CRL-








1682)




L2245
NEM subtracted human fetal




pUEX1



kidney cDNA







L2250
Human cerebral cortex
cerebral cortex






L2251
Human fetal lung
Fetal lung






L2252
Human placenta
placenta






L2255
GLC
corresponding non



pBLUESCRIPT TM




cancerous liver tissue



SK−


L2257
NIH_MGC_65
adenocarcinoma
colon


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2258
NIH_MGC_67
retinoblastoma
eye


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2259
NIH_MGC_68
large cell carcinoma
lung


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2260
NIH_MGC_69
large cell carcinoma,
lung


pCMV-




undifferentiated



SPORT6 (LIFE








TECHNOLOGIES TM)


L2261
NIH_MGC_70
epithelioid carcinoma
pancreas


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2262
NIH_MGC_72
melanotic melanoma
skin


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2263
NIH_MGC_66
adenocarcinoma
ovary


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2264
NIH_MGC_71
leiomyosarcoma
uterus


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2265
NIH_MGC_39
adenocarcinoma
pancreas


pOTB7


L2269
NCI_CGAP_Thy11
follicular carcinoma
thyroid


pAMP10


L2270
Lupski_dorsal_root_ganglion
dorsal root ganglia



pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L2277
BT0626

breast


puc18


L2279
BT0659

breast


puc18


L2281
BT0701

breast


puc18


L2283
BT0705

breast


puc18


L2285
BT0723

breast


puc18


L2289
BT0757

breast


puc18


L2291
BT0760

breast


puc18


L2293
BT0762

breast


puc18


L2294
BT0763

breast


puc18


L2300
BT0789

breast


puc18


L2301
BT0792

breast


puc18


L2308
CT0383

colon


puc18


L2317
CT0400

colon


puc18


L2323
CT0406

colon


puc18


L2328
CT0412

colon


puc18


L2332
CT0416

colon


puc18


L2333
CT0417

colon


puc18


L2336
CT0428

colon


puc18


L2338
CT0432

colon


puc18


L2339
CT0434

colon


puc18


L2346
CT0483

colon


puc18


L2348
CT0491

colon


puc18


L2352
UT0001

uterus_tumor


puc18


L2353
UT0003

uterus_tumor


puc18


L2354
UT0005

uterus_tumor


puc18


L2357
UT0021

uterus_tumor


puc18


L2359
UT0023

uterus_tumor


puc18


L2361
UT0028

uterus_tumor


puc18


L2364
UT0033

uterus_tumor


puc18


L2367
UT0039

uterus_tumor


puc18


L2368
UT0041

uterus_tumor


puc18


L2372
NN0034

nervous_normal


puc18


L2377
NN0054

nervous_normal


puc18


L2380
NN0068

nervous_normal


puc18


L2381
NN0070

nervous_normal


puc18


L2382
NN0073

nervous_normal


puc18


L2389
NN0087

nervous_normal


puc18


L2400
NN0116

nervous_normal


puc18


L2402
NN0118

nervous_normal


puc18


L2412
NN0136

nervous_normal


puc18


L2413
NN0141

nervous_normal


puc18


L2439
NN1022

nervous_normal


puc18


L2440
NN1023

nervous_normal


puc18


L2449
NN1063

nervous_normal


puc18


L2450
NN1065

nervous_normal


puc18


L2462
NN1089

nervous_normal


puc18


L2464
NN1104

nervous_normal


puc18


L2466
NN1111

nervous_normal


puc18


L2467
NN1112

nervous_normal


puc18


L2471
NN1123

nervous_normal


puc18


L2472
NN1124

nervous_normal


puc18


L2477
HT0408

head_neck


puc18


L2478
HT0445

head_neck


puc18


L2482
HT0497

head_neck


puc18


L2486
HT0527

head_neck


puc18


L2487
HT0542

head_neck


puc18


L2490
HT0545

head_neck


puc18


L2491
HT0559

head_neck


puc18


L2493
HT0576

head_neck


puc18


L2494
HT0577

head_neck


puc18


L2495
HT0594

head_neck


puc18


L2497
HT0618

head_neck


puc18


L2498
HT0619

head_neck


puc18


L2499
HT0622

head_neck


puc18


L2500
HT0623

head_neck


puc18


L2504
HT0636

head_neck


puc18


L2506
HT0638

head_neck


puc18


L2513
HT0678

head_neck


puc18


L2518
HT0697

head_neck


puc18


L2519
HT0698

head_neck


puc18


L2521
HT0702

head_neck


puc18


L2522
HT0704

head_neck


puc18


L2525
HT0710

head_neck


puc18


L2528
HT0713

head_neck


puc18


L2535
HT0723

head_neck


puc18


L2539
HT0727

head_neck


puc18


L2540
HT0728

head_neck


puc18


L2543
HT0734

head_neck


puc18


L2545
HT0736

head_neck


puc18


L2550
HT0743

head_neck


puc18


L2551
HT0744

head_neck


puc18


L2552
HT0745

head_neck


puc18


L2558
HT0756

head_neck


puc18


L2560
HT0758

head_neck


puc18


L2562
HT0760

head_neck


puc18


L2565
HT0764

head_neck


puc18


L2570
HT0771

head_neck


puc18


L2571
HT0773

head_neck


puc18


L2578
HT0785

head_neck


puc18


L2581
HT0790

head_neck


puc18


L2587
HT0797

head_neck


puc18


L2596
HT0807

head_neck


puc18


L2598
HT0809

head_neck


puc18


L2599
HT0810

head_neck


puc18


L2610
HT0837

head_neck


puc18


L2615
HT0843

head_neck


puc18


L2618
HT0847

head_neck


puc18


L2630
HT0865

head_neck


puc18


L2634
HT0872

head_neck


puc18


L2635
HT0875

head_neck


puc18


L2637
HT0877

head_neck


puc18


L2638
HT0878

head_neck


puc18


L2640
HT0881

head_neck


puc18


L2644
HT0886

head_neck


puc18


L2647
HT0894

head_neck


puc18


L2649
HT0905

head_neck


puc18


L2650
HT0934

head_neck


puc18


L2651
NIH_MGC_20
melanotic melanoma
skin


pOTB7


L2652
NIH_MGC_57
glioblastoma
brain


pDNR-LIB








(CLONTECH TM)


L2653
NIH_MGC_58
hypernephroma
kidney


pDNR-LIB








(CLONTECH TM)


L2654
NIH_MGC_9
adenocarcinoma cell line
ovary


pOTB7


L2655
NIH_MGC_55
from acute myelogenous
bone marrow


pDNR-LIB




leukemia



(CLONTECH TM)


L2657
NIH_MGC_54
from chronic myelogenous
bone marrow


pDNR-LIB




leukemia



(CLONTECH TM)


L2667
NT0013

nervous_tumor


puc18


L2668
NT0018

nervous_tumor


puc18


L2669
NT0022

nervous_tumor


puc18


L2670
NT0023

nervous_tumor


puc18


L2671
NT0024

nervous_tumor


puc18


L2673
NT0028

nervous_tumor


puc18


L2674
NT0029

nervous_tumor


puc18


L2675
NT0033

nervous_tumor


puc18


L2677
NT0039

nervous_tumor


puc18


L2681
NT0048

nervous_tumor


puc18


L2683
NT0053

nervous_tumor


puc18


L2686
NT0058

nervous_tumor


puc18


L2689
NT0073

nervous_tumor


puc18


L2696
NT0084

nervous_tumor


puc18


L2702
NT0098

nervous_tumor


puc18


L2705
NT0101

nervous_tumor


puc18


L2706
NT0102

nervous_tumor


puc18


L2708
NT0104

nervous_tumor


puc18


L2709
NT0105

nervous_tumor


puc18


L2716
NT0117

nervous_tumor


puc18


L2730
GN0021

placenta_normal


puc18


L2731
GN0023

placenta_normal


puc18


L2733
GN0037

placenta_normal


puc18


L2737
GN0047

placenta_normal


puc18


L2738
GN0049

placenta_normal


puc18


L2744
FT0004

prostate_tumor


puc18


L2754
FT0022

prostate_tumor


puc18


L2755
FT0023

prostate_tumor


puc18


L2756
FT0024

prostate_tumor


puc18


L2757
FT0025

prostate_tumor


puc18


L2758
FT0027

prostate_tumor


puc18


L2759
FT0028

prostate_tumor


puc18


L2763
FT0039

prostate_tumor


puc18


L2766
FT0042

prostate_tumor


puc18


L2767
FT0044

prostate_tumor


puc18


L2771
FT0050

prostate_tumor


puc18


L2777
FT0056

prostate_tumor


puc18


L2779
FT0058

prostate_tumor


puc18


L2788
FT0071

prostate_tumor


puc18


L2791
FT0077

prostate_tumor


puc18


L2793
FT0080

prostate_tumor


puc18


L2799
FT0096

prostate_tumor


puc18


L2800
FT0097

prostate_tumor


puc18


L2804
FT0103

prostate_tumor


puc18


L2809
FT0117

prostate_tumor


puc18


L2810
FT0119

prostate_tumor


puc18


L2811
FT0122

prostate_tumor


puc18


L2812
FT0123

prostate_tumor


puc18


L2814
FT0128

prostate_tumor


puc18


L2815
FT0129

prostate_tumor


puc18


L2817
FT0131

prostate_tumor


puc18


L2819
FT0134

prostate_tumor


puc18


L2831
FT0162

prostate_tumor


puc18


L2833
FT0164

prostate_tumor


puc18


L2836
FT0169

prostate_tumor


puc18


L2842
UM0009

uterus


puc18


L2843
UM0017

uterus


puc18


L2844
UM0018

uterus


puc18


L2845
UM0021

uterus


puc18


L2846
UM0022

uterus


puc18


L2848
UM0053

uterus


puc18


L2852
UM0077

uterus


puc18


L2854
UM0091

uterus


puc18


L2865
AN0004

amnion_normal


puc18


L2869
AN0012

amnion_normal


puc18


L2870
AN0013

amnion_normal


puc18


L2877
AN0027

amnion_normal


puc18


L2878
AN0029

amnion_normal


puc18


L2879
AN0032

amnion_normal


puc18


L2884
AN0041

amnion_normal


puc18


L2888
AN0056

amnion_normal


puc18


L2893
AN0062

amnion_normal


puc18


L2899
AN0094

amnion_normal


puc18


L2902
BN0036

breast_normal


puc18


L2903
BN0039

breast_normal


puc18


L2904
BN0042

breast_normal


puc18


L2905
BN0046

breast_normal


puc18


L2906
BN0047

breast_normal


puc18


L2909
BN0067

breast_normal


puc18


L2910
BN0070

breast_normal


puc18


L2914
BN0090

breast_normal


puc18


L2915
BN0098

breast_normal


puc18


L2918
BN0114

breast_normal


puc18


L2919
BN0115

breast_normal


puc18


L2924
BN0138

breast_normal


puc18


L2938
BN0174

breast_normal


puc18


L2962
BN0221

breast_normal


puc18


L2978
BN0247

breast_normal


puc18


L2985
BN0257

breast_normal


puc18


L2987
BN0259

breast_normal


puc18


L2991
BN0264

breast_normal


puc18


L2999
BN0273

breast_normal


puc18


L3001
BN0275

breast_normal


puc18


L3002
BN0276

breast_normal


puc18


L3010
BN0294

breast_normal


puc18


L3011
BN0295

breast_normal


puc18


L3012
BN0296

breast_normal


puc18


L3019
BN0303

breast_normal


puc18


L3020
BN0304

breast_normal


puc18


L3041
BN0332

breast_normal


puc18


L3058
EN0004

lung_normal


puc18


L3066
EN0018

lung_normal


puc18


L3071
EN0026

lung_normal


puc18


L3078
EN0042

lung_normal


puc18


L3080
ET0001

lung_tumor


puc18


L3081
ET0005

lung_tumor


puc18


L3089
ET0018

lung_tumor


puc18


L3092
ET0023

lung_tumor


puc18


L3093
ET0024

lung_tumor


puc18


L3095
ET0027

lung_tumor


puc18


L3104
ET0041

lung_tumor


puc18


L3109
ET0046

lung_tumor


puc18


L3111
ET0058

lung_tumor


puc18


L3117
ET0068

lung_tumor


puc18


L3118
ET0070

lung_tumor


puc18


L3119
ET0072

lung_tumor


puc18


L3127
ET0084

lung_tumor


puc18


L3128
MT0016

marrow


puc18


L3132
MT0022

marrow


puc18


L3134
MT0024

marrow


puc18


L3140
MT0031

marrow


puc18


L3144
MT0035

marrow


puc18


L3153
MT0049

marrow


puc18


L3154
MT0050

marrow


puc18


L3158
MT0057

marrow


puc18


L3160
MT0059

marrow


puc18


L3180
MT0101

marrow


puc18


L3181
MT0107

marrow


puc18


L3182
MT0108

marrow


puc18


L3184
MT0111

marrow


puc18


L3186
MT0113

marrow


puc18


L3199
OT0019

ovary


puc18


L3204
OT0034

ovary


puc18


L3207
OT0063

ovary


puc18


L3210
OT0067

ovary


puc18


L3211
OT0072

ovary


puc18


L3212
OT0076

ovary


puc18


L3213
OT0078

ovary


puc18


L3215
OT0083

ovary


puc18


L3216
OT0086

ovary


puc18


L3217
OT0091

ovary


puc18


L3226
FN0019

prostate_normal


puc18


L3250
FN0058

prostate_normal


puc18


L3255
FN0064

prostate_normal


puc18


L3262
FN0073

prostate_normal


puc18


L3264
FN0080

prostate_normal


puc18


L3271
FN0094

prostate_normal


puc18


L3274
FN0098

prostate_normal


puc18


L3278
FN0104

prostate_normal


puc18


L3280
FN0106

prostate_normal


puc18


L3281
FN0107

prostate_normal


puc18


L3295
FN0138

prostate_normal


puc18


L3297
FN0140

prostate_normal


puc18


L3300
FN0143

prostate_normal


puc18


L3311
FN0180

prostate_normal


puc18


L3312
FN0181

prostate_normal


puc18


L3316
FN0188

prostate_normal


puc18


L3327
SN0024

stomach_normal


puc18


L3330
SN0041

stomach_normal


puc18


L3336
SN0066

stomach_normal


puc18


L3352
TN0027

testis_normal


puc18


L3355
TN0032

testis_normal


puc18


L3357
TN0034

testis_normal


puc18


L3358
TN0035

testis_normal


puc18


L3359
TN0036

testis_normal


puc18


L3369
TN0065

testis_normal


puc18


L3372
TN0068

testis_normal


puc18


L3374
TN0070

testis_normal


puc18


L3377
TN0079

testis_normal


puc18


L3378
TN0080

testis_normal


puc18


L3385
Homo sapiens HeLa


HeLa




L3387
GKB
hepatocellular carcinoma



pBLUESCRIPT TM








SK−


L3388
GKC
hepatocellular carcinoma



pBLUESCRIPT TM








SK−


L3389
GKD
hepatocellular carcinoma



pBLUESCRIPT TM








SK−


L3391
NIH_MGC_53
carcinoma, cell line
bladder


pDNR-LIB








(CLONTECH TM)


L3401
AN0085

amnion_normal


puc18


L3402
AN0086

amnion_normal


puc18


L3403
AN0087

amnion_normal


puc18


L3404
AN0089

amnion_normal


puc18


L3421
BT0634

breast


puc18


L3431
CT0451

colon


puc18


L3432
CT0461

colon


puc18


L3435
CT0465

colon


puc18


L3443
CT0482

colon


puc18


L3445
CT0497

colon


puc18


L3450
CT0508

colon


puc18


L3459
FT0175

prostate_tumor


puc18


L3463
GN0016

placenta_normal


puc18


L3464
GN0018

placenta_normal


puc18


L3466
GN0020

placenta_normal


puc18


L3476
GN0051

placenta_normal


puc18


L3480
GN0057

placenta_normal


puc18


L3484
GN0067

placenta_normal


puc18


L3485
GN0070

placenta_normal


puc18


L3490
GN0075

placenta_normal


puc18


L3491
GN0076

placenta_normal


puc18


L3494
HT0539

head_neck


puc18


L3495
HT0570

head_neck


puc18


L3496
HT0572

head_neck


puc18


L3497
HT0600

head_neck


puc18


L3499
HT0617

head_neck


puc18


L3503
HT0870

head_neck


puc18


L3504
HT0873

head_neck


puc18


L3506
HT0879

head_neck


puc18


L3511
HT0900

head_neck


puc18


L3516
HT0913

head_neck


puc18


L3518
HT0915

head_neck


puc18


L3520
HT0918

head_neck


puc18


L3521
HT0919

head_neck


puc18


L3526
HT0931

head_neck


puc18


L3530
HT0939

head_neck


puc18


L3540
MT0126

marrow


puc18


L3546
NN0044

nervous_normal


puc18


L3554
OT0035

ovary


puc18


L3560
TN0023

testis_normal


puc18


L3561
TN0025

testis_normal


puc18


L3562
TN0030

testis_normal


puc18


L3563
TN0037

testis_normal


puc18


L3565
TN0045

testis_normal


puc18


L3566
TN0046

testis_normal


puc18


L3576
TN0086

testis_normal


puc18


L3585
TN0119

testis_normal


puc18


L3586
TN0120

testis_normal


puc18


L3592
TN0129

testis_normal


puc18


L3603
UM0093

uterus


puc18


L3605
UM0104

uterus


puc18


L3609
UT0007

uterus_tumor


puc18


L3612
UT0011

uterus_tumor


puc18


L3618
UT0050

uterus_tumor


puc18


L3625
UT0063

uterus_tumor


puc18


L3630
UT0071

uterus_tumor


puc18


L3631
UT0072

uterus_tumor


puc18


L3632
UT0074

uterus_tumor


puc18


L3634
NIH_MGC_56
primitive neuroectoderm
brain


pDNR-LIB








(CLONTECH TM)


L3635
NIH_MGC_62
melanotic melanoma, high
skin


pDNR-LIB




MDR



(CLONTECH TM)


L3636
NIH_MGC_73

brain


pDNR-LIB








(CLONTECH TM)


L3637
NIH_MGC_74

heart


pDNR-LIB








(CLONTECH TM)


L3638
NIH_MGC_78

pancreas


pDNR-LIB








(CLONTECH TM)


L3641
NIH_MGC_83

prostate


pDNR-LIB








(CLONTECH TM)


L3642
ADA
Adrenal gland



pBLUESCRIPT TM








SK−


L3643
ADB
Adrenal gland



pBLUESCRIPT TM








SK−


L3644
ADC
Adrenal gland



pBLUESCRIPT TM








SK−


L3645
Cu
adrenal cortico adenoma for



pBLUESCRIPT TM




Cushing''s syndrome



SK−


L3646
DCA




pTriplEx2


L3647
Human HO-1 melanoma cells







L3649
DCB




pTriplEx2


L3651
FHTA
hypothalamus



pTriplEx2


L3652
FHTB
hypothalamus



pTriplEx2


L3653
HTB
Hypothalamus



pBLUESCRIPT TM








SK−


L3655
HTC
Hypothalamus



pBLUESCRIPT TM








SK−


L3656
HTE
Hypothalamus



pBLUESCRIPT TM








SK−


L3657
HTF
Hypothalamus



pBLUESCRIPT TM








SK−


L3658
cdA
pheochromocytoma



pTriplEx2


L3659
CB
cord blood



pBLUESCRIPT TM


L3660
NP1
pituitary



pBLUESCRIPT TM








SK−


L3661
NPA
pituitary



pBLUESCRIPT TM








SK−


L3663
NIH_MGC_60
adenocarcinoma
prostate


pDNR-LIB








(CLONTECH TM)


L3664
NIH_MGC_61
embryonal carcinoma
testis


pDNR-LIB








(CLONTECH TM)


L3665
NIH_MGC_75

kidney


pDNR-LIB








(CLONTECH TM)


L3666
NIH_MGC_77

lung


pDNR-LIB








(CLONTECH TM)


L3667
NIH_MGC_79

placenta


pDNR-LIB








(CLONTECH TM)


L3668
AN0063

amnion_normal


puc18


L3672
AN0083

amnion_normal


puc18


L3673
AN0084

amnion_normal


puc18


L3684
BT0812

breast


puc18


L3693
CI0018

colon_ins


puc18


L3697
CS0012

colon_est


puc18


L3699
CT0437

colon


puc18


L3705
CT0486

colon


puc18


L3709
CT0515

colon


puc18


L3713
CT0524

colon


puc18


L3718
CT0532

colon


puc18


L3722
GN0030

placenta_normal


puc18


L3724
GN0034

placenta_normal


puc18


L3726
GN0038

placenta_normal


puc18


L3728
GN0077

placenta_normal


puc18


L3729
GN0079

placenta_normal


puc18


L3738
GN0092

placenta_normal


puc18


L3739
HT0540

head_neck


puc18


L3744
HT0916

head_neck


puc18


L3750
HT0945

head_neck


puc18


L3752
HT0947

head_neck


puc18


L3761
NN1141

nervous_normal


puc18


L3763
SN0036

stomach_normal


puc18


L3768
TN0073

testis_normal


puc18


L3778
TN0112

testis_normal


puc18


L3783
TN0136

testis_normal


puc18


L3790
TN0150

testis_normal


puc18


L3796
UT0042

uterus_tumor


puc18


L3802
UT0052

uterus_tumor


puc18


L3804
UT0073

uterus_tumor


puc18


L3805
UT0075

uterus_tumor


puc18


L3807
UT0077

uterus_tumor


puc18


L3808
UT0078

uterus_tumor


puc18


L3811
NPC
pituitary



pBLUESCRIPT TM








SK−


L3812
NPD
pituitary



pBLUESCRIPT TM








SK−


L3813
TP
pituitary tumor



pTriplEx2


L3814
BM
Bone marrow



pTriplEx2


L3815
MDS
Bone marrow



pTriplEx2


L3816
HEMBA1
whole embryo, mainly head



pME18SFL3


L3817
HEMBB1
whole embryo, mainly body



pME18SFL3


L3818
MAMMA1
mammary gland



pME18SFL3


L3819
NIH_MGC_76

liver


pDNR-LIB








(CLONTECH TM)


L3820
NIH_MGC_46
leiomyosarcoma cell line
uterus


pOTB7


L3821
NIH_MGC_48
primary B-cells from
B-cells


pOTB7




tonsils (cell line)






L3822
NIH_MGC_59
mucoepidermoid carcinoma
lung


pDNR-LIB








(CLONTECH TM)


L3823
NT2RM1


NT2

pUC19FL3


L3824
NT2RM2


NT2

pME18SFL3


L3825
NT2RM4


NT2

pME18SFL3


L3826
NT2RP1


NT2

pUC19FL3


L3827
NT2RP2


NT2

pME18SFL3


L3828
NT2RP3


NT2

pME18SFL3


L3829
NT2RP4


NT2

pME18SFL3


L3831
OVARC1
ovary, tumor tissue



pME18SFL3


L3832
PLACE1
placenta



pME18SFL3


L3833
PLACE2
placenta



pME18SFL3


L3834
PLACE3
placenta



pME18SFL3


L3835
PLACE4
placenta



pME18SFL3


L3836
SKNMC1


SK-N-MC

pME18SFL3


L3837
THYRO1
thyroid gland



pME18SFL3


L3839
Y79AA1


Y79

pME18SFL3


L3841
NIH_MGC_18
large cell carcinoma
lung


pOTB7


L3854
BT0817

breast


puc18


L3871
NIH_MGC_19
neuroblastoma
brain


pOTB7


L3872
NCI_CGAP_Skn1

skin, normal,


pCMV-





4 pooled sa


SPORT6 (LIFE








TECHNOLOGIES TM)


L3873
Human esophageal carcinoma
esophageal squamous cell



pGEM-T



mRNA
carcinoma



(Promega)


L3904
NCI_CGAP_Brn64
glioblastoma with EGFR
brain


pCMV-




amplification



SPORT6 (LIFE








TECHNOLOGIES TM)


L3905
NCI_CGAP_Brn67
anaplastic
brain


pCMV-




oligodendroglioma with



SPORT6 (LIFE




1p/19q loss



TECHNOLOGIES TM)


L4497
NCI_CGAP_Br22
invasive ductal carcinoma,
breast


pCMV-




3 pooled samples



SPORT6 (LIFE








TECHNOLOGIES TM)


L4500
NCI_CGAP_HN16
moderate to poorly
mouth


pAMP10




differentiated invasive








carcino






L4501
NCI_CGAP_Sub8




pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L4507
NCI_CGAP_Thy6
normal epithelium
thyroid


pAMP10


L4508
NCI_CGAP_Thy8
normal epithelium
thyroid


pAMP10


L4535
NCI_CGAP_Thy4
normal epithelium
thyroid


pAMP10


L4537
NCI_CGAP_Thy7
follicular adenoma (benign
thyroid


pAMP10




lesion)






L4556
NCI_CGAP_HN13
squamous cell carcinoma
tongue


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L4557
NCI_CGAP_Adr1
neuroblastoma
adrenal gland


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L4558
NCI_CGAP_Pan3

pancreas


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L4559
NCI_CGAP_Thy3
follicular carcinoma
thyroid


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L4560
NCI_CGAP_Ut7
tumor
uterus


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L4669
NCI_CGAP_Ov41
serous papillary tumor
ovary


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L4747
NCI_CGAP_Brn41
oligodendroglioma
brain


pT7T3D-Pac








(PHARMACIA TM)








with a








modified








polylinker


L4753
NCI_CGAP_HN15
leukoplakia of the buccal
mouth


pAMP10




mucosa






L4775
NCI_CGAP_Thy12
papillary carcinoma
thyroid


pAMP10


L5286
NCI_CGAP_Thy10
medullary carcinoma
thyroid


pAMP10


L5564
NCI_CGAP_HN20

normal


pAMP1





head/neck








tissue





L5565
NCI_CGAP_Brn66
glioblastoma with probably
brain


pCMV-




TP53 mutation and witho



SPORT6 (LIFE








TECHNOLOGIES TM)


L5566
NCI_CGAP_Brn70
anaplastic
brain


pCMV-




oligodendroglioma



SPORT6.ccdb


L5568
NCI_CGAP_HN21
nasopharyngeal carcinoma
head/neck


pAMP1


L5569
NCI_CGAP_HN17
normal epithelium
nasopharynx


pAMP10


L5570
NCI_CGAP_Co28
normal colonic mucosa
colon


pAMP1


L5572
NCI_CGAP_Co27
adenocarcinoma (mucinous
colon


pAMP1




component)






L5574
NCI_CGAP_HN19
normal epithelium
nasopharynx


pAMP10


L5575
NCI_CGAP_Brn65
glioblastoma without EGFR
brain


pCMV-




amplification



SPORT6 (LIFE








TECHNOLOGIES TM)


L5622
NCI_CGAP_Skn3

skin


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


L5623
NCI_CGAP_Skn4
squamous cell carcinoma
skin


pCMV-








SPORT6 (LIFE








TECHNOLOGIES TM)


N0003
Human Fetal Brain
Human Fetal Brain






N0004
Human Hippocampus
Human Hippocampus






N0006
Human Fetal Brain
Human Fetal Brain






N0007
Human Hippocampus
Human Hippocampus






N0009
Human Hippocampus,
Human Hippocampus







prescreened







N0011
Human Brain
Human Brain






S0001
Brain frontal cortex
Brain frontal cortex
Brain


LAMBDA








ZAP TM II


S0002
Monocyte activated
Monocyte-activated
blood
Cell Line

UNI-ZAP TM








XR


S0003
Human Osteoclastoma
Osteoclastoma
bone

disease
UNI-ZAP TM








XR


S0004
Prostate
Prostate BPH
Prostate


LAMBDA








ZAP TM II


S0005
Heart
Heart-left ventricle
Heart


pCDNA


S0006
Neuroblastoma
Human Neural Blastoma


disease
pCDNA


S0007
Early Stage Human Brain
Human Fetal Brain



UNI-ZAP TM








XR


S0008
Osteoclastoma
Osteoclastoma
bone

disease
UNI-ZAP TM








XR


S0010
Human Amygdala
Amygdala



UNI-ZAP TM








XR


S0011
STROMAL-
Osteoclastoma
bone

disease
UNI-ZAP TM



OSTEOCLASTOMA




XR


S0013
Prostate
Prostate
prostate


UNI-ZAP TM








XR


S0014
Kidney Cortex
Kidney cortex
Kidney


UNI-ZAP TM








XR


S0015
Kidney medulla
Kidney medulla
Kidney


UNI-ZAP TM








XR


S0016
Kidney Pyramids
Kidney pyramids
Kidney


UNI-ZAP TM








XR


S0020
Seven Trans Membrane Receptor
7TMD1







Family







S0021
Whole brain
Whole brain
Brain


ZAP








EXPRESS TM


S0022
Human Osteoclastoma Stromal
Osteoclastoma Stromal



UNI-ZAP TM



Cells - unamplified
Cells



XR


S0023
Human Kidney Cortex -
Human Kidney Cortex







unamplified







S0024
Human Kidney Medulla -
Human Kidney Medulla







unamplified







S0025
Human Kidney Pyramids -
Human Kidney Pyramids







unamplified







S0026
Stromal cell TF274
stromal cell
Bone marrow
Cell Line

UNI-ZAP TM








XR


S0027
Smooth muscle, serum treated
Smooth muscle
Pulmanary
Cell Line

UNI-ZAP TM





artery


XR


S0028
Smooth muscle, control
Smooth muscle
Pulmanary
Cell Line

UNI-ZAP TM





artery


XR


S0029
brain stem
Brain stem
brain


UNI-ZAP TM








XR


S0030
Brain pons
Brain Pons
Brain


UNI-ZAP TM








XR


S0031
Spinal cord
Spinal cord
spinal cord


UNI-ZAP TM








XR


S0032
Smooth muscle-ILb induced
Smooth muscle
Pulmanary
Cell Line

UNI-ZAP TM





artery


XR


S0035
Brain medulla oblongata
Brain medulla oblongata
Brain


UNI-ZAP TM








XR


S0036
Human Substantia Nigra
Human Substantia Nigra



UNI-ZAP TM








XR


S0037
Smooth muscle, IL1b induced
Smooth muscle
Pulmanary
Cell Line

UNI-ZAP TM





artery


XR


S0038
Human Whole Brain #2 - Oligo
Human Whole Brain #2



ZAP



dT >1.5 Kb




EXPRESS  TM


S0039
Hypothalamus
Hypothalamus
Brain


UNI-ZAP TM








XR


S0040
Adipocytes
Human Adipocytes from



UNI-ZAP TM




Osteoclastoma



XR


S0041
Thalamus
Human Thalamus



UNI-ZAP TM








XR


S0042
Testes
Human Testes



ZAP








EXPRESS  TM


S0044
Prostate BPH
prostate BPH
Prostate

disease
UNI-ZAP TM








XR


S0045
Endothelial cells-control
Endothelial cell
endothelial
Cell Line

UNI-ZAP TM





cell-lung


XR


S0046
Endothelial-induced
Endothelial cell
endothelial
Cell Line

UNI-ZAP TM





cell-lung


XR


S0048
Human Hypothalamus,
Human Hypothalamus,


disease
UNI-ZAP TM



Alzheimer''s
Alzheimer''s



XR


S0049
Human Brain, Striatum
Human Brain, Striatum



UNI-ZAP TM








XR


S0050
Human Frontal Cortex,
Human Frontal Cortex,


disease
UNI-ZAP TM



Schizophrenia
Schizophrenia



XR


S0051
Human
Human Hypothalamus,


disease
UNI-ZAP TM



Hypothalmus, Schizophrenia
Schizophrenia



XR


S0052
neutrophils control
human neutrophils
blood
Cell Line

UNI-ZAP TM








XR


S0053
Neutrophils IL-1 and LPS
human neutrophil induced
blood
Cell Line

UNI-ZAP TM



induced




XR


S0106
STRIATUM DEPRESSION

BRAIN

disease
UNI-ZAP TM








XR


S0110
Brain Amygdala Depression

Brain

disease
UNI-ZAP TM








XR


S0112
Hypothalamus

Brain


UNI-ZAP TM








XR


S0114
Anergic T-cell
Anergic T-cell

Cell Line

UNI-ZAP TM








XR


S0116
Bone marrow
Bone marrow
Bone marrow


UNI-ZAP TM








XR


S0118
Smooth muscle control 2
Smooth muscle
Pulmanary
Cell Line

UNI-ZAP TM





artery


XR


S0122
Osteoclastoma-normalized A
Osteoclastoma
bone

disease
pBLUESCRIPT TM


S0124
Smooth muscle-edited A
Smooth muscle
Pulmanary
Cell Line

UNI-ZAP TM





artery


XR


S0126
Osteoblasts
Osteoblasts
Knee
Cell Line

UNI-ZAP TM








XR


S0132
Epithelial-TNFa and INF induced
Airway Epithelial



UNI-ZAP TM








XR


S0134
Apoptotic T-cell
apoptotic cells

Cell Line

UNI-ZAP TM








XR


S0136
PERM TF274
stromal cell
Bone marrow
Cell Line

LAMBDA








ZAP TM II


S0140
eosinophil-IL5 induced
eosinophil
lung
Cell Line

UNI-ZAP TM








XR


S0142
Macrophage-oxLDL
macrophage-oxidized LDL
blood
Cell Line

UNI-ZAP TM




treated



XR


S0144
Macrophage (GM-CSF treated)
Macrophage (GM-CSF



UNI-ZAP TM




treated)



XR


S0146
prostate-edited
prostate BPH
Prostate


UNI-ZAP TM








XR


S0148
Normal Prostate
Prostate
prostate


UNI-ZAP TM








XR


S0150
LNCAP prostate cell line
LNCAP Cell Line
Prostate
Cell Line

UNI-ZAP TM








XR


S0152
PC3 Prostate cell line
PC3 prostate cell line



UNI-ZAP TM








XR


S0168
Prostate/LNCAP, subtraction I
PC3 prostate cell line



pBLUESCRIPT TM


S0174
Prostate-BPH subtracted II
Human Prostate BPH



pBLUESCRIPT TM


S0176
Prostate, normal, subtraction I
Prostate
prostate


UNI-ZAP TM








XR


S0180
Bone Marrow Stroma, TNF&LPS
Bone Marrow Stroma, TNF


disease
UNI-ZAP TM



ind
& LPS induced



XR


S0182
Human B Cell 8866
Human B-Cell 8866



UNI-ZAP TM








XR


S0184
7TM Receptor enriched, lib II
PBLS, 7TM receptor



Other




enriched






S0188
Prostate, BPH, Lib 2
Human Prostate BPH


disease
pSport1


S0190
Prostate BPH, Lib 2, subtracted
Human Prostate BPH



pSport1


S0192
Synovial Fibroblasts (control)
Synovial Fibroblasts



pSport1


S0194
Synovial hypoxia
Synovial Fibroblasts



pSport1


S0196
Synovial IL-1/TNF stimulated
Synovial Fibroblasts



pSport1


S0198
7TM-pbfd
PBLS, 7TM receptor



pCR II




enriched



[Invitrogen]


S0202
7TM-pbdd
PBLS, 7TM receptor



pCR II




enriched



[Invitrogen]


S0206
Smooth Muscle-HASTE
Smooth muscle
Pulmanary
Cell Line

pBLUESCRIPT  TM



normalized

artery





S0208
Messangial cell, frac 1
Messangial cell



pSport1


S0210
Messangial cell, frac 2
Messangial cell



pSport1


S0212
Bone Marrow Stromal Cell,
Bone Marrow Stromal



pSport1



untreated
Cell, untreated






S0214
Human Osteoclastoma, re-
Osteoclastoma
bone

disease
UNI-ZAP TM



excision




XR


S0216
Neutrophils IL-1 and LPS
human neutrophil induced
blood
Cell Line

UNI-ZAP TM



induced




XR


S0218
Apoptotic T-cell, re-excision
apoptotic cells

Cell Line

UNI-ZAP TM








XR


S0220
H. hypothalamus, frac A; re-
Hypothalamus
Brain


ZAP



excision




EXPRESS  TM


S0222
H. Frontal cortex, epileptic; re-
H. Brain, Frontal Cortex,
Brain

disease
UNI-ZAP TM



excision
Epileptic



XR


S0228
PSMIX
PBLS, 7TM receptor



pCR II




enriched



[Invitrogen]


S0242
Synovial Fibroblasts (Il1/TNF),
Synovial Fibroblasts



pSport1



subt







S0250
Human Osteoblasts II
Human Osteoblasts
Femur

disease
pCMVSport 2.0


S0252
7TM-PIMIX
PBLS, 7TM receptor



pCR II




enriched



[Invitrogen]


S0256
7TM-PHMIX
PBLS, 7TM receptor



pCR II




enriched



[Invitrogen]


S0260
Spinal Cord, re-excision
Spinal cord
spinal cord


UNI-ZAP TM








XR


S0262
PYCS
Human Antrum (PY_CS)



pCR II








[Invitrogen]


S0264
PPMIX
PPMIX (Human Pituitary)
Pituitary


pCR II








[Invitrogen]


S0266
PLMIX
PLMIX (Human Lung)
Lung


pCR II








[Invitrogen]


S0268
PRMIX
PRMIX (Human Prostate)
prostate


pCR II








[Invitrogen]


S0270
PTMIX
PTMIX (Human Thymus)
Thymus


pCR II








[Invitrogen]


S0274
PCMIX
PCMIX (Human
Brain


pCR II




Cerebellum)



[Invitrogen]


S0276
Synovial hypoxia-RSF subtracted
Synovial fobroblasts
Synovial


pSport1




(rheumatoid)
tissue





S0278
H Macrophage (GM-CSF
Macrophage (GM-CSF



UNI-ZAP TM



treated), re-excision
treated)



XR


S0280
Human Adipose Tissue, re-
Human Adipose Tissue



UNI-ZAP TM



excision




XR


S0282
Brain Frontal Cortex, re-excision
Brain frontal cortex
Brain


LAMBDA








ZAP TM II


S0284
7TMCTT (Testis)
7TMCTP (Placenta)
Testis


pCR II








[Invitrogen]


S0290
H7TMCTB (Brain)
7TMCTB (Brain)
Kidney


pCR II








[Invitrogen]


S0292
Osteoarthritis (OA-4)
Human Osteoarthritic
Bone

disease
pSport1




Cartilage






S0294
Larynx tumor
Larynx tumor
Larynx, vocal

disease
pSport1





cord





S0296
Normal lung
Normal lung
Lung


pSport1


S0298
Bone marrow stroma, treated
Bone marrow
Bone marrow


pSport1




stroma, treatedSB






S0300
Frontal lobe, dementia; re-excision
Frontal Lobe
Brain


UNI-ZAP TM




dementia/Alzheimer''s



XR


S0306
Larynx normal #10 261-273
Larynx normal



pSport1


S0308
Spleen/normal
Spleen normal



pSport1


S0310
Normal trachea
Normal trachea



pSport1


S0312
Human osteoarthritic; fraction II
Human osteoarthritic


disease
pSport1




cartilage






S0314
Human osteoarthritis; fraction I
Human osteoarthritic


disease
pSport1




cartilage






S0316
Human Normal
Human Normal Cartilage



pSport1



Cartilage, Fraction I







S0318
Human Normal Cartilage
Human Normal Cartilage



pSport1



Fraction II







S0320
Human Larynx
Larynx
Epiglottis


pSport1


S0322
Siebben Polyposis
Siebben Polyposis



pSport1


S0324
Human Brain
Brain
Cerebellum


pSport1


S0326
Mammary Gland
Mammary Gland
Whole


pSport1





mammary








gland





S0328
Palate carcinoma
Palate carcinoma
Uvula

disease
pSport1


S0330
Palate normal
Palate normal
Uvula


pSport1


S0332
Pharynx carcinoma
Pharynx carcinoma
Hypopharynx


pSport1


S0334
Human Normal Cartilage
Human Normal Cartilage



pSport1



Fraction III







S0336
Human Normal Cartilage
Human Normal Cartilage



pSport1



Fraction IV







S0338
Human Osteoarthritic Cartilage
Human osteoarthritic


disease
pSport1



Fraction III
cartilage






S0340
Human Osteoarthritic Cartilage
Human osteoarthritic


disease
pSport1



Fraction IV
cartilage






S0342
Adipocytes; re-excision
Human Adipocytes from



UNI-ZAP TM




Osteoclastoma



XR


S0344
Macrophage-oxLDL; re-excision
macrophage-oxidized LDL
blood
Cell Line

UNI-ZAP TM




treated



XR


S0346
Human Amygdala; re-excision
Amygdala



UNI-ZAP TM








XR


S0348
Cheek Carcinoma
Cheek Carcinoma


disease
pSport1


S0350
Pharynx Carcinoma
Pharynx carcinoma
Hypopharynx

disease
pSport1


S0352
Larynx Carcinoma
Larynx carcinoma


disease
pSport1


S0354
Colon Normal II
Colon Normal
Colon


pSport1


S0356
Colon Carcinoma
Colon Carcinoma
Colon

disease
pSport1


S0358
Colon Normal III
Colon Normal
Colon


pSport1


S0360
Colon Tumor II
Colon Tumor
Colon

disease
pSport1


S0362
Human Gastrocnemius
Gastrocnemius muscle



pSport1


S0364
Human Quadriceps
Quadriceps muscle



pSport1


S0366
Human Soleus
Soleus Muscle



pSport1


S0368
Human Pancreatic Langerhans
Islets of Langerhans



pSport1


S0370
Larynx carcinoma II
Larynx carcinoma


disease
pSport1


S0372
Larynx carcinoma III
Larynx carcinoma


disease
pSport1


S0374
Normal colon
Normal colon



pSport1


S0376
Colon Tumor
Colon Tumor


disease
pSport1


S0378
Pancreas normal PCA4 No
Pancreas Normal PCA4 No



pSport1


S0380
Pancreas Tumor PCA4 Tu
Pancreas Tumor PCA4 Tu


disease
pSport1


S0382
Larynx carcinoma IV
Larynx carcinoma


disease
pSport1


S0384
Tongue carcinoma
Tongue carcinoma


disease
pSport1


S0386
Human Whole Brain, re-excision
Whole brain
Brain


ZAP








EXPRESS TM


S0388
Human
Human Hypothalamus,


disease
UNI-ZAP TM



Hypothalamus, schizophrenia, re-
Schizophrenia



XR



excision







S0390
Smooth muscle, control; re-
Smooth muscle
Pulmanary
Cell Line

UNI-ZAP TM



excision

artery


XR


S0392
Salivary Gland
Salivary gland; normal



pSport1


S0394
Stomach; normal
Stomach; normal



pSport1


S0396
Uterus; normal
Uterus; normal



pSport1


S0398
Testis; normal
Testis; normal



pSport1


S0400
Brain; normal
Brain; normal



pSport1


S0402
Adrenal Gland, normal
Adrenal gland; normal



pSport1


S0404
Rectum normal
Rectum, normal



pSport1


S0406
Rectum tumour
Rectum tumour



pSport1


S0408
Colon, normal
Colon, normal



pSport1


S0410
Colon, tumour
Colon, tumour



pSport1


S0412
Temporal cortex-Alzheizmer;
Temporal cortex, alzheimer


disease
Other



subtracted







S0414
Hippocampus, Alzheimer
Hippocampus, Alzheimer



Other



Subtracted
Subtracted






S0418
CHME Cell Line; treated 5 hrs
CHME Cell Line; treated



pCMVSport 3.0


S0420
CHME Cell Line, untreated
CHME Cell line, untreatetd



pSport1


S0422
Mo7e Cell Line GM-CSF treated
Mo7e Cell Line GM-CSF



pCMVSport 3.0



(1 ng/ml)
treated (1 ng/ml)






S0424
TF-1 Cell Line GM-CSF Treated
TF-1 Cell Line GM-CSF



pSport1




Treated






S0426
Monocyte activated; re-excision
Monocyte-activated
blood
Cell Line

UNI-ZAP TM








XR


S0428
Neutrophils control; re-excision
human neutrophils
blood
Cell Line

UNI-ZAP TM








XR


S0430
Aryepiglottis Normal
Aryepiglottis Normal



pSport1


S0432
Sinus piniformis Tumour
Sinus piniformis Tumour



pSport1


S0434
Stomach Normal
Stomach Normal


disease
pSport1


S0436
Stomach Tumour
Stomach Tumour


disease
pSport1


S0438
Liver Normal Met5No
Liver Normal Met5No



pSport1


S0440
Liver Tumour Met 5 Tu
Liver Tumour



pSport1


S0442
Colon Normal
Colon Normal



pSport1


S0444
Colon Tumor
Colon Tumour


disease
pSport1


S0446
Tongue Tumour
Tongue Tumour



pSport1


S0448
Larynx Normal
Larynx Normal



pSport1


S0450
Larynx Tumour
Larynx Tumour



pSport1


S0452
Thymus
Thymus



pSport1


S0454
Placenta
Placenta
Placenta


pSport1


S0456
Tongue Normal
Tongue Normal



pSport1


S0458
Thyroid Normal (SDCA2 No)
Thyroid normal



pSport1


S0460
Thyroid Tumour
Thyroid Tumour



pSport1


S0462
Thyroid Thyroiditis
Thyroid Thyroiditis



pSport1


S0464
Larynx Normal
Larynx Normal



pSport1


S0466
Larynx Tumor
Larynx Tumor


disease
pSport1


S0468
Ea.hy.926 cell line
Ea.hy.926 cell line



pSport1


S0470
Adenocarcinoma
PYFD


disease
pSport1


S0472
Lung Mesothelium
PYBT



pSport1


S0474
Human blood platelets
Platelets
Blood


Other





platelets





S0665
Human Amygdala; re-excission
Amygdala



UNI-ZAP TM








XR


S3012
Smooth Muscle Serum Treated,
Smooth muscle
Pulmanary
Cell Line

pBLUESCRIPT TM



Norm

artery





S3014
Smooth muscle, serum
Smooth muscle
Pulmanary
Cell Line

pBLUESCRIPT TM



induced, re-exc

artery





S3018
TH1 cells
TH1 cells



UNI-ZAP TM








XR


S6014
H. hypothalamus, frac A
Hypothalamus
Brain


ZAP








EXPRESS TM


S6016
H. Frontal Cortex, Epileptic
H. Brain, Frontal Cortex,
Brain

disease
UNI-ZAP TM




Epileptic



XR


S6022
H. Adipose Tissue
Human Adipose Tissue



UNI-ZAP TM








XR


S6024
Alzheimers, spongy change
Alzheimer''s/Spongy
Brain

disease
UNI-ZAP TM




change



XR


S6026
Frontal Lobe, Dementia
Frontal Lobe
Brain


UNI-ZAP TM




dementia/Alzheimer''s



XR


S6028
Human Manic Depression Tissue
Human Manic depression
Brain

disease
UNI-ZAP TM




tissue



XR


T0001
Human Brown Fat
Brown Fat



pBLUESCRIPT TM








SK−


T0002
Activated T-cells
Activated T-Cell, PBL
Blood
Cell Line

pBLUESCRIPT TM




fraction



SK−


T0003
Human Fetal Lung
Human Fetal Lung



pBLUESCRIPT TM








SK−


T0004
Human White Fat
Human White Fat



pBLUESCRIPT TM








SK−


T0006
Human Pineal Gland
Human Pinneal Gland



pBLUESCRIPT TM








SK−


T0007
Colon Epithelium
Colon Epithelium



pBLUESCRIPT TM








SK−


T0008
Colorectal Tumor
Colorectal Tumor


disease
pBLUESCRIPT TM








SK−


T0010
Human Infant Brain
Human Infant Brain



Other


T0023
Human Pancreatic Carcinoma
Human Pancreatic


disease
pBLUESCRIPT TM




Carcinoma



SK−


T0039
HSA 172 Cells
Human HSA172 cell line



pBLUESCRIPT TM








SK−


T0040
HSC172 cells
SA172 Cells



pBLUESCRIPT TM








SK−


T0041
Jurkat T-cell G1 phase
Jurkat T-cell



pBLUESCRIPT TM








SK−


T0042
Jurkat T-Cell, S phase
Jurkat T-Cell Line



pBLUESCRIPT TM








SK−


T0047
T lymphocytes >70
T lymphocytes >70



pBLUESCRIPT TM








SK−


T0048
Human Aortic Endothelium
Human Aortic Endothilium



pBLUESCRIPT TM








SK−


T0049
Aorta endothelial cells + TNF-a
Aorta endothelial cells



pBLUESCRIPT TM








SK−


T0060
Human White Adipose
Human White Fat



pBLUESCRIPT TM








SK−


T0067
Human Thyroid
Human Thyroid



pBLUESCRIPT TM








SK−


T0067
Human Thyroid
Human Thyroid



PPBLUESCRIPT TM








SK−


T0068
Normal Ovary, Premenopausal
Normal Ovary,



pBLUESCRIPT TM




Premenopausal



SK−


T0069
Human Uterus, normal
Human Uterus, normal



pBLUESCRIPT TM








SK−


T0070
Human Adrenal Gland
Human Adrenal Gland



pBLUESCRIPT TM








SK−


T0071
Human Bone Marrow
Human Bone Marrow



pBLUESCRIPT TM








SK−


T0074
Human Adult Retina
Human Adult Retina



pBluescriptISK−


T0078
Human Liver, normal adult
Human Liver, normal Adult



pBLUESCRIPT TM








SK−


T0079
Human Kidney, normal Adult
Human Kidney, normal



pBLUESCRIPT TM




Adult



SK−


T0082
Human Adult Retina
Human Adult Retina



pBLUESCRIPT TM








SK−


T0082
Human Adult Retina
Human Adult Retina



PPBLUESCRIPT TM








SK−


T0086
Human Pancreatic Carcinoma -
Human Pancreatic


disease
pBLUESCRIPT TM



Screened
Carcinoma



SK−


T0087
Alzheimer''s, exon trap, 712P



disease
pAMP


T0090
Liver, normal




pBLUESCRIPT TM








SK−


T0091
Liver, hepatocellular carcinoma




pBLUESCRIPT TM








SK−


T0103
Human colon carcinoma (HCC)




pBLUESCRIPT TM



cell line




SK−


T0104
HCC cell line metastisis to liver




pBLUESCRIPT TM








SK−


T0109
Human (HCC) cell line liver




pBLUESCRIPT TM



(mouse) metastasis, remake




SK−


T0110
Human colon carcinoma (HCC)




pBLUESCRIPT TM



cell line, remake




SK−


T0112
Human (Caco-2) cell line,




pBLUESCRIPT TM



adenocarcinoma, colon




SK−


T0114
Human (Caco-2) cell line,




pBLUESCRIPT TM



adenocarcinoma, colon, remake




SK−


T0115
Human Colon Carcinoma (HCC)




pBLUESCRIPT TM



cell line




SK−









Table 5

Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B.1, column 9. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000 (world wide web at ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B.1, column 8, as determined using the Morbid Map database.








TABLE 5





OMIM



Reference
Description
















100678
ACAT2 deficiency


100690
Myasthenic syndrome, slow-channel congenital, 601462


100710
Myasthenic syndrome, slow-channel congenital, 601462


100730
Myasthenia gravis, neonatal transient


101000
Meningioma, NF2-related, sporadic Schwannoma, sporadic


102200
Somatotrophinoma


102480
Male infertility due to acrosin deficiency


102540
Cardiomyopathy, idiopathic dilated


102578
Leukemia, acute promyelocytic, PML/RARA type


102700
Severe combined immunodeficiency due to ADA deficiency


102770
Myoadenylate deaminase deficiency


102772
[AMP deaminase deficiency, erythrocytic]


103000
Hemolytic anemia due to adenylate kinase deficiency


103050
Autism, succinylpurinemic


103581
Albright hereditary osteodystrophy-2


103600
[Dysalbuminemic hyperthyroxinemia]


103720
Alcoholism, susceptibility to


103850
Aldolase A deficiency


103950
Emphysema due to alpha-2-macroglobulin deficiency


104150
[AFP deficiency, congenital]


104170
NAGA deficiency, mild


104311
Alzheimer disease-3


104500
Amelogenesis imperfecta-2, hypoplastic local type


104770
Amyloidosis, secondary, susceptibility to


105580
Anal canal carcinoma


105600
Dyserythropoietic anemia, congenital, type III


106100
Angioedema, hereditary


106150
Hypertension, essential, susceptibility to


106165
Hypertension, essential, 145500


106180
Myocardial infarction, susceptibility to


106210
Peters anomaly


106300
Ankylosing spondylitis


107250
Anterior segment mesenchymal dysgenesis


107271
CD59 deficiency


107300
Antithrombin III deficiency


107470
Atypical mycobacterial infection, familial disseminated,



209950


107670
Apolipoprotein A-II deficiency


107680
ApoA-I and apoC-III deficiency, combined


107720
Hypertriglyceridemia


107741
Hyperlipoproteinemia, type III


107776
Colton blood group, 110450


107777
Diabetes insipidus, nephrogenic, autosomal recessive,



222000


107910
Virilization, maternal and fetal, from placental aromatase



deficiency


107970
Arrhythmogenic right ventricular dysplasia-1


108120
Distal arthrogryposis-1


108725
Atherosclerosis, susceptibility to


108730
Brody myopathy, 601003


108800
Atrial septal defect, secundum type


108962
Hypertension, salt-resistant


108985
Atrophia areata


109150
Machado-Joseph disease


109270
Renal tubular acidosis, distal, 179800


109400
Basal cell nevus syndrome


109560
Leukemia/lymphoma, B-cell, 3


109565
Lymphoma, B-cell


109690
Asthma, nocturnal, susceptibility to


109700
Hemodialysis-related amyloidosis


110100
Blepharophimosis, epicanthus inversus, and ptosis, type 1


110700
Vivax malaria, susceptibility to


112250
Bone dysplasia with medullary fibrosarcoma


112261
Fibrodysplasia ossificans progressiva


112262
Fibrodysplasia ossificans progressiva, 135100


112410
Hypertension with brachydactyly


113100
Brachydactyly, type C


113520
Hyperleucinemia-isoleucinemia or hypervalinemia


113721
Breast cancer


113811
Epidermolysis bullosa, generalized atrophic benign, 226650


113900
Heart block, progressive familial, type I


114130
Osteoporosis


114208
Malignant hyperthermia susceptibility 5, 601887


114240
Muscular dystrophy, limb-girdle, type 2A, 253600


114290
Campomelic dysplasia with autosomal sex reversal


114350
Leukemia, acute myeloid


114400
Lynch cancer family syndrome II


114550
Hepatocellular carcinoma


114835
Monocyte carboxyesterase deficiency


115200
Cardiomyopathy, dilated, 1A


115470
Cat eye syndrome


115500
Acatalasemia


115650
Cataract, anterior polar-1


115660
Cataract, cerulean, type 1


115665
Cataract, congenital, Volkmann type


116600
Cataract, posterior polar


116800
Cataract, Marner type


116806
Colorectal cancer


116860
Cavernous angiomatous malformations


117700
[Hypoceruloplasminemia, hereditary]


118210
Charcot-Marie-Tooth neuropathy-2A


118425
Myotonia congenita, dominant, 160800


118470
[CETP deficiency]


118485
Polycystic ovary syndrome with hyperandrogenemia


118504
Epilepsy, benign neonatal, type 1, 121200


118511
Schizophrenia, neurophysiologic defect in


118800
Choreoathetosis, familial paroxysmal


119300
van der Woude syndrome


119530
Orofacial cleft-1


120070
Alport syndrome, autosomal recessive, 203780


120110
Metaphyseal chondrodysplasia, Schmid type


120120
Epidermolysis bullosa dystrophica, dominant, 131750


120131
Alport syndrome, autosomal recessive, 203780


120140
Osteoarthrosis, precocious


120150
Osteogenesis imperfecta, 4 clinical forms, 166200, 166210,



259420, 166220


120160
Osteogenesis imperfecta, 4 clinical forms, 166200, 166210,



259420, 166220


120180
Ehlers-Danlos syndrome, type III


120190
Ehlers-Danlos syndrome, type I, 130000


120215
Ehlers-Danlos syndrome, type I, 130000


120220
Bethlem myopathy, 158810


120240
Bethlem myopathy, 158810


120260
Epiphyseal dysplasia, multiple, type 2, 600204


120280
Stickler syndrome, type III


120290
OSMED syndrome, 215150


120435
Muir-Torre syndrome, 158320


120436
Muir-Torre family cancer syndrome, 158320


120550
C1q deficiency, type A


120570
C1q deficiency, type B


120575
C1q deficiency, type C


120580
C1r/C1s deficiency, combined


120620
SLE susceptibility


120700
C3 deficiency


120810
C4 deficiency


120820
C4 deficiency


120900
C5 deficiency


120920
Measles, susceptibility to


120940
C9 deficiency


120950
C8 deficiency, type I


120960
C8 deficiency, type II


121011
Deafness, autosomal dominant 3, 601544


121014
Heterotaxia, visceroatrial, autosomal recessive


121050
Contractural arachnodactyly, congenital


121300
Coproporphyria


121360
Myeloid leukemia, acute, M4Eo subtype


121800
Corneal dystrophy, crystalline, Schnyder


122720
Nicotine addiction, protection from


123000
Craniometaphyseal dysplasia


123100
Craniosynostosis, type 1


123101
Craniosynostosis, type 2


123270
[Creatine kinase, brain type, ectopic expression of]


123580
Cataract, congenital, autosomal dominant


123620
Cataract, cerulean, type 2, 601547


123660
Cataract, Coppock-like


123829
Melanoma


123940
White sponge nevus, 193900


124020
Mephenytoin poor metabolizer


124030
Parkinsonism, susceptibility to


124200
Darier disease (keratosis follicularis)


125264
Leukemia, acute nonlymphocytic


125270
Porphyria, acute hepatic


125370
Dentatorubro-pallidoluysian atrophy


125490
Dentinogenesis imperfecta-1


125660
Myopathy, desminopathic


125852
Insulin-dependent diabetes mellitus-2


126060
Anemia, megaloblastic, due to DHFR deficiency


126090
Hyperphenylalaninemia due to pterin-4a-carbinolamine



dehydratase deficiency, 264070


126337
Myxoid liposarcoma


126340
Xeroderma pigmentosum, group D, 278730


126391
DNA ligase I deficiency


126451
Schizophrenia, susceptibility to


126452
Autonomic nervous system dysfunction


126600
Doyne honeycomb retinal dystrophy


126650
Chloride diarrhea, congenital, Finnish type, 214700


128100
Dystonia-1, torsion


129010
Neuropathy, congenital hypomyelinating, 1


129490
Ectodermal dysplasia-3, anhidrotic


129500
Ectodermal dysplasia, hidrotic


129900
EEC syndrome-1


130410
Glutaricaciduria, type IIB


130500
Elliptocytosis-1


130650
Beckwith-Wiedemann syndrome


131100
Multiple endocrine neoplasia I


131195
Hereditary hemorrhagic telangiectasia-1, 187300


131210
Atherosclerosis, susceptibility to


131242
Shah-Waardenburg syndrome, 277580


131244
Hirschsprung disease-2, 600155


131400
Eosinophilia, familial


131440
Eosinophilic myeloproliferative disorder


131950
Epidermolysis bullosa, Ogna type


132700
Cylindromatosis


132800
Basal cell carcinoma


132810
Diphenylhydantoin toxicity


133170
Erythremia


133171
[Erythrocytosis, familial], 133100


133200
Erythrokeratodermia variabilis


133430
Breast cancer


133450
Neuroepithelioma


133510
Trichothiodystrophy


133530
Xeroderma pigmentosum, group G, 278780


133550
Dicarboxylicaminoaciduria, 222730


133700
Chondrosarcoma, 215300


133701
Exostoses, multiple, type 2


133780
Vitreoretinopathy, exudative, familial


134370
Membroproliferative glomerulonephritis


134570
Factor XIIIA deficiency


134580
Factor XIIIB deficiency


134637
Autoimmune lymphoproliferative syndrome


134638
Systemic lupus erythematosus, susceptibility, 152700


134790
Hyperferritinemia-cataract syndrome, 600886


134797
Shprintzen-Goldberg syndrome, 182212


134820
Dysfibrinogenemia, alpha type, causing bleeding diathesis


134830
Dysfibrinogenemia, beta type


134850
Dysfibrinogenemia, gamma type


134934
Thanatophoric dysplasia, types I and II, 187600


135300
Fibromatosis, gingival


135600
Ehlers-Danlos syndrome, type X


135700
Fibrosis of extraocular muscles, congenital, 1


135940
Ichthyosis vulgaris, 146700


136132
[Fish-odor syndrome], 602079


136350
Pfeiffer syndrome, 101600


136435
Ovarian dysgenesis, hypergonadotropic, with normal



karyotype, 233300


136530
Male infertility, familial


136550
Macular dystrophy, North Carolina type


136836
Fucosyltransferase-6 deficiency


137350
Amyloidosis, Finnish type, 105120


137600
Iridogoniodysgenesis syndrome


138030
[Hyperproglucagonemia]


138033
Diabetes mellitus, type II


138040
Cortisol resistance


138079
Hyperinsulinism, familial, 602485


138130
Hyperinsulinism-hyperammonemia syndrome


138140
Glucose transport defect, blood-brain barrier


138160
Diabetes mellitus, noninsulin-dependent


138190
Diabetes mellitus, noninsulin-dependent


138250
P5CS deficiency


138300
Hemolytic anemia due to glutathione reductase deficiency


138320
Hemolytic anemia due to glutathione peroxidase deficiency


138491
Startle disease, autosomal recessive


138570
Non-insulin dependent diabetes mellitus, susceptibility to


138571
Glycogen synthase, liver, deficiency of, 240600


138700
[Apolipoprotein H deficiency]


138720
Bernard-Soulier syndrome, type B


138760
[Glyoxalase II deficiency]


138971
Kostmann neutropenia, 202700


138981
Pulmonary alveolar proteinosis, 265120


139130
Hypertension, essential, susceptibility to, 145500


139150
Basal cell carcinoma


139190
Gigantism due to GHRF hypersecretion


139191
Growth hormone deficient dwarfism


139250
Isolated growth hormone deficiency, Illig type with absent



GH and Kowarski type with bioinactive GH


139320
Pituitary ACTH secreting adenoma


139330
Night blindness, congenital stationary


139350
Epidermolytic hyperkeratosis, 113800


139360
Pituitary ACTH-secreting adenoma


140100
[Anhaptoglobinemia]


141750
Alpha-thalassemia/mental retardation syndrome, type 1


141800
Methemoglobinemias, alpha-


141850
Thalassemia, alpha-


141900
Methemoglobinemias, beta-


142000
Thalassemia due to Hb Lepore


142200
HPFH, nondeletion type A


142250
HPFH, nondeletion type G


142270
Hereditary persistence of fetal hemoglobin


142335
Hereditary persistence of fetal hemoglobin, heterocellular,



Indian type


142360
Thrombophilia due to heparin cofactor II deficiency


142380
Hepatocellular carcinoma


142470
[Hereditary persistence of fetal hemoglobin, heterocellular]


142600
Hemolytic anemia due to hexokinase deficiency


142640
Thrombophilia due to elevated HRG


142680
Periodic fever, familial


142857
Pemphigoid, susceptibility to


142858
Beryllium disease, chronic, susceptibility to


142946
Holoprosencephaly-4


142959
Hand-foot-uterus syndrome, 140000


142989
Synpolydactyly, type II, 186000


143100
Huntington disease


143200
Wagner syndrome


143890
Hypercholesterolemia, familial


144120
Hyperimmunoglobulin G1 syndrome


144200
Epidermolytic palmoplantar keratoderma


145001
Hyperparathyroidism-jaw tumor syndrome


145260
Pseudohypoaldosteronism, type II


145410
Opitz G syndrome, type II


145505
Hypertension, essential


145981
Hypocalciuric hypercalcemia, type II


146150
Hypomelanosis of Ito


146200
Hypoparathyroidism, familial


146740
Neutropenia, alloimmune neonatal


146760
[IgG receptor I, phagocytic, familial deficiency of]


146790
Lupus nephritis, susceptibility to


147020
Agammaglobulinemia, 601495


147050
Atopy


147061
Allergy and asthma susceptibility


147110
IgG2 deficiency, selective


147141
Leukemia, acute lymphoblastic


147200
[Kappa light chain deficiency]


147280
Hepatocellular carcinoma


147440
Growth retardation with deafness and mental retardation


147450
Amytrophic lateral sclerosis, due to SOD1 deficiency,



105400


147545
Diabetes mellitus, noninsulin-dependent


147570
Interferon, immune, deficiency


147575
Myelodysplastic syndrome, preleukemic


147660
Interferon, alpha, deficiency


147670
Rabson-Mendenhall syndrome


147680
Severe combined immunodeficiency due to IL2 deficiency


147730
Interleukin-2 receptor, alpha chain, deficiency of


147781
Atopy, susceptibility to


147790
Leukemia, acute lymphocytic, with 4/11 translocation


147791
Jacobsen syndrome


148040
Epidermolysis bullosa simplex, Koebner, Dowling-Meara,



and Weber-Cockayne types, 131900, 131760, 131800


148041
Pachyonychia congenita, Jadassohn-Lewandowsky type,



167200


148043
Meesmann corneal dystrophy, 122100


148065
White sponge nevus, 193900


148066
Epidermolysis bullosa simplex, Koebner, Dowling-Meara,



and Weber-Cockayne types, 131900, 131760, 131800


148067
Nonepidermolytic palmoplantar keratoderma, 600962


148069
Pachyonychia congenita, Jackson-Lawler type, 167210


148070
Liver disease, susceptibility to, from hepatotoxins or viruses


148080
Epidermolytic hyperkeratosis, 113800


148370
Keratolytic winter erythema


148500
Tylosis with esophageal cancer


148900
Klippel-Feil syndrome with laryngeal malformation


150000
Exertional myoglobinuria due to deficiency of LDH-A


150100
Lactate dehydrogenase-B deficiency


150200
[Placental lactogen deficiency]


150210
Lactoferrin-deficient neutrophils, 245480


150230
Langer-Giedion syndrome


150240
Cutis laxa, marfanoid neonatal type


150250
Larsen syndrome, autosomal dominant


150270
Laryngeal adductor paralysis


150292
Epidermolysis bullosa, Herlitz junctional type, 226700


150310
Epidermolysis bullosa, Herlitz junctional type, 226700


151385
Leukemia, acute myeloid


151390
Leukemia, acute T-cell


151400
Leukemia/lymphoma, B-cell, 1


151410
Leukemia, chronic myeloid


151440
Leukemia, T-cell acute lymphoblastoid


151670
Hepatic lipase deficiency


152200
Coronary artery disease, susceptibility to


152427
Long QT syndrome-2


152445
Vohwinkel syndrome, 124500


152760
Hypogonadotropic hypogonadism due to GNRH deficiency,



227200


152780
Hypogonadism, hypergonadotropic


152790
Precocious puberty, male, 176410


153454
Ehlers-Danlos syndrome, type VI, 225400


153455
Cutis laxa, recessive, type I, 219100


153700
Macular dystrophy, vitelliform type


153840
Macular dystrophy, atypical vitelliform


153880
Macular dystrophy, dominant cystoid


153900
Stargardt disease-2


154275
Malignant hyperthermia susceptibility 2


154276
Malignant hyperthermia susceptibility 3


154400
Acrofacial dysostosis, Nager type


154500
Treacher Collins mandibulofacial dysostosis


154545
Chronic infections, due to opsonin defect


154550
Carbohydrate-deficient glycoprotein syndrome, type Ib,



602579


154705
Marfan syndrome, type II


155555
[Red hair/fair skin]


155600
Malignant melanoma, cutaneous


156225
Muscular dystrophy, congenital merosin-deficient


156232
Mesomelic dysplasia, Kantaputra type


156570
Methylcobalamin deficiency, cbl G type


156600
Microcoria, congenital


156845
Tietz syndrome, 103500


156850
Cataract, congenital, with microphthalmia


157140
Dementia, frontotemporal, with parkinsonism, 601630


157147
Abetalipoproteinemia, 200100


157170
Holoprosencephaly-2


157640
PEO with mitochondrial DNA deletions, type 1


157655
Lactic acidosis due to defect in iron-sulfur cluster of



complex I


157900
Moebius syndrome


158590
Spinal muscular atrophy-4


159000
Muscular dystrophy, limb-girdle, type 1A


159001
Muscular dystrophy, limb-girdle, type 1B


159440
Charcot-Marie-Tooth neuropathy-1B, 118200


159555
Leukemia, myeloid/lymphoid or mixed-lineage


159595
Leukemia, transient, of Down syndrome


160760
Cardiomyopathy, familial hypertrophic, 1, 192600


160777
Griscelli disease, 214450


160781
Cardiomyopathy, hypertrophic, mid-left ventricular chamber



type


160900
Myotonic dystrophy


160980
Carney myxoma-endocrine complex


161015
Mitochondrial complex I deficiency, 252010


162100
Neuralgic amyotrophy with predilection for brachial plexus


162150
Obestiy with impaired prohormone processing, 600955


162200
Neurofibromatosis, type 1


162400
Neuropathy, hereditary sensory and autonomic, type 1


163729
Hypertension, pregnancy-induced


163890
Parkinson disease, type 1, 601508


163950
Noonan syndrome-1


164009
Leukemia, acute promyelocytic, NUMA/RARA type


164040
Leukemia, acute promyelocytic, NPM/RARA type


164160
Obesity, severe, due to leptin deficiency


164200
Oculodentodigital dysplasia


164500
Spinocerebellar ataxia-7


164731
Ovarian carcinoma, 167000


164759
Ovarian carcinoma


164761
Medullary thyroid carcinoma, 155240


164770
Myeloid malignancy, predisposition to


164790
Colorectal cancer


164860
Renal cell carcinoma, papillary, familial and sporadic


164920
Piebaldism


164953
Liposarcoma


165240
Pallister-Hall syndrome, 146510


165320
Hepatocellular carcinoma


165500
Optic atrophy 1


166600
Osteopetrosis, AD, type II


166800
Otosclerosis


167000
Ovarian cancer, serous


167250
Paget disease of bone


167409
Optic nerve coloboma with renal disease, 120330


167410
Rhabdomyosarcoma, alveolar, 268220


167415
Hypothyroidism, congenital, due to thyroid dysgenesis or



hypoplasia


168000
Paraganglioma, familial nonchromaffin, 1


168360
Paraneoplastic sensory neuropathy


168450
Hypoparathyroidism, autosomal dominant


168461
Multiple myeloma, 254250


168468
Metaphyseal chondrodysplasia, Murk Jansen type, 156400


168470
Humoral hypercalcemia of malignancy


168500
Parietal foramina


169600
Hailey-Hailey disease


170261
Bare lymphocyte syndrome, type I, due to TAP2 deficiency


170500
Myotonia congenita, atypical acetazolamide-responsive


170650
Periodontitis, juvenile


170995
Zellweger syndrome-2


171050
Colchicine resistance


171060
Cholestasis, progressive familial intrahepatic, type III,



602347


171190
Hypertension, essential, 145500


171650
Lysosomal acid phosphatase deficiency


171760
Hypophosphatasia, adult, 146300


171860
Hemolytic anemia due to phosphofructokinase deficiency


172400
Hemolytic anemia due to glucosephosphate isomerase



deficiency


172411
Colorectal cancer, resistance to


172430
Enolase deficiency


172471
Glycogenosis, hepatic, autosomal


172490
Phosphorylase kinase deficiency of liver and muscle, 261750


173110
Pituitary hormone deficiency, combined


173350
Plasminogen Tochigi disease


173360
Thrombophilia due to excessive plasminogen activator



inhibitor


173370
Plasminogen activator deficiency


173470
Glanzmann thrombasthenia, type B


173610
Platelet alpha/delta storage pool deficiency


173850
Polio, susceptibility to


173870
Xeroderma pigmentosum


173910
Polycystic kidney disease, adult, type II


174000
Medullary cystic kidney disease, AD


174810
Osteolysis, familial expansile


174900
Polyposis, juvenile intestinal


175100
Turcot syndrome, 276300


176000
Porphyria, acute intermittent


176100
Porphyria cutanea tarda


176260
Episodic ataxia/myokymia syndrome, 160120


176261
Jervell and Lange-Nielsen syndrome, 220400


176270
Prader-Willi syndrome


176300
[Dystransthyretinemic hyperthyroxinemia]


176310
Leukemia, acute pre-B-cell


176450
Sacral agenesis-1


176640
Creutzfeldt-Jakob disease, 123400


176730
Diabetes mellitus, rare form


176801
Metachromatic leukodystrophy due to deficiency of SAP-1


176830
Obesity, adrenal insufficiency, and red hair


176860
Purpura fulminans, neonatal


176880
Protein S deficiency


176930
Dysprothrombinemia


176943
Apert syndrome, 101200


176947
Selective T-cell defect


176960
Pituitary tumor, invasive


177070
Spherocytosis, hereditary, Japanese type


177400
Apnea, postanesthetic


177900
Psoriasis susceptibility-1


178300
Ptosis, hereditary congenital, 1


178600
Pulmonary hypertension, familial primary


178640
Pulmonary alveolar proteinosis, congenital, 265120


179095
Male infertility


179450
Ragweed sensitivity


179605
Retinitis pigmentosa, digenic


179615
Reticulosis, familial histiocytic, 267700


179616
Severe combined immunodeficiency, B cell-negative,



601457


179755
Renal cell carcinoma, papillary, 1


179820
[Hyperproreninemia]


180020
Retinal cone dystrophy-1


180069
Retinal dystrophy, autosomal recessive, childhood-onset


180071
Retinitis pigmentosa, autosomal recessive


180072
Night blindness, congenital stationary, type 3, 163500


180090
Retinitis pigmentosa, autosomal recessive


180100
Retinitis pigmentosa-1


180104
Retinitis pigmentosa-9


180105
Retinitis pigmentosa-10


180200
Osteosarcoma, 259500


180240
Leukemia, acute promyelocytic


180250
Retinol binding protein, deficiency of


180297
Anemia, hemolytic, Rh-null, suppressor type, 268150


180380
Night blindness, congenital stationery, rhodopsin-related


180381
Oguchi disease-2, 258100


180385
Leukemia, acute T-cell


180721
Retinitis pigmentosa, digenic


180840
Susceptibility to IDDM


180860
Russell-Silver syndrome


180901
Malignant hyperthermia susceptibility 1, 145600


181030
Salivary gland pleomorphic adenoma


181031
Oguchi disease-1, 258100


181405
Scapuloperoneal spinal muscular atrophy, New England type


181430
Scapuloperoneal syndrome, myopathic type


181460
Schistosoma mansoni, susceptibility/resistance to


181510
Schizophrenia


181600
Sclerotylosis


182138
Anxiety-related personality traits


182279
Prader-Willi syndrome


182280
Small-cell cancer of lung


182290
Smith-Magenis syndrome


182380
Glucose/galactose malabsorption


182381
Renal glucosuria, 253100


182452
Lung cancer, small cell


182500
Cataract, congenital


182600
Spastic paraplegia-3A


182601
Spastic paraplegia-4


182860
Pyropoikilocytosis


182870
Spherocytosis-1


182900
Spherocytosis-2


183600
Split hand/foot malformation, type 1


185000
Stomatocytosis I


185430
Atherosclerosis, susceptibility to


185470
Myopathy due to succinate dehydrogenase deficiency


185800
Symphalangism, proximal


186580
Arthrocutaneouveal granulomatosis


186740
Immunodeficiency due to defect in CD3-gamma


186770
Leukemia, T-cell acute lymphocytic


186780
CD3, zeta chain, deficiency


186830
Immunodeficiency, T-cell receptor/CD3 complex


186855
Leukemia-2, T-cell acute lymphoblastic


186860
Leukemia/lymphoma, T-cell


186880
Leukemia/lymphoma, T-cell


186921
Leukemia, T-cell acute lymphoblastic


186940
[CD4(+) lymphocyte deficiency]


187040
Leukemia-1, T-cell acute lymphoblastic


187680
6-mercaptopurine sensitivity


188025
Thrombocytopenia, Paris-Trousseau type


188070
Bleeding disorder due to defective thromboxane A2 receptor


188400
Velocardiofacial syndrome, 192430


188450
Goiter, adolescent multinodular


188540
Hypothyroidism, nongoitrous


188550
Thyroid papillary carcinoma


188826
Sorsby fundus dystrophy, 136900


189800
Preeclampsia/eclampsia


189980
Leukemia, chronic myeloid


190000
Atransferrinemia


190020
Bladder cancer, 109800


190040
Meningioma, SIS-related


190070
Colorectal adenoma


190100
Geniospasm


190160
Thyroid hormone resistance, 274300, 188570


190182
Colon cancer


190195
Ichthyosiform erythroderma, congenital, 242100


190198
Leukemia, T-cell acute lymphoblastic


190300
Tremor, familial essential, 1


190450
Hemolytic anemia due to triosephosphate isomerase



deficiency


190605
Triphalangeal thumb-polysyndactyly syndrome


190685
Down syndrome


190900
Colorblindness, tritan


191010
Cardiomyopathy, familial hypertrophic, 3, 115196


191030
Nemaline myopathy-1, 161800


191044
Cardiomyopathy, familial hypertrophic


191045
Cardiomyopathy, familial hypertrophic, 2, 115195


191092
Tuberous sclerosis-2


191100
Tuberous sclerosis-1


191170
Colorectal cancer, 114500


191181
Cervical carcinoma


191290
Segawa syndrome, recessive


191315
Insensitivity to pain, congenital, with anhidrosis, 256800


191540
[Urate oxidase deficiency]


192090
Ovarian carcinoma


192340
Diabetes insipidus, neurohypophyseal, 125700


192500
Jervell and Lange-Nielsen syndrome, 220400


192974
Neonatal alloimmune thrombocytopenia


193100
Hypophosphatemic rickets, autosomal dominant


193235
Vitreoretinopathy, neovascular inflammatory


193300
Renal cell carcinoma


193400
von Willebrand disease


193500
Rhabdomyosarcoma, alveolar, 268220


194070
Wilms tumor, type 1


194071
Wilms tumor, type 2


194190
Wolf-Hirschhorn syndrome


200150
Choreoacanthocytosis


200990
Acrocallosal syndrome


201450
Acyl-CoA dehydrogenase, medium chain, deficiency of


201460
Acyl-CoA dehydrogenase, long chain, deficiency of


201470
Acyl-CoA dehydrogenase, short-chain, deficiency of


201475
VLCAD deficiency


201810
3-beta-hydroxysteroid dehydrogenase, type II, deficiency


201910
Adrenal hyperplasia, congenital, due to 21-hydroxylase



deficiency


202110
Adrenal hyperplasia, congenital, due to 17-alpha-



hydroxylase deficiency


203100
Waardenburg syndrome/ocular albinism, digenic, 103470


203200
Albinism, ocular, autosomal recessive


203300
Hermansky-Pudlak syndrome


203310
Ocular albinism, autosomal recessive


203500
Alkaptonuria


203740
Alpha-ketoglutarate dehydrogenase deficiency


203750
3-ketothiolase deficiency


203800
Alstrom syndrome


204500
Ceroid-lipofuscinosis, neuronal 2, classic late infantile


205100
Amyotrophic lateral sclerosis, juvenile


205900
Anemia, Diamond-Blackfan


207750
Hyperlipoproteinemia, type Ib


207800
Argininemia


208100
Arthrogryposis multiplex congenita, neurogenic


208250
Jacobs syndrome


208400
Aspartylglucosaminuria


208900
Ataxia-telangiectasia


209900
Bardet-Biedl syndrome 2


209901
Bardet-Biedl syndrome 1


210900
Bloom syndrome


211420
Breast cancer, ductal


212138
Carnitine-acylcarnitine translocase deficiency


214300
Klippel-Feil syndrome


214400
Charcot-Marie-Tooth neuropathy-4A


214500
Chediak-Higashi syndrome


215700
Citrullinemia


216550
Cohen syndrome


216900
Achromatopsia


216950
C1r/C1s deficiency, combined


217000
C2 deficiency


217030
C3b inactivator deficiency


217050
C6 deficiency


217070
C7 deficiency


217095
Conotruncal cardiac anomalies


217300
Cornea plana congenita, recessive


217800
Macular corneal dystrophy


218000
Andermann syndrome


218030
Apparent mineralocorticoid excess, hypertension due to


219800
Cystinosis, nephropathic


221770
Polycystic lipomembranous osteodysplasia with sclerosing



leukencephalopathy


221820
Gliosis, familial progressive subcortical


222100
Diabetes mellitus, insulin-dependent-1


222600
Atelosteogenesis II, 256050


222700
Lysinuric protein intolerance


222745
DECR deficiency


222800
Hemolytic anemia due to bisphosphoglycerate mutase



deficiency


222900
Sucrose intolerance


223000
Lactase deficiency, adult, 223100


223360
Dopamine-beta-hydroxylase deficiency


223900
Dysautonomia, familial


224100
Congenital dyserythropoietic anemia II


224120
Dyserythropoietic anemia, contenital, type I


225500
Ellis-van Creveld syndrome


226450
Epidermolysis bullosa inversa, junctional


227220
[Eye color, brown]


227400
Thromboembolism susceptibility due to factor V Leiden


227500
Factor VII deficiency


227600
Factor X deficiency


227645
Fanconi anemia, type C


227646
Fanconi anemia, type D


227650
Fanconi anemia, type A


228960
[Kininogen deficiency]


229300
Friedreich ataxia


229600
Fructose intolerance


229700
Fructose-bisphosphatase deficiency


229800
[Fructosuria]


230000
Fucosidosis


230200
Galactokinase deficiency with cataracts


230350
Galactose epimerase deficiency


230400
Galactosemia


230450
Hemolytic anemia due to gamma-glutamylcysteine



synthetase deficiency


230800
Gaucher disease


231550
Achalasia-addisonianism-alacrimia syndrome


231670
Glutaricaciduria, type I


231675
Glutaricaciduria, type IIC


231680
Glutaricaciduria, type IIA


231950
Glutathioninuria


232000
Propionicacidemia, type I or pccA type


232050
Propionicacidemia, type II or pccB type


232300
Glycogen storage disease II


232400
Glycogen storage disease IIIa


232500
Glycogen storage disease IV


232600
McArdle disease


232700
Glycogen storage disease VI


232800
Glycogen storage disease VII


233100
[Renal glucosuria]


233700
Chronic granulomatous disease due to deficiency of NCF-1


233710
Chronic granulomatous disease due to deficiency of NCF-2


234000
Factor XII deficiency


234200
Neurodegeneration with brain iron accumulation


235200
Hemochromatosis


235800
[Histidinemia]


236100
Holoprosencephaly-1


236200
Homocystinuria, B6-responsive and nonresponsive types


236250
Homocystinuria due to MTHFR deficiency


236700
McKusick-Kaufman syndrome


236730
Urofacial syndrome


237300
Carbamoylphosphate synthetase I deficiency


238300
Hyperglycinemia, nonketotic, type I


238310
Hyperglycinemia, nonketotic, type II


238600
Chylomicronemia syndrome, familial


238970
HHH syndrome


239100
Van Buchem disease


239500
Hyperprolinemia, type I


240300
Autoimmune polyglandular disease, type I


240400
Scurvy


243500
Isovalericacidemia


245000
Papillon-Lefevre syndrome


245050
Ketoacidosis due to SCOT deficiency


245200
Krabbe disease


245349
Lacticacidemia due to PDX1 deficiency


245900
Norum disease


246450
HMG-CoA lyase deficiency


246530
Leukotriene C4 synthase deficiency


246600
Pancreatic lipase deficiency


246900
Lipoamide dehydrogenase deficiency


247200
Miller-Dieker lissencephaly syndrome


247640
Leukemia, acute lymphoblastic


248510
Mannosidosis, beta-


248600
Maple syrup urine disease, type Ia


248610
Maple syrup urine disease, type II


248611
Maple syrup urine disease, type Ib


249000
Meckel syndrome


249100
Familial Mediterranean fever


249270
Thiamine-responsive megaloblastic anemia


250100
Metachromatic leukodystrophy


250250
Cartilage-hair hypoplasia


250790
Methemoglobinemia due to cytochrome b5 deficiency


250800
Methemoglobinemia, type I


250850
Hypermethioninemia, persistent, autosomal dominant, due to



methionine adenosyltransferase I/III deficiency


251000
Methylmalonicaciduria, mutase deficiency type


251170
Mevalonicaciduria


251600
Microphthalmia, autosomal recessive


252500
Mucolipidosis II


252800
Mucopolysaccharidosis Ih


252900
Sanfilippo syndrome, type A


252940
Sanfilippo syndrome, type D


253000
Mucopolysaccharidosis IV A


253200
Maroteaux-Lamy syndrome, several forms


253250
Mulibrey nanism


253270
Multiple carboxylase deficiency, biotin-responsive


253601
Miyoshi myopathy, 254130


253700
Muscular dystrophy, limb-girdle, type 2C


253800
Walker-Warburg syndrome, 236670


254210
Myasthenia gravis, familial infantile


254770
Epilepsy, juvenile myoclonic


254780
Myoclonus epilepsy, Lafora type


255800
Schwartz-Jampel syndrome


256030
Nemaline myopathy-2


256100
Nephronophthisis, juvenile


256540
Galactosialidosis


256550
Sialidosis, type I


256700
Neuroblastoma


256731
Ceroid-lipofuscinosis, neuronal-5, variant late infantile


256850
Giant axonal neuropathy-1


257200
Niemann-Pick disease, type A


257220
Niemann-Pick disease, type C


258501
3-methylglutaconicaciduria, type III


258870
Gyrate atrophy of choroid and retina with ornithinemia, B6



responsive or unresponsive


258900
Oroticaciduria


259700
Osteopetrosis, recessive


259730
Renal tubular acidosis-osteopetrosis syndrome


259770
Osteoporosis-pseudoglioma syndrome


259900
Hyperoxaluria, primary, type 1


261510
Pseudo-Zellweger syndrome


261515
Peroxisomal bifunctional enzyme deficiency


261600
Phenylketonuria


261640
Phenylketonuria due to PTS deficiency


261670
Myopathy due to phosphoglycerate mutase deficiency


262000
Bjornstad syndrome


263200
Polycystic kidney disease, autosomal recessive


263700
Porphyria, congenital erythropoietic


264300
Pseudohermaphroditism, male, with gynecomastia


264470
Adrenoleukodystrophy, pseudoneonatal


264700
Pseudo-vitamin D dependency rickets 1


266100
Pyridoxine dependency with seizures


266150
Pyruvate carboxylase deficiency


266200
Anemia, hemolytic, due to PK deficiency


266300
[Hair color, red]


266600
Inflammatory bowel disease-1


267750
Knobloch syndrome


268800
Sandhoff disease, infantile, juvenile, and adult forms


268900
[Sarcosinemia]


269920
Salla disease


270100
Situs inversus viscerum


270200
Sjogren-Larsson syndrome


270800
Spastic paraplegia-5A


271245
Spinocerebellar ataxia-8, infantile, with sensory neuropathy


271900
Canavan disease


272750
GM2-gangliosidosis, AB variant


272800
Tay-Sachs disease


273300
Male germ cell tumor


273800
Thrombocytopenia, neonatal alloimmune


274180
Thromboxane synthase deficiency


274270
Thymine-uraciluria


274600
Pendred syndrome


275200
Thyroid adenoma, hyperfunctioning


275350
Transcobalamin II deficiency


276000
Pancreatitis, hereditary, 167800


276600
Tyrosinemia, type II


276700
Tyrosinemia, type I


276710
Tyrosinemia, type III


276900
Usher syndrome, type 1A


276901
Usher syndrome, type 2


276902
Usher syndrome, type 3


276903
Usher syndrome, type 1B


276904
Usher syndrome, type 1C


277700
Werner syndrome


277730
Wernicke-Korsakoff syndrome, susceptibility to


277900
Wilson disease


278000
Wolman disease


278250
Wrinkly skin syndrome


278300
Xanthinuria, type I


278700
Xeroderma pigmentosum, group A


278760
Xeroderma pigmentosum, group F


300000
Opitz G syndrome, type I


300008
Nephrolithiasis, type I, 310468


300011
Menkes disease, 309400


300031
Mental retardation, X-linked, FRAXF type


300032
Alpha-thalassemia/mental retardation syndrome, type 2,



301040


300037
Simpson dysmorphia syndrome, 312870


300039
Deafness, X-linked 3, conductive, with stapes fixation,



304400


300044
Wernicke-Korsakoff syndrome, susceptibility to


300046
Mental retardation, X-linked 23, nonspecific


300047
Mental retardation, X-linked 20


300048
Intestinal pseudoobstruction, neuronal, X-linked


300049
Nodular heterotopia, bilateral periventricular


300055
Mental retardation with psychosis, pyramidal signs, and



macroorchidism


300062
Mental retardation, X-linked 14


300066
Deafness, X-linked 6, sensorineural


300067
Subcortical laminar heterotopia, X-linked dominant


300071
Night blindness, congenital stationary, type 2


300075
Coffin-Lowry syndrome, 303600


300076
Wood neuroimmunologic syndrome


300077
Mental retardation, X-linked 29


300088
Epilepsy, female restricted, with mental retardation


300100
Adrenoleukodystrophy


300104
Mental retardation, X-linked nonspecific, 309541


300110
Night blindness, congenital stationary, X-linked incomplete,



300071


300121
Subcortical laminal heteropia, X-linked, 300067


300123
Mental retardation with isolated growth hormone deficiency


300126
Dyskeratosis congenita-1, 305000


300127
Mental retardation, X-linked, 60


300136
Diabetes mellitus, insulin-dependent, X-linked, susceptibility



to


300300
XLA and isolated growth hormone deficiency, 307200


300310
Agammaglobulinemia, type 2, X-linked


300500
Ocular albinism, Nettleship-Falls type


300600
Ocular albinism, Forsius-Eriksson type


300650
Ocular albinism with sensorineural deafness


301000
Thrombocytopenia, X-linked, 313900


301200
Amelogenesis imperfecta


301201
Amelogenesis imperfecta-3, hypoplastic type


301220
Partington syndrome II


301300
Anemia, sideroblastic/hypochromic


301310
Anemia, sideroblastic, with spinocerebellar ataxia


301500
Fabry disease


301590
Anophthalmos-1


301830
Arthrogryposis, X-linked (spinal muscular atrophy, infantile,



X-linked)


301835
Arts syndrome


301845
Bazex syndrome


301900
Borjeson-Forssman-Lehmann syndrome


302060
Noncompaction of left ventricular myocardium, isolated


302350
Nance-Horan syndrome


302801
Charcot-Marie-Tooth neuropathy, X-linked-2, recessive


302950
Chondrodysplasia punctata, X-linked recessive, 302940


302960
Chondrodysplasia punctata, X-linked dominant


303400
Cleft palate, X-linked


303630
Alport syndrome, 301050


303631
Leiomyomatosis, diffuse, with Alport syndrome


303700
Colorblindness, blue monochromatic


303800
Colorblindness, deutan


303900
Colorblindness, protan


304020
Cone dystrophy, progressive X-linked, 1


304040
Charcot-Marie-Tooth neuropathy, X-linked-1, dominant,



302800


304050
Aicardi syndrome


304110
Craniofrontonasal dysplasia


304340
Mental retardation, X-linked, syndromic-5, with Dandy-



Walker malformation, basal ganglia disease, and seizures


304500
Deafness, X-linked 2, perceptive congenital


304700
Mohr-Tranebjaerg syndrome


304800
Diabetes insipidus, nephrogenic


305100
Anhidrotic ectodermal dysplasia


305400
Aarskog-Scott syndrome


305435
Heterocellular hereditary persistence of fetal hemoglobin,



Swiss type


305450
FG syndrome


305900
Favism


306000
Glycogenosis, X-linked hepatic, type I


306100
Gonadal dysgenesis, XY female type


306400
Chronic granulomatous disease, X-linked


306700
Hemophilia A


306900
Hemophilia B


306995
[Homosexuality, male]


307150
Hypertrichosis, congenital generalized


307700
Hypoparathyroidism, X-linked


307800
Hypophosphatemia, hereditary


308000
HPRT-related gout


308230
Immunodeficiency, X-linked, with hyper-IgM


308240
Lymphoproliferative syndrome, X-linked


308300
Incontinentia pigmenti, sporadic type


308310
Incontinentia pigmenti, familial


308380
Severe combined immunodeficiency, X-linked, 300400


308700
Kallmann syndrome


308800
Keratosis follicularis spinulosa decalvans


308840
Spastic paraplegia, 312900


309000
Lowe syndrome


309200
Manic-depressive illness, X-linked


309300
Megalocornea, X-linked


309470
Mental retardation, X-linked, syndromic-3, with spastic



diplegia


309500
Renpenning syndrome-1


309510
Mental retardation, X-linked, syndromic-1, with dystonic



movements, ataxia, and seizures


309530
Mental retardation, X-linked 1, non-dysmorphic


309545
Mental retardation, X-linked nonspecific, with aphasia


309548
Mental retardation, X-linked, FRAXE type


309555
Gustavson syndrome


309585
Mental retardation, X-linked, syndromic-6, with



gynecomastia and obesity


309600
Allan-Herndon syndrome


309605
Mental retardation, X-linked, syndromic-4, with congenital



contractures and low fingertip arches


309610
Mental retardation, X-linked, syndromic-2, with



dysmorphism and cerebral atrophy


309620
Mental retardation-skeletal dysplasia


309850
Brunner syndrome


309900
Mucopolysaccharidosis II


310300
Emery-Dreifuss muscular dystrophy


310400
Myotubular myopathy, X-linked


310460
Myopia-1


310490
Cowchock syndrome


310500
Night blindness, congenital stationary, type 1


311050
Optic atrophy, X-linked


311200
Oral-facial-digital syndrome 1


311250
Ornithine transcarbamylase deficiency


311300
Otopalatodigital syndrome, type I


311360
Ovarian failure, premature


311510
Waisman parkinsonism-mental retardation syndrome


311800
Myoglobinuria/hemolysis due to PGK deficiency


311850
Phosphoribosyl pyrophosphate synthetase-related gout


311870
Muscle glycogenosis


312000
Panhypopituitarism, X-linked


312040
N syndrome, 310465


312060
Properdin deficiency, X-linked


312080
Pelizaeus-Merzbacher disease


312170
Pyruvate dehydrogenase deficiency


312600
Retinitis pigmentosa-2


312610
Retinitis pigmentosa-3


312700
Retinoschisis


312760
Turner syndrome


313350
Split hand/foot malformation, type 2


313400
Spondyloepiphyseal dysplasia tarda


313700
Perineal hypospadias


313850
Thoracoabdominal syndrome


314200
[Euthyroidal hyper- and hypothyroxinemia]


314250
Dystonia-3, torsion, with parkinsonism, Filipino type


314300
Goeminne TKCR syndrome


314400
Cardiac valvular dysplasia-1


314580
Wieacker-Wolff syndrome


314850
McLeod phenotype


600020
Prostate cancer, 176807


600035
Schizencephaly


600040
Colorectal cancer


600044
Thrombocythemia, essential, 187950


600045
Xeroderma pigmentosum, group E, subtype 2


600048
Breast cancer-3


600059
Retinitis pigmentosa-13


600065
Leukocyte adhesion deficiency, 116920


600079
Colon cancer


600095
Split hand/foot malformation, type 3


600101
Deafness, autosomal dominant 2


600105
Retinitis pigmentosa-12, autosomal recessive


600119
Muscular dystrophy, Duchenne-like, type 2


600138
Retinitis pigmentosa-11


600140
Rubenstein-Taybi syndrome, 180849


600143
Epilepsy, progressive, with mental retardation


600151
Bardet-Biedl syndrome 3


600160
Melanoma, 155601


600163
Long QT syndrome-3


600173
SCID, autosomal recessive, T-negative/B-positive type


600175
Spinal muscular atrophy, congenital nonprogressive, of



lower limbs


600179
Leber congenital amaurosis, type I, 204000


600184
Carnitine acetyltransferase deficiency


600185
Pancreatic cancer


600194
Ichthyosis bullosa of Siemens, 146800


600202
Dyslexia, specific, 2


600211
Cleidocranial dysplasia, 119600


600221
Venous malformations, multiple cutaneous and mucosal,



600195


600223
Spinocerebellar ataxia-4


600225
Phenylketonuria, atypical, due to GCH1 deficiency, 233910


600228
Pseudohypoaldosteronism, type I, 264350


600231
Palmoplantar keratoderma, Bothnia type


600234
HMG-CoA synthease-2 deficiency


600243
Temperature-sensitive apoptosis


600258
Colorectal cancer, hereditary nonpolyposis, type 3


600259
Turcot syndrome with glioblastoma, 276300


600261
Ehlers-Danlos-like syndrome


600266
Resistance/susceptibility to TB, etc.


600273
Polycystic kidney disease, infantile severe, with tuberous



sclerosis


600276
Cerebral arteriopathy with subcortical infarcts and



leukoencephalopathy, 125310


600281
Non-insulin-dependent diabetes mellitus, 125853


600309
Atrioventricular canal defect-1


600310
Pseudoachondroplasia, 177170


600318
Diabetes mellitus, insulin-dependent, 3


600319
Diabetes mellitus, insulin-dependent, 4


600320
Insulin-dependent diabetes mellitus-5


600321
Diabetes mellitus, insulin-dependent, 7


600332
Rippling muscle disease-1


600354
Spinal muscular atrophy-1, 253300


600359
Bartter syndrome, type 2


600363
Spastic paraplegia-6


600364
Cone dystrophy-3, 602093


600374
Bardet-Biedl syndrome 4


600414
Adrenoleukodystrophy, neonatal, 202370


600415
Ataxia with isolated vitamin E deficiency, 277460


600429
[Ii blood group, 110800]


600509
Persistent hyperinsulinemic hypoglycemia of infancy,



256450


600510
Pigment dispersion syndrome


600511
Schizophrenia-3


600512
Epilepsy, partial


600525
Trichodontoosseous syndrome, 190320


600528
CPT deficiency, hepatic, type I, 255120


600536
Myopathy, congenital


600542
Chondrosarcoma, extraskeletal myxoid


600584
Atrial septal defect with atrioventricular conduction defects,



108900


600593
Craniosynostosis, Adelaide type


600617
Lipoid adrenal hyperplasia, 201710


600618
Leukemia, acute lymphoblastic


600623
Prostate cancer, 176807


600624
Cone-rod retinal dystrophy-1


600631
Enuresis, nocturnal, 1


600650
Myopathy due to CPT II deficiency, 255110


600652
Deafness, autosomal dominant 4


600669
Epilepsy, generalized, idiopathic


600678
Cancer susceptibility


600698
Salivary adenoma


600700
Lipoma


600701
Lipoma


600722
Ceroid lipofuscinosis, neuronal, variant juvenile type, with



granular osmiophilic deposits


600725
Holoprosencephaly-3, 142945


600757
Orofacial cleft-3


600759
Alzheimer disease-4


600760
Pseudohypoaldosteronism, type I, 264350


600761
Pseudohypoaldosteronism, type I, 264350


600792
Deafness, autosomal recessive 5


600795
Dementia, familial, nonspecific


600807
Bronchial asthma


600808
Enuresis, nocturnal, 2


600811
Xeroderma pigmentosum, group E, DDB-negative subtype,



278740


600835
AIDS, resistance to


600837
Hirschsprung disease, 142623


600839
Bartter syndrome, 241200


600850
Schizophrenia disorder-4


600852
Retinitis pigmentosa-17


600856
Beckwith-Wiedemann syndrome, 130650


600881
Cataract, congenital, zonular, with sutural opacities


600882
Charcot-Marie-Tooth neuropathy-2B


600883
Diabetes mellitus, insulin-dependent, 8


600884
Cardiomyopathy, familial dilated 1B


600887
Endometrial carcinoma


600897
Cataract, zonular pulverulent-1, 116200


600900
Muscular dystrophy, limb-girdle, type 2E


600918
Cystinuria, type III


600919
Long QT syndrome-4 with sinus bradycardia


600923
Porphyria variegata, 176200


600937
Persistent hyperinsulinemic hypoglycemia of infancy,



256450


600946
Short stature, autosomal dominant, with normal serum



growth hormone binding protein


600956
Persistent Mullerian duct syndrome, type II, 261550


600957
Persistent Mullerian duct syndrome, type I, 261550


600958
Cardiomyopathy, familial hypertrophic, 4, 115197


600965
Deafness, autosomal dominant 6


600968
Gitelman syndrome, 263800


600971
Deafness, autosomal recessive 6


600974
Deafness, autosomal recessive 7


600975
Glaucoma 3, primary infantile, B


600977
Cone dystrophy, progressive


600983
Pseudohypoaldosteronism type I, autosomal dominant,



177735


600993
Pancreatic cancer


600994
Deafness, autosomal dominant 5


600995
Nephrotic syndrome, idiopathic, steroid-resistant


600996
Arrhythmogenic right ventricular dysplasia-2


600998
Bleeding diathesis due to GNAQ deficiency


601002
5-oxoprolinuria, 266130


601011
Spinocerebellar ataxia-6, 183086


601071
Deafness, autosomal recessive 9


601072
Deafness, autosomal recessive 8


601090
Iridogoniodysgenesis, 601631


601097
Neuropathy, recurrent, with pressure palsies, 162500


601105
Pycnodysostosis, 265800


601107
Dubin-Johnson syndrome, 237500


601130
Tolbutamide poor metabolizer


601145
Epilepsy, progressive myoclonic 1, 254800


601146
Brachydactyly, type C, 113100


601154
Cardiomyopathy, dilated, 1E


601199
Neonatal hyperparathyroidism, 239200


601202
Cataract, anterior polar-2


601208
Insulin-dependent diabetes mellitus-11


601226
Progressive external ophthalmoplegia, type 2


601238
Cerebellar ataxia, Cayman type


601267
HIV infection, susceptibility/resistence to


601277
Ichthyosis, lamellar, type 2


601282
Muscular dystrophy with epidermolysis bullosa simplex,



226670


601284
Hereditary hemorrhagic telangiectasia-2, 600376


601295
Bile acid malabsorption, primary


601309
Basal cell carcinoma, sporadic


601313
Polycystic kidney disease, adult type I, 173900


601316
Deafness, autosomal dominant 10


601318
Diabetes mellitus, insulin-dependent, 13


601362
DiGeorge syndrome/velocardiofacial syndrome complex-2


601363
Wilms tumor, type 4


601369
Deafness, autosomal dominant 9


601373
HIV infection, susceptibility/resistance to


601382
Charcot-Marie-Tooth neuropathy-4B


601385
Prostate cancer


601386
Deafness, autosomal recessive 12


601387
Breast cancer


601399
Platelet disorder, familial, with associated myeloid



malignancy


601402
Leukemia, myeloid, acute


601406
B-cell non-Hodgkin lymphoma, high-grade


601410
Diabetes mellitus, transient neonatal


601411
Muscular dystrophy, limb-girdle, type 2F, 601287


601412
Deafness, autosomal dominant 7


601414
Retinitis pigmentosa-18


601455
Hereditary motor and sensory neuropathy, Lom type


601458
Inflammatory bowel disease-2


601471
Moebius syndrome-2


601472
Charcot-Marie-Tooth neuropathy-2D


601493
Cardiomyopathy, dilated 1C


601494
Cardiomyopathy, familial, dilated-2


601498
Peroxisomal biogenesis disorder, complementation group 4


601517
Spinocerebellar ataxia-2, 183090


601518
Prostate cancer, hereditary, 1, 176807


601542
Rieger syndrome, type 1, 180500


601545
Lissencephaly-1


601556
Spinocerebellar ataxia-1, 164400


601567
Combined factor V and VIII deficiency, 227300


601596
Charcot-Marie-Tooth neuropathy, demyelinating


601604
Mycobacterial and salmonella infections, susceptibility to


601606
Trichoepithelioma, multiple familial


601607
Rhabdoid tumors


601620
Holt-Oram syndrome, 142900


601621
Ulnar-mammary syndrome, 181450


601622
Saethre-Chotzen syndrome, 101400


601623
Angelman syndrome


601649
Blepharophimosis, epicanthus inversus, and ptosis, type 2


601650
Paraganglioma, familial nonchromaffin, 2


601652
Glaucoma 1A, primary open angle, juvenile-onset, 137750


601653
Branchiootic syndrome


601666
Insulin-dependent diabetes mellitus-15


601669
Hirschsprung disease, one form


601676
Acute insulin response


601680
Distal arthrogryposis, type 2B


601682
Glaucoma 1C, primary open angle


601687
Meesmann corneal dystrophy, 122100


601690
Platelet-activating factor acetylhydrolase deficiency


601691
Retinitis pigmentosa-19, 601718


601692
Reis-Bucklers corneal dystrophy


601718
Retinitis pigmentosa-19


601728
Bannayan-Zonana syndrome, 153480


601744
Systemic lupus erythematosus, susceptibility to, 1


601757
Rhizomelic chondrodysplasia punctata, type 1, 215100


601768
Leukemia, acute myeloid


601769
Osteoporosis, involutional


601771
Glaucoma 3A, primary infantile, 231300


601777
Cone dystrophy, progressive


601780
Ceroid-lipofuscinosis, neuronal-6, variant late infantile


601785
Carbohydrate-deficient glycoprotein syndrome, type I,



212065


601800
[Hair color, brown]


601843
Hypothyroidism, congenital, 274400


601844
Pseudohypoaldosteronism type II


601846
Muscular dystrophy with rimmed vacuoles


601847
Progressive intrahepatic cholestasis-2


601850
Retinitis pigmentosa-deafness syndrome


601863
Bare lymphocyte syndrome, complementation group C


601868
Deafness, autosomal dominant 13


601884
[High bone mass]


601885
Cataract, zonular pulverulent-2


601889
Lymphoma, diffuse large cell


601916
Pancreatic cancer


601920
Alagille syndrome, 118450


601928
Monilethrix, 158000


601941
Insulin-dependent diabetes mellitus-6


601954
Muscular dystrophy, limb-girdle, type 2G


601969
Medulloblastoma, 155255


601975
Ectodermal dysplasia/skin fragility syndrome


601990
Neuroblastoma


602014
Hypomagnesemia with secondary hypocalcemia


602023
Bartter syndrome, type 3


602025
Obesity/hyperinsulinism, susceptibility to


602028
Multiple myeloma


602066
Convulsions, infantile and paroxysmal choreoathetosis


602067
Cardiomyopathy, dilated, 1F


602078
Fibrosis of extraocular muscles, congenital, 2


602080
Paget disease of bone-2


602081
Speech-language disorder-1


602082
Corneal dystrophy, Thiel-Behnke type


602084
Endometrial carcinoma


602085
Postaxial polydactyly, type A2


602086
Arrhythmogenic right ventricular dysplasia-3


602087
Arrhythmogenic right ventricular dysplasia-4


602088
Nephronophthisis, infantile


602089
Hemangioma, capillary, hereditary


602091
Marfan syndrome, atypical


602092
Deafness, autosomal recessive 18


602094
Lipodystrophy, familial partial


602096
Alzheimer disease-5


602099
Amytrophic lateral sclerosis-5


602116
Glioma


602117
Prader-Willi syndrome


602121
Deafness, autosomal dominant nonsyndromic sensorineural,



1, 124900


602134
Tremor, familial essential, 2


602136
Refsum disease, infantile, 266510


602153
Monilethrix, 158000


602216
Peutz-Jeghers syndrome, 175200


602221
Stem-cell leukemia/lymphoma syndrome


602225
Cone-rod retinal dystrophy-2, 120970


602229
Waardenburg-Shah syndrome, 277580


602232
Epilepsy, benign neonatal, type 2, 121201


602235
Epilepsy, benign, neonatal, type 1, 121200


602279
Oculopharyngeal muscular dystorphy, 164300


602280
Retinitis pigmentosa-14, 600132


602363
Ellis-van Creveld-like syndrome


602397
Cholestasis, benign recurrent intrahepatic, 243300


602403
Alzheimer disease, susceptibility to


602404
Parkinson disease, type 3


602421
Sweat chloride elevation without CF


602447
Coronary artery disease, susceptibility to


602460
Deafness, autosomal dominant 15, 602459


602475
Ossification of posterior longitudinal ligament of spine


602476
Febrile convulsions, familial, 1


602477
Febrile convulsions, familial, 2


602491
Hyperlipidemia, familial combined, 1


602522
Bartter syndrome, infantile, with sensorineural deafness


602544
Parkinson disease, juvenile, type 2, 600116


602568
Homocystinuria-megaloblastic anemia, cbl E type, 236270


602574
Deafness, autosomal dominant 12, 601842


602575
Nail-patella syndrome with open-angle glaucoma, 137750


602616
Carbohydrate-deficient glycoprotein syndrome, type II,



212066


602629
Dystonia-6, torsion


602631
Rhabdomyosarcoma, 268210


602666
Deafness, autosomal recessive 3, 600316


602669
Anterior segment mesenchymal dysgenesis and cataract,



107250


602685
Mental retardation, severe, with spasticity and tapetoretinal



degeneration


602716
Nephrosis-1, congenital, Finnish type, 256300


602759
Prostate cancer, hereditary, 2, 176807


602771
Muscular dystrophy, congenital, with early spine rigidity


602772
Retinitis pitmentosa-24


602782
Faisalabad histiocytosis


602783
Spastic paraplegia-7









Mature Polypeptides

The present invention also encompasses mature forms of a polypeptide having the amino acid sequence of SEQ ID NO:Y and/or the amino acid sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional activities of the full-length or mature form of the polypeptide (e.g., biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating disorders such as immune, cardiovascular, cancer, and other proliferative disorders), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.
According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.
In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1A.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the predicted mature form of the polypeptide as delineated in columns 14 and 15 of Table 1A. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional activities of the full-length or mature form of the polypeptide (e.g., biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating disorders such as immune, cardiovascular, cancer, and other proliferative disorders), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynucleotides encoding proteins comprising, or consisting of, the predicted mature form of polypeptides of the invention (e.g., polynucleotides having the sequence of SEQ ID NO:X (Table 1A, column 5), the sequence delineated in columns 7 and 8 of Table 1A, and a sequence encoding the mature polypeptide delineated in columns 14 and 15 of Table 1A (e.g., the sequence of SEQ ID NO:X encoding the mature polypeptide delineated in columns 14 and 15 of Table 1A) are also encompassed by the invention, as are fragments or variants of these polynucleotides (such as, fragments as described herein, polynucleotides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polynucleotides, and nucleic acids which hybridizes under stringent conditions to the complementary strand of the polynucleotide).
As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 15 residues of the predicted cleavage point (i.e., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 more or less contiguous residues of SEQ ID NO:Y at the N-terminus when compared to the predicted mature form of the polypeptide (e.g., the mature polypeptide delineated in columns 14 and 15 of Table 1A). Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
Polynucleotide and Polypeptide Variants
The present invention is also directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID NO:X that encodes the polypeptide sequence as defined in columns 11, 12, 13, 14, and/or 15 of Table 1A, nucleotide sequences encoding the polypeptide sequence as defined in columns 11, 12, 13, 14, and/or 15 of Table 1A, the nucleotide sequence of SEQ ID NO:X encoding the polypeptide sequence as defined in Table 1B.1 (such as the sequence defined in column 5 of Table 1B.1), nucleotide sequences encoding the polypeptide as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1), the nucleotide sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, the nucleotide sequence as defined in column 6 of Table 1C, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1C, the cDNA sequence contained in ATCCTM Deposit No:Z, nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in ATCCTM Deposit No:Z, and/or nucleotide sequences encoding a mature (secreted) polypeptide encoded by the cDNA sequence contained in ATCCTM Deposit No:Z.
The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide as defined in columns 11, 12, 13, 14, and/or 15 of Table 1A, the polypeptide sequence as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1), a polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined in column 6 of Table 1C, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, the polypeptide sequence encoded by the cDNA sequence contained in ATCCTM Deposit No:Z and/or a mature (secreted) polypeptide encoded by the cDNA sequence contained in ATCCTM Deposit No:Z.
“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence described in SEQ ID NO:X or contained in the cDNA sequence of ATCCTM Deposit No:Z; (b) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCCTM Deposit No:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCCTM Deposit No:Z which encodes a mature polypeptide (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)); (d) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCCTM Deposit No:Z, which encodes a biologically active fragment of a polypeptide; (e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCCTM Deposit No:Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID NO:Y (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)) or a mature polypeptide of the amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA contained in ATCCTM Deposit No:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1) or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1) or the complementary strand thereto, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
In a preferred embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides. In another preferred embodiment, polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z; (b) the amino acid sequence of a mature (secreted) form of a polypeptide having the amino acid sequence of SEQ ID NO:Y (e.g., as delineated in columns 14 and 15 of Table 1A) or a mature form of the amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z mature; (c) the amino acid sequence of a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCCTM Deposit No:Z.
The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence encoded by the cDNA contained in ATCCTM Deposit No:Z, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C, the amino acid sequence as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1), an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to in Tables 1B.1 or 2 as the ORF (open reading frame), or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence delineated in columns 14 and 15) or a fragment thereof, Table 1B.1 (e.g., the amino acid sequence identified in column 6) or a fragment thereof, Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C or a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z, or a fragment thereof, the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
Thus, the invention further includes polypeptide variants which show a biological or functional activity of the polypeptides of the invention (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating disorders such as immune or cardiovascular disorders). Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues); and (4) in situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).
Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in fact, encode a polypeptide having functional activity. By a polypeptide having “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein and/or a mature (secreted) protein of the invention. Such functional activities include, but are not limited to, biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.
The functional activity of the polypeptides, and fragments, variants, derivatives, and analogs of the invention, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind or compete with a full-length polypeptide of the present invention for binding to an anti-polypeptide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a polypeptide of the present invention to bind to a substrate(s) of the polypeptide of the invention can be routinely assayed using techniques known in the art.
In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants, derivatives, and analogs thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in ATCCTM Deposit No:Z, the nucleic acid sequence referred to in Tables 1B.1 and 1B.2 (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 1A (e.g., the nucleic acid sequence delineated in columns 7 and 8), the nucleic acid sequence disclosed in Table 2 (e.g., the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides “having functional activity.” In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, serum albumin (preferably human serum albumin) or a fragment thereof, or leader or secretory sequence, or a sequence facilitating purification, or (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course it is highly preferable for a polypeptide to have an amino acid sequence which, for example, comprises the amino acid sequence of a polypeptide of SEQ ID NO:Y, the amino acid sequence of the mature (e.g., secreted) polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X, an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ ID NO:X, an amino acid sequence encoded by cDNA contained in ATCCTM Deposit No:Z, and/or the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z, or a fragment thereof, which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in ATCCTM Deposit No:Z or fragments thereof; wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynucleotide and Polypeptide Fragments
The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a “polynucleotide fragment” refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in ATCCTM Deposit No:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the mature (secreted) polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the mature amino acid sequence as defined in columns 14 and 15 of Table 1A or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto.
The polynucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in ATCCTM Deposit No:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context “about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length) are also encompassed by the invention.
Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity; such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in ATCCTM Deposit No:Z, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1C, column 6. Additional, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand of a sequence delineated in column 6 of Table 1C. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C which correspond to the same ATCCTM Deposit No:Z (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of the amino acid sequence contained in SEQ ID NO:Y, is a portion of the mature form of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, is a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO:X, is a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, is a portion of the amino acid sequence of a mature (secreted) polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, and/or is a portion of an amino acid sequence encoded by the cDNA contained in ATCCTM Deposit No:Z. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA and SEQ ID NO:Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities; such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
The present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, and/or a mature polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z). In particular, N-terminal deletions may be described by the general formula m−q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q−6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, and/or a mature polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z). In particular, C-terminal deletions may be described by the general formula 1−n, where n is any whole integer ranging from 6 to q−1, and where n corresponds to the position of amino acid residue in a polypeptide of the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA contained in ATCCTM Deposit No:Z, and/or the complement thereof, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in ATCCTM Deposit No:Z, or the polynucleotide sequence as defined in column 6 of Table 1C, may be analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2) or the cDNA contained in ATCCTM Deposit No:Z may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, Wis. 53715 USA, world wide web at dnastar.com/).
Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide regions that may be routinely obtained using the DNASTAR computer algorithm include, but are not limited to, Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions; Eisenberg alpha- and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., 1, 2, 3 or 4) of the features set out above. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.
Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g. biological activity such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) of the polypeptide sequence of which the amino acid sequence is a fragment. By a polypeptide displaying a “functional activity” is meant a polypeptide capable of one or more known functional activities associated with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimerization, as described herein.
Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity. By a polypeptide demonstrating a “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.
The functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the antigenic fragments of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Epitopes and Antibodies
The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereto; the polypeptide sequence encoded by the cDNA contained in ATCCTM Deposit No:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in ATCCTM Deposit No:Z under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lower stringency hybridization conditions defined supra.
The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)
In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
Non-limiting examples of epitopes of polypeptides that can be used to generate antibodies of the invention include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y specified in Table 1B.1 (such as the sequence specified in column 6 of Table 1B.1). These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index that is included in the DNAStar suite of computer programs. By “comprise” it is intended that a polypeptide contains at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y shown in Table 1B.1 (such as the sequence specified in column 6 of Table 1B.1), but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterologous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein. In particular embodiments, epitope portions of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in Table 1B.1 (such as the sequence specified in column 6 of Table 1B.1).
Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides. By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-x of human serum albumin, where x is an integer from 1 to 585 and the albumin fragment has human serum albumin activity. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1−z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Fusion Proteins
Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as targeting molecules once fused to other proteins.
Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
In certain preferred embodiments, proteins of the invention are fusion proteins comprising an amino acid sequence that is an N and/or C-terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence of the invention. Polynucleotides encoding these proteins are also encompassed by the invention.
Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
As one of skill in the art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, can be combined with heterologous polypeptide sequences. For example, the polypeptides of the present invention may be fused with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), or albumin (including, but not limited to, native or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).) For example, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A 0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995). Polynucleotides comprising or alternatively consisting of nucleic acids which encode these fusion proteins are also encompassed by the invention.
Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a polypeptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).
Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
Recombinant and Synthetic Production of Polypeptides of the Invention
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCTM Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBLUESCRIPTTM vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from STRATAGENETM Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from PHARMACIATM Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from STRATAGENETM; and pSVK3, pBPV, pMSG and pSVL available from PHARMACIATM. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.
Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors is the availability of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169 (1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.
The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication Number WO 96/29411; International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (121I, 123I, 125I, 131I), carbon (14C), sulfur (35S), tritium (3H), indium (111In, 112In, 113mIn, 115mIn), technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd) molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh and 97Ru.
In specific embodiments, a polypeptide of the present invention or fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177Lu, 90Y, 166Ho, and 153Sm, to polypeptides. In a preferred embodiment the radiometal ion associated with the macrocyclic chelators is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 90Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
As mentioned, the proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation that exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monomethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO2CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichlorophenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in ATCCTM Deposit No:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in ATCCTM Deposit No:Z). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.
In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.
The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z, and/or an epitope, of the present invention) as determined by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgG1. In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.
Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures. Preferred epitopes of the invention include the predicted epitopes shown in Table 1B.1 (such as epitopes shown in column 7 of Table 1B.1), as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6M, 5×10−7 M, 107 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); incorporated by reference herein in its entirety.
As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.
The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCCTM. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCCTM #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human×mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as ABGENIXTM, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein.
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suppliers, including, for example Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169 (1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entireties by reference herein.
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).
As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP 394,827; and Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al., J. Biochem. 270:3958-3964 (1995). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. See, for example, EP A 232,262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Assays for Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
Antibodies of the invention may be characterized using immunocytochemistry methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
Therapeutic Uses
Table 1D: In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Table 1A and Table 1D (in the same row as the disease or disorder to be treated is listed in the “Preferred Indications” column of Table 1D) in an amount effective to treat, prevent, or ameliorate the disease or disorder.
As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to treat the associated disease.
The present invention encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, diagnose, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in preventing, treating, diagnosing, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.
In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.
The “Preferred Indication” column describes diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
The recitation of “Cancer” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).
In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.
The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.
The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.
The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.
The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.
The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.
The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).
The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.
The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.
The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.
The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.
The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.
Table 1E also provides information regarding biological activities and preferred therapeutic uses (i.e. see, “Preferred Indications” column) for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information regarding assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA ATCCTM Deposit No:Z”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention as well as indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, diagnosing, preventing, and/or treating.
Tables 1E.1 and 1E.2 also provide information regarding biological activities and preferred therapeutic uses (i.e. see, “Preferred Indications” column) for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Tables 1E.2 also provide information regarding assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column of Table 1E.1 (“Gene No.”) provides the gene number in the application for each clone identifier. The second column of Table 1E.1 (“cDNA Clone ID”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, and 1C. The third column of Table 1E.1 (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, 1B.1, 1B.2, and 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides).
In Table 1E.2, each of the biological activities of Table 1E.1 are listed by “Biological Activity Number” and the corresponding “Biological Activity” and are followed by an “Exemplary Activity Assay” column and a “Preferred Indication” column; however, for some biological activities no “Exemplary Activity Assay” or “Preferred Indication” is given. The “Exemplary Activity Assay” column describes the biological activity listed in the column that precedes it and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The “Preferred Indication” column also refers to the biological activity listed in the preceding column and describes disease(s) or disorder(s) that may be detected, diagnosed, prevented, treated, or ameliorated by the nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof).
The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to detect, prevent, diagnose, prognosticate, treat, inhibit and/or ameliorate diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein, such as immune, cardiovascular, cancer, and other proliferative diseases and disorders. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions, such as immune, cardiovascular, cancer, and other proliferative diseases and disorders. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions, and/or as described elsewhere herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such as, for example, a predicted linear epitope shown in Table 1B.1 (such as an epitope shown in column 7 of Table 1B.1); or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to detect, diagnose, inhibit, prevent, treat, prognosticate, and/or ameliorate diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein (such as immune, cardiovascular, cancer, and other proliferative diseases and disorders). The treatment and/or prevention of diseases, disorders, or conditions (such as immune, cardiovascular, cancer, and other proliferative diseases and disorders) associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diseases and disorders (such as immune, cardiovascular, cancer, and other proliferative diseases and disorders) related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, and 10−15 M.
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder (such an immune, cardiovascular, cancer, and other proliferative disease or disorder) associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
In a preferred embodiment, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; BIOLISTICTM, DUPONTTM), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
In a specific embodiment, viral vectors which contain nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; BIOLISTICTM, DUPONTTM), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Diagnosis and Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, prognosticate, or monitor diseases, disorders, and/or conditions (such as immune, cardiovascular, cancer, and other proliferative diseases, disorders, and conditions) associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disease or disorder (such an immune, cardiovascular, cancer, or other proliferative disease or disorders), comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disease or disorder. With respect to cancer and other hyperproliferative diseases and disorders (such as immunogenic cancer or cancer of the cardiovascular system), the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer or other hyperproliferative disease (such as immunogenic cancer or cancer of the cardiovascular system).
Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
One facet of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
Kits
The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody that does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (SIGMATM, St. Louis, Mo.).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Uses of the Polynucleotides
Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome, thus each polynucleotide of the present invention can routinely be used as a chromosome marker using techniques known in the art. Table 1B.1, column 8 provides the chromosome location of some of the polynucleotides of the invention.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably at least 15 bp (e.g., 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can optionally be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to SEQ ID NO:X will yield an amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA libraries, and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its entirety).
Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).
For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.
Thus, the present invention also provides a method for chromosomal localization which involves (a) preparing PCR primers from the polynucleotide sequences in Table 1B.1 and/or 2 and SEQ ID NO:X and (b) screening somatic cell hybrids containing individual chromosomes.
The polynucleotides of the present invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping. For a review of these techniques and others known in the art, see, e.g. Dear, “Genome Mapping: A Practical Approach,” IRL Press at Oxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry. 3:483-492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by reference in its entirety.
Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Table 1B.1 (such as column 9 of Table 1B.1) provides an OMIM reference identification number of diseases associated with the cytologic band disclosed in Table 1B.1 (such as column 8 of Table 1B.1), as determined using techniques described herein and by reference to Table 5. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
Thus, once coinheritance is established, differences in a polynucleotide of the invention and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker. Diagnostic and prognostic methods, kits and reagents encompassed by the present invention are briefly described below and more thoroughly elsewhere herein (see e.g., the sections labeled “Antibodies”, “Diagnostic Assays”, and “Methods for Detecting Diseases”).
Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder. Additional non-limiting examples of diagnostic methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., Example 12).
In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the invention, where each probe has one strand containing a 31′mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.
Where a diagnosis of a related disorder, including, for example, diagnosis of a tumor, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
By “measuring the expression level of polynucleotides of the invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder or being determined by averaging levels from a population of individuals not having a related disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source that contains polypeptide of the present invention or the corresponding mRNA. As indicated, biological samples include body fluids (such as semen, lymph, vaginal pool, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
The method(s) provided above may preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides of the invention are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the invention attached may be used to identify polymorphisms between the isolated polynucleotide sequences of the invention, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, such as for example, in neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, digestive disorders, metabolic disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supra are hereby incorporated by reference in their entirety herein.
The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides of the invention are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (Tm) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
The compounds of the present invention have uses which include, but are not limited to, detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., “The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra)
For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO 91/15580). However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5′ end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells. (International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness is not be limited to treatment, prevention, and/or prognosis of proliferative disorders of cells and tissues of hematopoietic origin, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.
In addition to the foregoing, a polynucleotide of the present invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. The oligonucleotide described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide of the present invention antigens. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease, and in particular, for the treatment of proliferative diseases and/or conditions. Non-limiting antisense and triple helix methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the section labeled “Antisense and Ribozyme (Antagonists)”).
Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled “Gene Therapy Methods”, and Examples 16, 17 and 18).
The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention, specific to tissues, including but not limited to those shown in Table 1B.2. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Additional non-limiting examples of such uses are further described herein.
The polynucleotides of the present invention are also useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays) or cell type(s) (e.g., immunocytochemistry assays). In addition, for a number of disorders of the above tissues or cells, significantly higher or lower levels of gene expression of the polynucleotides/polypeptides of the present invention may be detected in certain tissues (e.g., tissues expressing polypeptides and/or polynucleotides of the present invention, for example, those disclosed in Table 1B.2 (such as in column 5 of Table 1B.2), and/or cancerous and/or wounded tissues) or bodily fluids (e.g., semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
Thus, the invention provides a diagnostic method of a disorder, which involves: (a) assaying gene expression level in cells or body fluid of an individual; (b) comparing the gene expression level with a standard gene expression level, whereby an increase or decrease in the assayed gene expression level compared to the standard expression level is indicative of a disorder.
In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
Uses of the Polypeptides
Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
Polypeptides and antibodies directed to polypeptides of the invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistry assays).
Antibodies can be used to assay levels of polypeptides encoded by polynucleotides of the invention in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
In addition to assaying levels of polypeptide of the present invention in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc, (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F, 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which express the polypeptide encoded by a polynucleotide of the invention. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., polypeptides encoded by polynucleotides of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention in association with toxins or cytotoxic prodrugs.
By “toxin” is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi, or other radioisotopes such as for example 103Pd, 133Xe, 131I, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, 90Yttrium, 117Tin, 186Rhenium, 166Holmium, and 188Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 90Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 111In. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 131I.
Techniques known in the art may be applied to label polypeptides of the invention (including antibodies). Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).
Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Moreover, polypeptides of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor suppressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an antibody directed to a polypeptide of the present invention can bind, and/or neutralize the polypeptide, and/or reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the biological activities described herein.
Diagnostic Assays
The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described in the legends for Tables 1D, 1E.1, and 1F, and as indicated in the “Preferred Indications” columns in Table 1D, 1E.1, and 1F; and, also as described herein under the section heading “Biological Activities.” Such disorders also include, but are not limited to, those related to biological activities described in Tables 1E, 1E.1, and 1E.2.
For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding the polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2 (such as column 5) (Tissue Distribution Library Code).
By “assaying the expression level of the gene encoding the polypeptide” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of polypeptides of the invention compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur using any art-known method.
Assaying polypeptide levels in a biological sample can occur using antibody-based techniques. For example, polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of interest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
In a preferred embodiment, antibodies, or fragments of antibodies directed to any one or all of the predicted epitope domains of the polypeptides of the invention (shown in Table 1B.1 such as column 7 of Table 1B.1) may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
In an additional preferred embodiment, antibodies, or fragments of antibodies directed to a conformational epitope of a polypeptide of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
The antibodies (or fragments thereof), and/or polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The antibody (or fragment thereof) or polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the gene product, or conserved variants or peptide fragments, or polypeptide binding, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Immunoassays and non-immunoassays for gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled antibody or detectable polypeptide of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
By “solid phase support or carrier” is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
The binding activity of a given lot of antibody or antigen polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
In addition to assaying polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, polypeptide or polynucleotide can also be detected in vivo by imaging. For example, in one embodiment of the invention, polypeptides and/or antibodies of the invention are used to image diseased cells, such as neoplasms. In another embodiment, polynucleotides of the invention (e.g., polynucleotides complementary to all or a portion of an mRNA) and/or antibodies (e.g., antibodies directed to any one or a combination of the epitopes of a polypeptide of the invention, antibodies directed to a conformational epitope of a polypeptide of the invention, or antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell) are used to image diseased or neoplastic cells.
Antibody labels or markers for in vivo imaging of polypeptides of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where in vivo imaging is used to detect enhanced levels of polypeptides for diagnosis in humans, it may be preferable to use human antibodies or “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).
Additionally, any polypeptides of the invention whose presence can be detected, can be administered. For example, polypeptides of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
A polypeptide-specific antibody or antibody fragment that has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the antigenic protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
With respect to antibodies, one of the ways in which an antibody of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect polypeptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Methods for Detecting Diseases
In general, a disease may be detected in a patient based on the presence of one or more proteins of the invention and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a disease or disorder, including cancer and/or as described elsewhere herein. In addition, such proteins may be useful for the detection of other diseases and cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding polypeptides of the invention, which is also indicative of the presence or absence of a disease or disorder, including cancer. In general, polypeptides of the invention should be present at a level that is at least three fold higher in diseased tissue than in normal tissue.
There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, supra. In general, the presence or absence of a disease in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of a binding agent(s) immobilized on a solid support to bind to and remove the polypeptide of the invention from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include polypeptides of the invention and portions thereof, or antibodies, to which the binding agent binds, as described above.
The solid support may be any material known to those of skill in the art to which polypeptides of the invention may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for the suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
Gene Therapy Methods
Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of the polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
In one embodiment, the polynucleotide of the present invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, LIPOFECTINTM or precipitating agents and the like. However, the polynucleotide of the present invention can also be delivered in liposome formulations and LIPOFECTINTM formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from STRATAGENETM; pSVK3, pBPV, pMSG and pSVL available from PHARMACIATM; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotide of the present invention.
Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, LIPOFECTINTM, precipitating agents, etc. Such methods of delivery are known in the art.
In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.
Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark LIPOFECTINTM, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (BOEHRINGERTM).
Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphosphatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding a polypeptide of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a polypeptide of the present invention.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses a polypeptide of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis. 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).
Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest that is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express a polypeptide of the invention.
Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein incorporated by reference. This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.
The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
The polynucleotide encoding a polypeptide of the present invention may contain a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, BIOLISTICTM injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
Therapeutic compositions useful in systemic administration include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising polypeptides of the invention for targeting the vehicle to a particular site.
Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cattle, horses and pigs, with humans being particularly preferred.
Biological Activities
Polynucleotides or polypeptides, or agonists or antagonists of the present invention, can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or antagonists could be used to treat the associated disease.
Members of the secreted family of proteins are believed to be involved in biological activities associated with, for example, cellular signaling. Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with aberrant activity of secreted polypeptides.
In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, treatment, and/or amelioration of diseases and/or disorders relating to the endocrine system, the nervous system (See, for example, “Neurological Disorders” section below), the immune system (See, for example, “Immune Activity” section below), the gastrointestinal system (e.g., Crohn's disease, pancreatitis, gallstones, antibiotic-associated colitis, duodenitis, gastrointestinal neoplasms, and as described in the “Gastrointestinal Disorders” section below), or the cardiovascular system (e.g., atherosclerosis, stroke, myocardial infarction, hypertension, and as described in the “Cardiovascular Disorders” section below). In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, treatment, and/or amelioration of cancer and other hyperproliferative diseases and/or disorders (e.g., as described in the “Hyperproliferative Disorders”).
In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed including one, two, three, four, five, or more tissues disclosed in Table 1B.2 (such as column 5) (Tissue Distribution Library Code).
Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, detection, prevention, prognostication, and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.
More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, treatment and/or amelioration of diseases and/or disorders associated with the following system or systems.
Immune Activity
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells. Polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat and/or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells.
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, diagnosing, prognosticating, treating and/or ameliorating immunodeficiencies, including both congenital and acquired immunodeficiencies. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein disorders, ataxia telangiectasia, Digeorge Syndrome, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria. Examples of B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.
In specific embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using the polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof.
Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polypeptides or polynucleotides of the invention, or antagonists or agonists thereof.
Other immunodeficiencies that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chédiak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including C1, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.
In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, detecting, diagnosing, prognosticating, treating and/or ameliorating autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
Autoimmune diseases or disorders that may be prevented, detected, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, hypothyroidism, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), inflammatory myopathies, insulin resistance, and insulin-resistant diabetes mellitus.
Additional disorders that are likely to have an autoimmune component that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Grave's Disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmia, bullous pemphigoid, pemphigus, polyendocrinopathies, purpura, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus (SLE), autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
Additional disorders that are likely to have an autoimmune component that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated with the compositions of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
Additional disorders that may have an autoimmune component that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.
In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention. In a specific preferred embodiment, rheumatoid arthritis is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
In another specific preferred embodiment, systemic lupus erythematosus is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
In another specific preferred embodiment IgA nephropathy is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention
In preferred embodiments, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a immunosuppressive agent(s).
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases, disorders, and/or conditions of hematopoietic cells. Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively, Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof. Moreover, these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
Additionally, polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate IgE concentrations in vitro or in vivo.
Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention have uses in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of inflammatory conditions. For example, since polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis; blood brain permeability, ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, septic shock, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).
Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, polynucleotides, polypeptides, and antibodies of the invention, as well as agonists or antagonists thereof, have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.
In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate organ transplant rejections and graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.
In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat, autoimmune and inflammatory diseases (e.g., immune complex-induced vasculitis, glomerulonephritis, hemolytic anemia, myasthenia gravis, type II collagen-induced arthritis, experimental allergic and hyperacute xenograft rejection, rheumatoid arthritis, and systemic lupus erythematosus (SLE).
Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate and/or diagnose inflammation. For example, the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, diagnose and/or prognose inflammatory conditions, both chronic and acute conditions, including, but not limited to, chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)
In other embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
Polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.
In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance tumor-specific immune responses.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Plasmodium (malaria), and Borrelia burgdorferi. 
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania. 
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
In one embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell responsiveness to pathogens.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an activator of T cells.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to induce higher affinity antibodies.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to increase serum immunoglobulin concentrations.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to accelerate recovery of immunocompromised individuals.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, said enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodeficiency.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in the pretreatment of bone marrow samples prior to transplant.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence/immunodeficiency such as observed among SCID patients.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania. 
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.
In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in one or more of the applications described herein, as they may apply to veterinary medicine.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.
The polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit complement mediated cell lysis.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed to treat adult respiratory distress syndrome (ARDS).
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.
In a specific embodiment, polynucleotides or polypeptides, and/or agonists thereof are used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, polynucleotides or polypeptides, and/or agonists thereof may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseases and disorders that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with polynucleotides or polypeptides, and/or agonists of the present invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease (“CVID”; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.
In a specific embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled “Hyperproliferative Disorders” elsewhere herein.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of activating T cells.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an inhibitor of graft versus host disease or transplant rejection.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for B cell and/or T cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.
In another embodiment, administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention, may be used to treat or prevent IgE-mediated allergic reactions including, but not limited to, asthma, rhinitis, and eczema.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
Antagonists of the invention include, for example, binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the polypeptides of the present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of the invention include, for example, binding or stimulatory antibodies, and soluble forms of the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.
In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741). Administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention to such animals is useful for the generation of monoclonal antibodies against the polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention.
Blood-Related Disorders
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or thrombolytic activity, polynucleotides or polypeptides, and/or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.
In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used for the prevention of occlusion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular cannulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hematopoietic activity (the formation of blood cells). For example, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of anemias and leukopenias described below. Alternatively, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of leukocytoses, such as, for example eosinophilia.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate blood dyscrasia.
Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias. Anemias that may be treated detect, prevented, diagnosed, prognosticated, treated, and/or ameliorated by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary sideroblastic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens. The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfadrugs. Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S—C disease, and hemoglobin E disease). Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating thalassemias, including, but not limited to major and minor forms of alpha-thalassemia and beta-thalassemia.
In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.
The effect of the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.
Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.
In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells. Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are not limited to, eosinophilia, and accumulations of macrophages. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating leukopenia. In other specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating leukocytosis.
Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating decreases in neutrophil numbers, known as neutropenia. Neutropenias that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoimmune diseases, conditions in which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but not limited lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndrome, severe combined immunodeficiency, ataxia telangiectsia).
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders associated with macrophage numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease.
In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.
In yet another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating leukemias and lymphomas including, but not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous, myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukemia), chronic myelocytic (myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies of undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myeloid metaplasia, thrombocythemia, (including both primary and secondary thrombocythemia) and chronic myelocytic leukemia.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as a treatment prior to surgery, to increase blood cell production.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils and macrophages.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis. In another specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase cytokine production.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating primary hematopoietic disorders.
Hyperproliferative Disorders
In certain embodiments, polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used to treat or detect hyperproliferative disorders, including neoplasms. Polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, Polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
Examples of hyperproliferative disorders that can be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
Similarly, other hyperproliferative disorders can also be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
In another preferred embodiment, polynucleotides or polypeptides, or agonists or antagonists of the present invention are used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.)
Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Hyperplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate, nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.
Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, opthalmomandibulomelic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
Additional pre-neoplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to those described herein. In a further preferred embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.
Additionally, polynucleotides, polypeptides, and/or agonists or antagonists of the invention may affect apoptosis, and therefore, would be useful in treating a number of diseases associated with increased cell survival or the inhibition of apoptosis. For example, diseases associated with increased cell survival or the inhibition of apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
In preferred embodiments, polynucleotides, polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
Hyperproliferative diseases and/or disorders that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
Similarly, other hyperproliferative disorders can also be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
Another preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.
Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the polynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.
Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, LIPOFECTINTM, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.
The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the described disorders. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnosis, prognosis, monitoring, or therapeutic purposes without undue experimentation.
In particular, the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing cell proliferative and/or differentiation disorders as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14, 5×10−15M, and 10−15M.
Moreover, polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).
Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med. Hypotheses. 50(5):423-33 (1998), Chem Biol Interact. April 24; 111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int J Tissue React; 20(1):3-15 (1998), which are all hereby incorporated by reference).
Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewhere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998; 231:125-41, which is hereby incorporated by reference). Such therapeutic effects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
Renal Disorders
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate disorders of the renal system. Renal disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.
Kidney diseases which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders of the kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections, urinary retention, and acute or chronic unilateral obstructive uropathy.)
In addition, compositions of the invention can be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).
Compositions of the invention can also be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, BIOLISTICTM injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.
Cardiovascular Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate cardiovascular diseases and disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.
Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease.
Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, BIOLISTICTM injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.
Respiratory Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diseases and/or disorders of the respiratory system.
Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic interstitial pneumonitis, organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wegener's granulomatosis (granulomatous vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).
Additional diseases and disorders of the respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.; candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal workers' pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis, and benign pneumoconiosis), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe disease, Hand-Schüller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult respiratory distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.
Anti Angiogenesis Activity
The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating other disorders, besides cancers, which involve angiogenesis. These disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.
Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists of the invention are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
Moreover, Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue that normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
Particularly preferred embodiments of the invention may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a mucoadhesive polymer that binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.
Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.
Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.
Additionally, disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, disorders and/or states, which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with the polynucleotides, polypeptides, agonists and/or agonists of the invention include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vasculogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.
In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.
Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.
Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angiostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.
Diseases at the Cellular Level
Diseases associated with increased cell survival or the inhibition of apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using polynucleotides or polypeptides, as well as antagonists or agonists of the present invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
In preferred embodiments, polynucleotides, polypeptides, and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote dermal reestablishment subsequent to dermal loss.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that polynucleotides or polypeptides, agonists or antagonists of the present invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, can be used to promote skin strength and to improve the appearance of aged skin.
It is believed that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Polynucleotides or polypeptides, agonists or antagonists of the present invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may have a cytoprotective effect on the small intestine mucosa. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to treat gastric and duodenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with polynucleotides or polypeptides, agonists or antagonists of the present invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat diseases associate with the under expression.
Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to prevent and heal damage to the lungs due to various pathological states. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and bronchiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of alveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary dysplasia, in premature infants.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetrachloride and other hepatotoxins known in the art).
In addition, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
Neural Activity and Neurological Diseases
The polynucleotides, polypeptides and agonists or antagonists of the invention may be used for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
In one embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with cerebral infarction.
In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.
In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a heart attack. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a heart attack.
The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci., 4:17-42 (1981); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Further, polypeptides or polynucleotides of the invention may play a role in neuronal survival; synapse formation; conductance; neural differentiation, etc. Thus, compositions of the invention (including polynucleotides, polypeptides, and agonists or antagonists) may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders. The compositions of the invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders. Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, compositions of the invention may also play a role in the treatment, prevention and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders.
Additionally, polypeptides, polynucleotides and/or agonists or antagonists of the invention, may be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct), leukomalacia, periventricular, and vascular headache (e.g., cluster headache or migraines).
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate neurological cell proliferation and/or differentiation. Therefore, polynucleotides, polypeptides, agonists and/or antagonists of the invention may be used to treat and/or detect neurologic diseases. Moreover, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used as a marker or detector of a particular nervous system disease or disorder.
Examples of neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include brain diseases, such as metabolic brain diseases which includes phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central nervous system infections such as AIDS Dementia Complex, Brain Abscess, subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and cerebral malaria.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include meningitis such as arachnoiditis, aseptic meningtitis such as viral meningtitis which includes lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseases such as amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities such as holoprosencephaly, neural tube defects such as anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and spina bifida occulta.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations (such as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment and tinnitus, language disorders such as aphasia which include agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wernicke Aphasia, articulation disorders, communicative disorders such as speech disorders which include dysarthria, echolalia, mutism and stuttering, voice disorders such as aphonia and hoarseness, decerebrate state, delirium, fasciculation, hallucinations, meningism, movement disorders such as angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle rigidity such as stiff-man syndrome, muscle spasticity, paralysis such as facial paralysis which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, opthalmoplegia such as diplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressive external opthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders such as hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as trismus, unconsciousness such as coma, persistent vegetative state and syncope and vertigo, neuromuscular diseases such as amyotonia congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Adie's Syndrome, Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility disorders which includes amblyopia, nystagmus, oculomotor nerve paralysis, opthalmoplegia such as Duane's Syndrome, Horner's Syndrome, Chronic Progressive External Opthalmoplegia which includes Kearns Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as diabetic foot.
Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).
Endocrine Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.
Hormones secreted by the glands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.
Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and endometriosis).
Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma-islet cell tumor syndrome; disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.
In specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides, agonists and antagonists, may be used to diagnose, prognose, treat, prevent, or ameliorate diseases and disorders associated with aberrant glucose metabolism or glucose uptake into cells.
In a specific embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists and/or antagonists thereof may be used to diagnose, prognose, treat, prevent, and/or ameliorate type I diabetes mellitus (insulin dependent diabetes mellitus, IDDM).
In another embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists and/or antagonists thereof may be used to diagnose, prognose, treat, prevent, and/or ameliorate type II diabetes mellitus (insulin resistant diabetes mellitus).
Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to diagnose, prognose, treat, prevent, and/or ameliorate conditions associated with (type I or type II) diabetes mellitus, including, but not limited to, diabetic ketoacidosis, diabetic coma, nonketotic hyperglycemic-hyperosmolar coma, seizures, mental confusion, drowsiness, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section), dyslipidemia, kidney disease (e.g., renal failure, nephropathy other diseases and disorders as described in the “Renal Disorders” section), nerve damage, neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infections (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section, especially of the urinary tract and skin), carpal tunnel syndrome and Dupuytren's contracture.
In other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to regulate the animal's weight. In specific embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin. In still other embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin-like growth factor.
In addition, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.
Moreover, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate endocrine diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
Reproductive System Disorders
The polynucleotides or polypeptides, or agonists or antagonists of the invention may be used for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and/or disorders of the reproductive system. Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).
Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
Additionally, the compositions of the invention may be useful in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squamous cell carcinomas, carcinoma in situ, verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders, such as priapism, Peyronie's disease, erectile dysfunction, and impotence.
Moreover, diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.
Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
Further, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.
Disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the polypeptides, polynucleotides, or agonists or antagonists of the invention may be useful as a marker or detector of, as well as in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of congenital uterine abnormalities, such as bicornuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus.
Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutismo, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometrioid carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy. Additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders, and obstruction of the intestine.
Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
Other disorders and/or diseases of the female reproductive system that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by the polynucleotides, polypeptides, and agonists or antagonists of the present invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.
Infectious Disease
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.
Similarly, bacterial and fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacteria, bacterial families, and fungi: Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia, Clostridium (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pasteurellacea, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Chlamydiaceae, Syphilis, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal families can cause diseases or symptoms, including, but not limited to: antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and acute inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneous abortions, birth defects, pneumonia, lung infections, ear infections, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections, nosocomial infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, diphtheria, botulism, and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to detect, prevent, diagnose, treat, and/or ameliorate malaria.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
Regeneration
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotides or polypeptides, as well as agonists or antagonists of the present invention.
Gastrointestinal Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate gastrointestinal diseases and disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and structuring, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adenocarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess).
Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T. solium).
Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).
Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal diseases (ileal neoplasms, ileitis), immunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections, cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [cecal volvulus], intussusception), intestinal perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.
Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).
Chemotaxis
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
It is also contemplated that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could be used as an inhibitor of chemotaxis.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
Additionally, the receptor to which the polypeptide of the present invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of the polypeptide of the present invention thereby effectively generating agonists and antagonists of the polypeptide of the present invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptide of the present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).
Other preferred fragments are biologically active fragments of the polypeptide of the present invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.
In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues.
Also, one could identify molecules that bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention.
Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the present invention, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.
Targeted Delivery
In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.
As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.
By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin.
Drug Screening
Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.
This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.
Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.
Antisense and Ribozyme (Antagonists)
In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to cDNA sequences contained in cDNA ATCCTM Deposit No:Z identified for example, in Table 1A. In one embodiment, antisense sequence is generated internally, by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, mM MgCl2, 10 mM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).
For example, the 5′ coding portion of a polynucleotide that encodes the polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding the polypeptide of the present invention or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of the present invention. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335. Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of polynucleotide sequences described herein could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA of the present invention, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
While antisense nucleotides complementary to the coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.
Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of SEQ ID NO:X. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.
The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.
The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.
The antagonist/agonist may also be employed to treat the diseases described herein.
Thus, the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.
Binding Peptides and Other Molecules
The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind polypeptides of the invention, and the binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the polypeptides of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.
This method comprises the steps of:
a. contacting polypeptides of the invention with a plurality of molecules; and
b. identifying a molecule that binds the polypeptides of the invention.
The step of contacting the polypeptides of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the polypeptides on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptides. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized polypeptides of the invention. The molecules having a selective affinity for the polypeptides can then be purified by affinity selection. The nature of the solid support, process for attachment of the polypeptides to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.
Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be “probed” by the polypeptides of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the polypeptides and the individual clone. Prior to contacting the polypeptides with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for polypeptides of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the polypeptides of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound polypeptides from a mixture of the polypeptides of the invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the polypeptides of the invention or the plurality of polypeptides are bound to a solid support.
The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind polypeptides of the invention. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.
Examples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.
By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.
Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolidones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.
Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.
In a specific embodiment, screening to identify a molecule that binds polypeptides of the invention can be carried out by contacting the library members with polypeptides of the invention immobilized on a solid phase and harvesting those library members that bind to the polypeptides of the invention. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.
In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to polypeptides of the invention.
Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term “biased” is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.
Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.
As mentioned above, in the case of a binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.
The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.
Other Activities
A polypeptide, polynucleotide, agonist, or antagonist of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. The polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
Polypeptides, polynucleotides, agonists, or antagonists of the present invention may be used to treat weight disorders, including but not limited to, obesity, cachexia, wasting disease, anorexia, and bulimia.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.
Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCCTM Deposit No:Z. X can be any integer as defined in Table 1A. Also preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Clone Sequence and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A. Further preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A. Similarly preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in column 5, “ORF (From-To)”, in Table 1B.1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in columns 8 and 9, “NT From” and “NT To” respectively, in Table 2.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCCTM Deposit No:Z.
Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCCTM Deposit No:Z.
A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in column 5, “ORF (From-To)”, in Table 1B.1.
A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in columns 8 and 9, “NT From” and “NT To”, respectively, in Table 2.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCCTM Deposit No:Z.
Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCCTM Deposit No:Z, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
Also preferred is a composition of matter comprising a DNA molecule which comprises the cDNA contained in ATCCTM Deposit No:Z.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides of the cDNA sequence contained in ATCCTM Deposit No:Z.
Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of an open reading frame sequence encoded by cDNA contained in ATCCTM Deposit No:Z.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCCTM Deposit No:Z.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCCTM Deposit No:Z.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by cDNA contained in ATCCTM Deposit No:Z.
A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCCTM Deposit No:Z; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.
Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of the cDNA contained in ATCCTM Deposit No:Z.
The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; or the cDNA contained in ATCCTM Deposit No:Z which encodes a protein, wherein the method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of cDNA contained in ATCCTM Deposit No:Z.
The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCCTM Deposit No:Z. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a DNA microarray or “chip” of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 500, 1000, 2000, 3000, or 4000 nucleotide sequences, wherein at least one sequence in said DNA microarray or “chip” is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1A and/or Tables 1B.1 and 1B.2; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA “Clone ID” in Table 1A and/or Tables 1B.1 and 1B.2. Further preferred is the above isolated polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO:Y in the range of positions beginning with the residue at about the position of the First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid of the Open Reading Frame as set forth for SEQ ID NO:Y in Table 1A.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a polypeptide encoded by contained in ATCCTM Deposit No:Z
Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a portion of said polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or the polypeptide sequence of SEQ ID NO:Y.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of a polypeptide encoded by cDNA contained in ATCCTM Deposit No:Z.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.
Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.
Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleic acid sequence identified in Tables 1A, 1B.1, 1B.2 or 2 encoding a polypeptide, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
Also preferred is a polypeptide molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z.
Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecules into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a human protein comprising an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; an amino acid sequence of SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table 1A and said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y is defined in Table 1A; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with ATCCTM Deposit No:Z; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCCTM Deposit No:Z. The isolated polypeptide produced by this method is also preferred.
Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to increase the level of said protein activity in said individual.
Also preferred is an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA sequence included in ATCCTM Deposit No:Z, which is hybridizable to SEQ ID NO:X; (b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA sequence included in ATCCTM Deposit No:Z, which is hybridizable to SEQ ID NO:X; (c) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence included in ATCCTM Deposit No:Z, which is hybridizable to SEQ ID NO:X; (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded by the cDNA sequence included in ATCCTM Deposit No:Z, which is hybridizable to SEQ ID NO:X; (e) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA sequence included in ATCCTM Deposit No:Z, which is hybridizable to SEQ ID NO:X, having biological activity; (f) a polynucleotide which is a variant of SEQ ID NO:X; (g) a polynucleotide which is an allelic variant of SEQ ID NO:X; (h) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y; and (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.
Also preferred is the isolated nucleic acid molecule as described in preferred embodiment I, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein. The nucleotide sequence encoding a secreted protein may further comprise sequential nucleotide deletions from either the C-terminus or the N-terminus.
Also preferred is the isolated nucleic acid molecule as described in preferred embodiment I, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:Y or the polypeptide encoded by the cDNA sequence included in ATCCTM Deposit No:Z, which is hybridizable to SEQ ID NO:X. The nucleotide sequence encoding the sequence identified as SEQ ID NO:Y or the polypeptide encoded by the cDNA sequence included in ATCCTM Deposit No:Z may further comprise sequential nucleotide deletions from either the C-terminus or the N-terminus.
Also preferred is the isolated nucleic acid molecule as described in preferred embodiment I, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:X or the cDNA sequence included in ATCCTM Deposit No:Z, which is hybridizable to SEQ ID NO:X.
Also preferred is a recombinant vector comprising the isolated nucleic acid molecule described in preferred embodiment I.
Also preferred is a recombinant host cell comprising the isolated nucleic acid molecule described in preferred embodiment I and the method of making the recombinant host cell. The recombinant host cell may further comprise vector sequences.
Also preferred is a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising determining the presence or absence of a mutation in the polynucleotide described in preferred embodiment I and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of the mutation.
An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence included in ATCCTM Deposit No:Z; (b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence included in ATCCTM Deposit No:Z, having biological activity; (c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence included in ATCCTM Deposit No:Z; (d) a polypeptide epitope of SEQ ID NO:Y or the encoded sequence included in ATCCTM Deposit No:Z; (e) a secreted form of SEQ ID NO:Y or the encoded sequence included in ATCCTM Deposit No:Z; (f) a full length protein of SEQ ID NO:Y or the encoded sequence included in ATCCTM Deposit No:Z; (g) a variant of SEQ ID NO:Y; (h) an allelic variant of SEQ ID NO:Y; and (i) a species homologue of SEQ ID NO:Y.
Also preferred is the isolated polypeptide as described in preferred embodiment II, wherein the secreted form or the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
Also preferred is an isolated antibody that binds specifically to the isolated polypeptide described in preferred embodiment II.
Also preferred is a recombinant host cell that expresses the isolated polypeptide described in preferred embodiment II.
Also preferred is a method of making an isolated polypeptide that comprises culturing the recombinant host cells that expresses the isolated polypeptide described in preferred embodiment II and recovering said polypeptide. Further envisioned is the polypeptide produced by this method.
Also preferred is a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or amount of expression of the polypeptide described in preferred embodiment II in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
Also preferred is a method for identifying a binding partner to the polypeptide described in preferred embodiment II comprising: (a) contacting the polypeptide with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide
The gene corresponding to the cDNA sequence of SEQ ID NO:X.
A method of identifying an activity in a biological assay, wherein the method comprises: (a) expressing SEQ ID NO:X in a cell; (b) isolating the supernatent; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatent having the activity. Preferred embodiment IV may further include the product produced by the method.
Also preferred is a method for preventing, treating, or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of the polynucleotide described in preferred embodiment I or the polypeptide described in preferred embodiment II.
Also preferred is a method of treatment of an individual in need of a decreased level of a protein activity, which method comprised administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to decrease the level of said protein activity in said individual.
Also preferred is a method of treatment of an individual in need of a specific delivery of toxic compositions to diseased cells (e.g., tumors, leukemias or lymphomas), which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide of the invention, including, but not limited to a binding agent, or antibody of the claimed invention that are associated with toxin or cytotoxic prodrugs.
Having generally described the invention, the same will be more readily understood by reference to the examples disclosed herein, which are provided by way of illustration and are not intended as limiting.
In specific embodiments of the invention, for each “Contig ID” listed in the second column of Table 2, preferably excluded are one or more polynucleotides comprising, or alternatively consisting of, a nucleotide sequence referenced in the fifth column of Table 2 and described by the general formula of a−b, whereas a and b are uniquely determined for the corresponding SEQ ID NO:X referred to in column 3 of Table 2. Further specific embodiments are directed to polynucleotide sequences excluding one, two, three, four, or more of the specific polynucleotide sequences referred to in the fifth column of Table 2. In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.
Table 6
Table 6 summarizes some of the ATCCTM Deposits, Deposit dates, and ATCCTM designation numbers of deposits made with the ATCCTM in connection with the present application. These deposits were made in addition to those described in the Table 1A.









TABLE 6







ATCC TM


ATCC TM Deposits
Deposit Date
Designation Number







LP01, LP02, LP03, LP04,
May 20, 1997
209059, 209060, 209061,


LP05, LP06, LP07, LP08,

209062, 209063, 209064,


LP09, LP10, LP11

209065, 209066, 209067,




209068, 209069


LP12
Jan. 12, 1998
209579


LP13
Jan. 12, 1998
209578


LP14
Jul. 16, 1998
203067


LP15
Jul. 16, 1998
203068


LP16
Feb. 1, 1999
203609


LP17
Feb. 1, 1999
203610


LP20
Nov. 17, 1998
203485


LP21
Jun. 18, 1999
PTA-252


LP22
Jun. 18, 1999
PTA-253


LP23
Dec. 22, 1999
PTA-1081









EXAMPLES
Example 1
Isolation of a Selected cDNA Clone from the Deposited Sample
Each ATCCTM Deposit No:Z is contained in a plasmid vector. Table 1A identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The following correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table 1A as being isolated in the vector “LAMBDA ZAPTM,” the corresponding deposited clone is in “pBLUESCRIPTTM.”











Vector Used to Construct Library
Corresponding Deposited Plasmid







LAMBDA ZAP TM
pBLUESCRIPT TM (pBS)


UNI-ZAP TM XR
pBLUESCRIPT TM (pBS)


ZAP EXPRESS TM
pBK


lafmid BA
plafmid BA


pSport1
pSport1


pCMVSport 2.0
pCMVSport 2.0


pCMVSport 3.0
pCMVSport 3.0


pCR ® 2.1
pCR ® 2.1









Vectors LAMBDA ZAPTM (U.S. Pat. Nos. 5,128,256 and 5,286,636), UNI-ZAPTM XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), ZAP EXPRESSTM (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPTTM (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENETM Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from STRATAGENETM. pBS comes in 4 forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region (“S” is for Sad and “K” is for KpnI which are the first sites on each respective end of the linker). “+” or “−” refer to the orientation of the f1 origin of replication (“ori”), such that in one orientation, single stranded rescue initiated from the f1 on generates sense strand DNA and in the other, antisense.
Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIESTM, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIESTM. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993)). Vector lafmid BA (Bento Soares, Columbia University, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIESTM. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991)). Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 1A, as well as the corresponding plasmid vector sequences designated above.
The deposited material in the sample assigned the ATCCTM Deposit Number cited by reference to Tables 1A, 6, and 7 for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCCTM Deposit Number contain at least a plasmid for each ATCCTM Deposit No:Z.










TABLE 7








ATCC TM


Libraries owned by Catalog
Catalog Description
Vector
Deposit







HUKA HUKB HUKC HUKD
Human Uterine Cancer
Lambda ZAP II
LP01


HUKE HUKF HUKG





HCNA HCNB
Human Colon
Lambda Zap II
LP01


HFFA
Human Fetal Brain, random
Lambda Zap II
LP01



primed




HTWA
Resting T-Cell
Lambda ZAP II
LP01


HBQA
Early Stage Human Brain,
Lambda ZAP II
LP01



random primed




HLMB HLMF HLMG HLMH
breast lymph node CDNA
Lambda ZAP II
LP01


HLMI HLMJ HLMM HLMN
library




HCQA HCQB
human colon cancer
Lamda ZAP II
LP01


HMEA HMEC HMED
Human Microvascular
Lambda ZAP II
LP01


HMEE HMEF HMEG HMEI
Endothelial Cells, fract. A




HMEJ HMEK HMEL





HUSA HUSC
Human Umbilical Vein
Lambda ZAP II
LP01



Endothelial Cells, fract. A




HLQA HLQB
Hepatocellular Tumor
Lambda ZAP II
LP01


HHGA HHGB HHGC HHGD
Hemangiopericytoma
Lambda ZAP II
LP01


HSDM
Human Striatum Depression,
Lambda ZAP II
LP01



re-rescue




HUSH
H Umbilical Vein Endothelial
Lambda ZAP II
LP01



Cells, frac A, re-excision




HSGS
Salivary gland, subtracted
Lambda ZAP II
LP01


HFXA HFXB HFXC HFXD
Brain frontal cortex
Lambda ZAP II
LP01


HFXE HFXF HFXG HFXH





HPQA HPQB HPQC
PERM TF274
Lambda ZAP II
LP01


HFXJ HFXK
Brain Frontal Cortex, re-
Lambda ZAP II
LP01



excision




HCWA HCWB HCWC
CD34 positive cells (Cord
ZAP Express
LP02


HCWD HCWE HCWF
Blood)




HCWG HCWH HCWI HCWJ





HCWK





HCUA HCUB HCUC
CD34 depleted Buffy Coat
ZAP Express
LP02



(Cord Blood)




HRSM
A-14 cell line
ZAP Express
LP02


HRSA
A1-CELL LINE
ZAP Express
LP02


HCUD HCUE HCUF HCUG
CD34 depleted Buffy Coat
ZAP Express
LP02


HCUH HCUI
(Cord Blood), re-excision




HBXE HBXF HBXG
H. Whole Brain #2, re-
ZAP Express
LP02



excision




HRLM
L8 cell line
ZAP Express
LP02


HBXA HBXB HBXC HBXD
Human Whole Brain #2 -
ZAP Express
LP02



Oligo dT >1.5 Kb




HUDA HUDB HUDC
Testes
ZAP Express
LP02


HHTM HHTN HHTO
H. hypothalamus, frac A; re-
ZAP Express
LP02



excision




HHTL
H. hypothalamus, frac A
ZAP Express
LP02


HASA HASD
Human Adult Spleen
Uni-ZAP XR
LP03


HFKC HFKD HFKE HFKF
Human Fetal Kidney
Uni-ZAP XR
LP03


HFKG





HE8A HE8B HE8C HE8D
Human 8 Week Whole
Uni-ZAP XR
LP03


HE8E HE8F HE8M HE8N
Embryo




HGBA HGBD HGBE HGBF
Human Gall Bladder
Uni-ZAP XR
LP03


HGBG HGBH HGBI





HLHA HLHB HLHC HLHD
Human Fetal Lung III
Uni-ZAP XR
LP03


HLHE HLHF HLHG HLHH





HLHQ





HPMA HPMB HPMC HPMD
Human Placenta
Uni-ZAP XR
LP03


HPME HPMF HPMG HPMH





HPRA HPRB HPRC HPRD
Human Prostate
Uni-ZAP XR
LP03


HSIA HSIC HSID HSIE
Human Adult Small Intestine
Uni-ZAP XR
LP03


HTEA HTEB HTEC HTED
Human Testes
Uni-ZAP XR
LP03


HTEE HTEF HTEG HTEH





HTEI HTEJ HTEK





HTPA HTPB HTPC HTPD
Human Pancreas Tumor
Uni-ZAP XR
LP03


HTPE





HTTA HTTB HTTC HTTD
Human Testes Tumor
Uni-ZAP XR
LP03


HTTE HTTF





HAPA HAPB HAPC HAPM
Human Adult Pulmonary
Uni-ZAP XR
LP03


HETA HETB HETC HETD
Human Endometrial Tumor
Uni-ZAP XR
LP03


HETE HETF HETG HETH





HETI





HHFB HHFC HHFD HHFE
Human Fetal Heart
Uni-ZAP XR
LP03


HHFF HHFG HHFH HHFI





HHPB HHPC HHPD HHPE
Human Hippocampus
Uni-ZAP XR
LP03


HHPF HHPG HHPH





HCE1 HCE2 HCE3 HCE4
Human Cerebellum
Uni-ZAP XR
LP03


HCE5 HCEB HCEC HCED





HCEE HCEF HCEG





HUVB HUVC HUVD HUVE
Human Umbilical Vein,
Uni-ZAP XR
LP03



Endo. remake




HSTA HSTB HSTC HSTD
Human Skin Tumor
Uni-ZAP XR
LP03


HTAA HTAB HTAC HTAD
Human Activated T-Cells
Uni-ZAP XR
LP03


HTAE





HFEA HFEB HFEC
Human Fetal Epithelium
Uni-ZAP XR
LP03



(Skin)




HJPA HJPB HJPC HJPD
HUMAN JURKAT
Uni-ZAP XR
LP03



MEMBRANE BOUND





POLYSOMES




HESA
Human epithelioid sarcoma
Uni-Zap XR
LP03


HLTA HLTB HLTC HLTD
Human T-Cell Lymphoma
Uni-ZAP XR
LP03


HLTE HLTF





HFTA HFTB HFTC HFTD
Human Fetal Dura Mater
Uni-ZAP XR
LP03


HRDA HRDB HRDC HRDD
Human Rhabdomyosarcoma
Uni-ZAP XR
LP03


HRDE HRDF





HCAA HCAB HCAC
Cem cells cyclohexamide
Uni-ZAP XR
LP03



treated




HRGA HRGB HRGC HRGD
Raji Cells, cyclohexamide
Uni-ZAP XR
LP03



treated




HSUA HSUB HSUC HSUM
Supt Cells, cyclohexamide
Uni-ZAP XR
LP03



treated




HT4A HT4C HT4D
Activated T-Cells, 12 hrs.
Uni-ZAP XR
LP03


HE9A HE9B HE9C HE9D
Nine Week Old Early Stage
Uni-ZAP XR
LP03


HE9E HE9F HE9G HE9H
Human




HE9M HE9N





HATA HATB HATC HATD
Human Adrenal Gland Tumor
Uni-ZAP XR
LP03


HATE





HT5A
Activated T-Cells, 24 hrs.
Uni-ZAP XR
LP03


HFGA HFGM
Human Fetal Brain
Uni-ZAP XR
LP03


HNEA HNEB HNEC HNED
Human Neutrophil
Uni-ZAP XR
LP03


HNEE





HBGB HBGD
Human Primary Breast
Uni-ZAP XR
LP03



Cancer




HBNA HBNB
Human Normal Breast
Uni-ZAP XR
LP03


HCAS
Cem Cells, cyclohexamide
Uni-ZAP XR
LP03



treated, subtra




HHPS
Human Hippocampus,
pBS
LP03



subtracted




HKCS HKCU
Human Colon Cancer,
pBS
LP03



subtracted




HRGS
Raji cells, cyclohexamide
pBS
LP03



treated, subtracted




HSUT
Supt cells, cyclohexamide
pBS
LP03



treated, differentially





expressed




HT4S
Activated T-Cells, 12 hrs,
Uni-ZAP XR
LP03



subtracted




HCDA HCDB HCDC HCDD
Human Chondrosarcoma
Uni-ZAP XR
LP03


HCDE





HOAA HOAB HOAC
Human Osteosarcoma
Uni-ZAP XR
LP03


HTLA HTLB HTLC HTLD
Human adult testis, large
Uni-ZAP XR
LP03


HTLE HTLF
inserts




HLMA HLMC HLMD
Breast Lymph node cDNA
Uni-ZAP XR
LP03



library




H6EA H6EB H6EC
HL-60, PMA 4 H
Uni-ZAP XR
LP03


HTXA HTXB HTXC HTXD
Activated T-Cell
Uni-ZAP XR
LP03


HTXE HTXF HTXG HTXH
(12 hs)/Thiouridine





labelledEco




HNFA HNFB HNFC HNFD
Human Neutrophil, Activated
Uni-ZAP XR
LP03


HNFE HNFF HNFG HNFH





HNFJ





HTOB HTOC
HUMAN TONSILS,
Uni-ZAP XR
LP03



FRACTION 2




HMGB
Human OB MG63 control
Uni-ZAP XR
LP03



fraction I




HOPB
Human OB HOS control
Uni-ZAP XR
LP03



fraction I




HORB
Human OB HOS treated (10 nM
Uni-ZAP XR
LP03



E2) fraction I




HSVA HSVB HSVC
Human Chronic Synovitis
Uni-ZAP XR
LP03


HROA
HUMAN STOMACH
Uni-ZAP XR
LP03


HBJA HBJB HBJC HBJD
HUMAN B CELL
Uni-ZAP XR
LP03


HBJE HBJF HBJG HBJH
LYMPHOMA




HBJI HBJJ HBJK





HCRA HCRB HCRC
human corpus colosum
Uni-ZAP XR
LP03


HODA HODB HODC HODD
human ovarian cancer
Uni-ZAP XR
LP03


HDSA
Dermatofibrosarcoma
Uni-ZAP XR
LP03



Protuberance




HMWA HMWB HMWC
Bone Marrow Cell Line
Uni-ZAP XR
LP03


HMWD HMWE HMWF
(RS4; 11)




HMWG HMWH HMWI





HMWJ





HSOA
stomach cancer (human)
Uni-ZAP XR
LP03


HERA
SKIN
Uni-ZAP XR
LP03


HMDA
Brain-medulloblastoma
Uni-ZAP XR
LP03


HGLA HGLB HGLD
Glioblastoma
Uni-ZAP XR
LP03


HEAA
H. Atrophic Endometrium
Uni-ZAP XR
LP03


HBCA HBCB
H. Lymph node breast Cancer
Uni-ZAP XR
LP03


HPWT
Human Prostate BPH, re-
Uni-ZAP XR
LP03



excision




HFVG HFVH HFVI
Fetal Liver, subtraction II
pBS
LP03


HNFI
Human Neutrophils,
pBS
LP03



Activated, re-excision




HBMB HBMC HBMD
Human Bone Marrow, re-
pBS
LP03



excision




HKML HKMM HKMN
H. Kidney Medulla, re-
pBS
LP03



excision




HKIX HKIY
H. Kidney Cortex, subtracted
pBS
LP03


HADT
H. Amygdala Depression,
pBS
LP03



subtracted




H6AS
HI-60, untreated, subtracted
Uni-ZAP XR
LP03


H6ES
HL-60, PMA 4 H, subtracted
Uni-ZAP XR
LP03


H6BS
HL-60, RA 4 h, Subtracted
Uni-ZAP XR
LP03


H6CS
HL-60, PMA 1 d, subtracted
Uni-ZAP XR
LP03


HTXJ HTXK
Activated T-
Uni-ZAP XR
LP03



cell(12 h)/Thiouridine-re-





excision




HMSA HMSB HMSC HMSD
Monocyte activated
Uni-ZAP XR
LP03


HMSE HMSF HMSG HMSH





HMSI HMSJ HMSK





HAGA HAGB HAGC HAGD
Human Amygdala
Uni-ZAP XR
LP03


HAGE HAGF





HSRA HSRB HSRE
STROMAL -
Uni-ZAP XR
LP03



OSTEOCLASTOMA




HSRD HSRF HSRG HSRH
Human Osteoclastoma
Uni-ZAP XR
LP03



Stromal Cells - unamplified




HSQA HSQB HSQC HSQD
Stromal cell TF274
Uni-ZAP XR
LP03


HSQE HSQF HSQG





HSKA HSKB HSKC HSKD
Smooth muscle, serum treated
Uni-ZAP XR
LP03


HSKE HSKF HSKZ





HSLA HSLB HSLC HSLD
Smooth muscle, control
Uni-ZAP XR
LP03


HSLE HSLF HSLG





HSDA HSDD HSDE HSDF
Spinal cord
Uni-ZAP XR
LP03


HSDG HSDH





HPWS
Prostate-BPH subtracted II
pBS
LP03


HSKW HSKX HSKY
Smooth Muscle-HASTE
pBS
LP03



normalized




HFPB HFPC HFPD
H. Frontal cortex, epileptic; re-
Uni-ZAP XR
LP03



excision




HSDI HSDJ HSDK
Spinal Cord, re-excision
Uni-ZAP XR
LP03


HSKN HSKO
Smooth Muscle Serum
pBS
LP03



Treated, Norm




HSKG HSKH HSKI
Smooth muscle, serum
pBS
LP03



induced, re-exc




HFCA HFCB HFCC HFCD
Human Fetal Brain
Uni-ZAP XR
LP04


HFCE HFCF





HPTA HPTB HPTD
Human Pituitary
Uni-ZAP XR
LP04


HTHB HTHC HTHD
Human Thymus
Uni-ZAP XR
LP04


HE6B HE6C HE6D HE6E
Human Whole Six Week Old
Uni-ZAP XR
LP04


HE6F HE6G HE6S
Embryo




HSSA HSSB HSSC HSSD
Human Synovial Sarcoma
Uni-ZAP XR
LP04


HSSE HSSF HSSG HSSH





HSSI HSSJ HSSK





HE7T
7 Week Old Early Stage
Uni-ZAP XR
LP04



Human, subtracted




HEPA HEPB HEPC
Human Epididymus
Uni-ZAP XR
LP04


HSNA HSNB HSNC HSNM
Human Synovium
Uni-ZAP XR
LP04


HSNN





HPFB HPFC HPFD HPFE
Human Prostate Cancer,
Uni-ZAP XR
LP04



Stage C fraction




HE2A HE2D HE2E HE2H
12 Week Old Early Stage
Uni-ZAP XR
LP04


HE2I HE2M HE2N HE2O
Human




HE2B HE2C HE2F HE2G
12 Week Old Early Stage
Uni-ZAP XR
LP04


HE2P HE2Q
Human, II




HPTS HPTT HPTU
Human Pituitary, subtracted
Uni-ZAP XR
LP04


HAUA HAUB HAUC
Amniotic Cells - TNF
Uni-ZAP XR
LP04



induced




HAQA HAQB HAQC HAQD
Amniotic Cells - Primary
Uni-ZAP XR
LP04



Culture




HWTA HWTB HWTC
wilm's tumor
Uni-ZAP XR
LP04


HBSD
Bone Cancer, re-excision
Uni-ZAP XR
LP04


HSGB
Salivary gland, re-excision
Uni-ZAP XR
LP04


HSJA HSJB HSJC
Smooth muscle-ILb induced
Uni-ZAP XR
LP04


HSXA HSKB HSXC HSXD
Human Substantia Nigra
Uni-ZAP XR
LP04


HSHA HSHB HSHC
Smooth muscle, IL1b induced
Uni-ZAP XR
LP04


HOUA HOUB HOUC HOUD
Adipocytes
Uni-ZAP XR
LP04


HOUE





HPWA HPWB HPWC
Prostate BPH
Uni-ZAP XR
LP04


HPWD HPWE





HELA HELB HELC HELD
Endothelial cells-control
Uni-ZAP XR
LP04


HELE HELF HELG HELH





HEMA HEMB HEMC
Endothelial-induced
Uni-ZAP XR
LP04


HEMD HEME HEMF HEMG





HEMH





HBIA HBIB HBIC
Human Brain, Striatum
Uni-ZAP XR
LP04


HHSA HHSB HHSC HHSD
Human
Uni-ZAP XR
LP04


HHSE
Hypothalmus, Schizophrenia




HNGA HNGB HNGC HNGD
neutrophils control
Uni-ZAP XR
LP04


HNGE HNGF HNGG HNGH





HNGI HNGJ





HNHA HNHB HNHC HNHD
Neutrophils IL-1 and LPS
Uni-ZAP XR
LP04


HNHE HNHF HNHG HNHH
induced




HNHI HNHJ





HSDB HSDC
STRIATUM DEPRESSION
Uni-ZAP XR
LP04


HHPT
Hypothalamus
Uni-ZAP XR
LP04


HSAT HSAU HSAV HSAW
Anergic T-cell
Uni-ZAP XR
LP04


HSAX HSAY HSAZ





HBMS HBMT HBMU
Bone marrow
Uni-ZAP XR
LP04


HBMV HBMW HBMX





HOEA HOEB HOEC HOED
Osteoblasts
Uni-ZAP XR
LP04


HOEE HOEF HOEJ





HAIA HAIB HAIC HAID
Epithelial-TNFa and INF
Uni-ZAP XR
LP04


HALE HALF
induced




HTGA HTGB HTGC HTGD
Apoptotic T-cell
Uni-ZAP XR
LP04


HMCA HMCB HMCC
Macrophage-oxLDL
Uni-ZAP XR
LP04


HMCD HMCE





HMAA HMAB HMAC
Macrophage (GM-CSF
Uni-ZAP XR
LP04


HMAD HMAE HMAF
treated)




HMAG





HPHA
Normal Prostate
Uni-ZAP XR
LP04


HPIA HPIB HPIC
LNCAP prostate cell line
Uni-ZAP XR
LP04


HPJA HPJB HPJC
PC3 Prostate cell line
Uni-ZAP XR
LP04


HOSE HOSF HOSG
Human Osteoclastoma, re-
Uni-ZAP XR
LP04



excision




HTGE HTGF
Apoptotic T-cell, re-excision
Uni-ZAP XR
LP04


HMAJ HMAK
H Macrophage (GM-CSF
Uni-ZAP XR
LP04



treated), re-excision




HACB HACC HACD
Human Adipose Tissue, re-
Uni-ZAP XR
LP04



excision




HFPA
H. Frontal Cortex, Epileptic
Uni-ZAP XR
LP04


HFAA HFAB HFAC HFAD
Alzheimer's, spongy change
Uni-ZAP XR
LP04


HFAE





HFAM
Frontal Lobe, Dementia
Uni-ZAP XR
LP04


HMIA HMIB HMIC
Human Manic Depression
Uni-ZAP XR
LP04



Tissue




HTSA HTSE HTSF HTSG
Human Thymus
pBS
LP05


HTSH





HPBA HPBB HPBC HPBD
Human Pineal Gland
pBS
LP05


HPBE





HSAA HSAB HSAC
HSA 172 Cells
pBS
LP05


HSBA HSBB HSBC HSBM
HSC172 cells
pBS
LP05


HJAA HJAB HJAC HJAD
Jurkat T-cell G1 phase
pBS
LP05


HJBA HJBB HJBC HJBD
Jurkat T-Cell, S phase
pBS
LP05


HAFA HAFB
Aorta endothelial cells +
pBS
LP05



TNF-a




HAWA HAWB HAWC
Human White Adipose
pBS
LP05


HTNA HTNB
Human Thyroid
pBS
LP05


HONA
Normal Ovary,
pBS
LP05



Premenopausal




HARA HARB
Human Adult Retina
pBS
LP05


HLJA HLJB
Human Lung
pCMVSport 1
LP06


HOFM HOFN HOFO
H. Ovarian Tumor, II,
pCMVSport 2.0
LP07



OV5232




HOGA HOGB HOGC
OV 10-3-95
pCMVSport 2.0
LP07


HCGL
CD34+cells, II
pCMVSport 2.0
LP07


HDLA
Hodgkin's Lymphoma I
pCMVSport 2.0
LP07


HDTA HDTB HDTC HDTD
Hodgkin's Lymphoma II
pCMVSport 2.0
LP07


HDTE





HKAA HKAB HKAC HKAD
Keratinocyte
pCMVSport2.0
LP07


HKAE HKAF HKAG HKAH





HCIM
CAPFINDER, Crohn's
pCMVSport 2.0
LP07



Disease, lib 2




HKAL
Keratinocyte, lib 2
pCMVSport2.0
LP07


HKAT
Keratinocyte, lib 3
pCMVSport2.0
LP07


HNDA
Nasal polyps
pCMVSport2.0
LP07


HDRA
H. Primary Dendritic Cells, lib 3
pCMVSport2.0
LP07


HOHA HOHB HOHC
Human Osteoblasts II
pCMVSport2.0
LP07


HLDA HLDB HLDC
Liver, Hepatoma
pCMVSport3.0
LP08


HLDN HLDO HLDP
Human Liver, normal
pCMVSport3.0
LP08


HMTA
pBMC stimulated w/ poly I/C
pCMVSport3.0
LP08


HNTA
NTERA2, control
pCMVSport3.0
LP08


HDPA HDPB HDPC HDPD
Primary Dendritic Cells, lib 1
pCMVSport3.0
LP08


HDPF HDPG HDPH HDPI





HDPJ HDPK





HDPM HDPN HDPO HDPP
Primary Dendritic cells, frac 2
pCMVSport3.0
LP08


HMUA HMUB HMUC
Myoloid Progenitor Cell Line
pCMVSport3.0
LP08


HHEA HHEB HHEC HHED
T Cell helper I
pCMVSport3.0
LP08


HHEM HHEN HHEO HHEP
T cell helper II
pCMVSport3.0
LP08


HEQA HEQB HEQC
Human endometrial stromal
pCMVSport3.0
LP08



cells




HJMA HJMB
Human endometrial stromal
pCMVSport3.0
LP08



cells-treated with





progesterone




HSWA HSWB HSWC
Human endometrial stromal
pCMVSport3.0
LP08



cells-treated with estradiol




HSYA HSYB HSYC
Human Thymus Stromal Cells
pCMVSport3.0
LP08


HLWA HLWB HLWC
Human Placenta
pCMVSport3.0
LP08


HRAA HRAB HRAC
Rejected Kidney, lib 4
pCMVSport3.0
LP08


HMTM
PCR, pBMC I/C treated
PCRII
LP09


HMJA
H. Meniingima, M6
pSport 1
LP10


HMKA HMKB HMKC
H. Meningima, M1
pSport 1
LP10


HMKD HMKE





HUSG HUSI
Human umbilical vein
pSport 1
LP10



endothelial cells, IL-4





induced




HUSX HUSY
Human Umbilical Vein
pSport 1
LP10



Endothelial Cells, uninduced




HOFA
Ovarian Tumor I, OV5232
pSport 1
LP10


HCFA HCFB HCFC HCFD
T-Cell PHA 16 hrs
pSport 1
LP10


HCFL HCFM HCFN HCFO
T-Cell PHA 24 hrs
pSport 1
LP10


HADA HADC HADD HADE
Human Adipose
pSport 1
LP10


HADF HADG





HOVA HOVB HOVC
Human Ovary
pSport 1
LP10


HTWB HTWC HTWD
Resting T-Cell Library, II
pSport 1
LP10


HTWE HTWF





HMMA
Spleen metastic melanoma
pSport 1
LP10


HLYA HLYB HLYC HLYD
Spleen, Chronic lymphocytic
pSport 1
LP10


HLYE
leukemia




HCGA
CD34+ cell, I
pSport 1
LP10


HEOM HEON
Human Eosinophils
pSport 1
LP10


HTDA
Human Tonsil, Lib 3
pSport 1
LP10


HSPA
Salivary Gland, Lib 2
pSport 1
LP10


HCHA HCHB HCHC
Breast Cancer cell line, MDA
pSport 1
LP10



36




HCHM HCHN
Breast Cancer Cell line,
pSport 1
LP10



angiogenic




HCIA
Crohn's Disease
pSport 1
LP10


HDAA HDAB HDAC
HEL cell line
pSport 1
LP10


HABA
Human Astrocyte
pSport 1
LP10


HUFA HUFB HUFC
Ulcerative Colitis
pSport 1
LP10


HNTM
NTERA2 + retinoic acid, 14
pSport 1
LP10



days




HDQA
Primary Dendritic
pSport 1
LP10



cells, CapFinder2, frac 1




HDQM
Primary Dendritic Cells,
pSport 1
LP10



CapFinder, frac 2




HLDX
Human Liver,
pSport 1
LP10



normal, CapFinder




HULA HULB HULC
Human Dermal Endothelial
pSport1
LP10



Cells, untreated




HUMA
Human Dermal Endothelial
pSport1
LP10



cells, treated




HCJA
Human Stromal Endometrial
pSport1
LP10



fibroblasts, untreated




HCJM
Human Stromal endometrial
pSport1
LP10



fibroblasts, treated w/





estradiol




HEDA
Human Stromal endometrial
pSport1
LP10



fibroblasts, treated with





progesterone




HFNA
Human ovary tumor cell
pSport1
LP10



OV350721




HKGA HKGB HKGC HKGD
Merkel Cells
pSport1
LP10


HISA HISB HISC
Pancreas Islet Cell Tumor
pSport1
LP10


HLSA
Skin, burned
pSport1
LP10


HBZA
Prostate, BPH, Lib 2
pSport 1
LP10


HBZS
Prostate BPH, Lib 2,
pSport 1
LP10



subtracted




HFIA HFIB HFIC
Synovial Fibroblasts (control)
pSport 1
LP10


HFIH HFII HFIJ
Synovial hypoxia
pSport 1
LP10


HFIT HFIU HFIV
Synovial IL-1/TNF stimulated
pSport 1
LP10


HGCA
Messangial cell, frac 1
pSport1
LP10


HMVA HMVB HMVC
Bone Marrow Stromal Cell,
pSport1
LP10



untreated




HFIX HFIY HFIZ
Synovial Fibroblasts
pSport1
LP10



(Il1/TNF), subt




HFOX HFOY HFOZ
Synovial hypoxia-RSF
pSport1
LP10



subtracted




HMQA HMQB HMQC
Human Activated Monocytes
Uni-ZAP XR
LP11


HMQD





HLIA HLIB HLIC
Human Liver
pCMVSport 1
LP012


HHBA HHBB HHBC HHBD
Human Heart
pCMVSport 1
LP012


HHBE





HBBA HBBB
Human Brain
pCMVSport 1
LP012


HLJA HLJB HLJC HLJD
Human Lung
pCMVSport 1
LP012


HLJE





HOGA HOGB HOGC
Ovarian Tumor
pCMVSport 2.0
LP012


HTJM
Human Tonsils, Lib 2
pCMVSport 2.0
LP012


HAMF HAMG
KMH2
pCMVSport 3.0
LP012


HAJA HAJB HAJC
L428
pCMVSport 3.0
LP012


HWBA HWBB HWBC
Dendritic cells, pooled
pCMVSport 3.0
LP012


HWBD HWBE





HWAA HWAB HWAC
Human Bone Marrow,
pCMVSport 3.0
LP012


HWAD HWAE
treated




HYAA HYAB HYAC
B Cell lymphoma
pCMVSport 3.0
LP012


HWHG HWHH HWHI
Healing groin wound, 6.5
pCMVSport 3.0
LP012



hours post incision




HWHP HWHQ HWHR
Healing groin wound; 7.5
pCMVSport 3.0
LP012



hours post incision




HARM
Healing groin wound - zero hr
pCMVSport 3.0
LP012



post-incision (control)




HBIM
Olfactory epithelium;
pCMVSport 3.0
LP012



nasalcavity




HWDA
Healing Abdomen wound;
pCMVSport 3.0
LP012



70&90 min post incision




HWEA
Healing Abdomen Wound; 15
pCMVSport 3.0
LP012



days post incision




HWJA
Healing Abdomen
pCMVSport 3.0
LP012



Wound; 21&29 days




HNAL
Human Tongue, frac 2
pSport1
LP012


HMJA
H. Meniingima, M6
pSport1
LP012


HMKA HMKB HMKC
H. Meningima, M1
pSport1
LP012


HMKD HMKE





HOFA
Ovarian Tumor I, OV5232
pSport1
LP012


HCFA HCFB HCFC HCFD
T-Cell PHA 16 hrs
pSport1
LP012


HCFL HCFM HCFN HCFO
T-Cell PHA 24 hrs
pSport1
LP012


HMMA HMMB HMMC
Spleen metastic melanoma
pSport1
LP012


HTDA
Human Tonsil, Lib 3
pSport1
LP012


HDBA
Human Fetal Thymus
pSport1
LP012


HDUA
Pericardium
pSport1
LP012


HBZA
Prostate, BPH, Lib 2
pSport1
LP012


HWCA
Larynx tumor
pSport1
LP012


HWKA
Normal lung
pSport1
LP012


HSMB
Bone marrow stroma, treated
pSport1
LP012


HBHM
Normal trachea
pSport1
LP012


HLFC
Human Larynx
pSport1
LP012


HLRB
Siebben Polyposis
pSport1
LP012


HNIA
Mammary Gland
pSport1
LP012


HNJB
Palate carcinoma
pSport1
LP012


HNKA
Palate normal
pSport1
LP012


HMZA
Pharynx carcinoma
pSport1
LP012


HABG
Cheek Carcinoma
pSport1
LP012


HMZM
Pharynx Carcinoma
pSport1
LP012


HDRM
Larynx Carcinoma
pSport1
LP012


HVAA
Pancreas normal PCA4 No
pSport1
LP012


HICA
Tongue carcinoma
pSport1
LP012


HUKA HUKB HUKC HUKD
Human Uterine Cancer
Lambda ZAP II
LP013


HUKE





HFFA
Human Fetal Brain, random
Lambda ZAP II
LP013



primed




HTUA
Activated T-cell labeled with
Lambda ZAP II
LP013



4-thioluri




HBQA
Early Stage Human Brain,
Lambda ZAP II
LP013



random primed




HMEB
Human microvascular
Lambda ZAP II
LP013



Endothelial cells, fract. B




HUSH
Human Umbilical Vein
Lambda ZAP II
LP013



Endothelial cells, fract. A, re-





excision




HLQC HLQD
Hepatocellular tumor, re-
Lambda ZAP II
LP013



excision




HTWJ HTWK HTWL
Resting T-cell, re-excision
Lambda ZAP II
LP013


HF6S
Human Whole 6 week Old
pBLUESCRIPT TM
LP013



Embryo (II), subt




HHPS
Human Hippocampus,
pBLUESCRIPT TM
LP013



subtracted




HL1S
LNCAP, differential
pBLUESCRIPT TM
LP013



expression




HLHS HLHT
Early Stage Human Lung,
pBLUESCRIPT TM
LP013



Subtracted




HSUS
Supt cells, cyclohexamide
pBLUESCRIPT TM
LP013



treated, subtracted




HSUT
Supt cells, cyclohexamide
pBLUESCRIPT TM
LP013



treated, differentially





expressed




HSDS
H. Striatum Depression,
pBLUESCRIPT TM
LP013



subtracted




HPTZ
Human Pituitary, Subtracted
pBLUESCRIPT TM
LP013



VII




HSDX
H. Striatum Depression, subt
pBLUESCRIPT TM
LP013



II




HSDZ
H. Striatum Depression, subt
pBLUESCRIPT TM
LP013


HPBA HPBB HPBC HPBD
Human Pineal Gland
pBLUESCRIPT TM
LP013


HPBE

SK−



HRTA
Colorectal Tumor
pBLUESCRIPT TM
LP013




SK−



HSBA HSBB HSBC HSBM
HSC172 cells
pBLUESCRIPT TM
LP013




SK−



HJAA HJAB HJAC HJAD
Jurkat T-cell G1 phase
pBLUESCRIPT TM
LP013




SK−



HJBA HJBB HJBC HJBD
Jurkat T-cell, S1 phase
pBLUESCRIPT TM
LP013




SK−



HTNA HTNB
Human Thyroid
pBLUESCRIPT TM
LP013




SK−



HAHA HAHB
Human Adult Heart
Uni-ZAP XR
LP013


HE6A
Whole 6 week Old Embryo
Uni-ZAP XR
LP013


HFCA HFCB HFCC HFCD
Human Fetal Brain
Uni-ZAP XR
LP013


HFCE





HFKC HFKD HFKE HFKF
Human Fetal Kidney
Uni-ZAP XR
LP013


HFKG





HGBA HGBD HGBE HGBF
Human Gall Bladder
Uni-ZAP XR
LP013


HGBG





HPRA HPRB HPRC HPRD
Human Prostate
Uni-ZAP XR
LP013


HTEA HTEB HTEC HTED
Human Testes
Uni-ZAP XR
LP013


HTEE





HTTA HTTB HTTC HTTD
Human Testes Tumor
Uni-ZAP XR
LP013


HTTE





HYBA HYBB
Human Fetal Bone
Uni-ZAP XR
LP013


HFLA
Human Fetal Liver
Uni-ZAP XR
LP013


HHFB HHFC HHFD HHFE
Human Fetal Heart
Uni-ZAP XR
LP013


HHFF





HUVB HUVC HUVD HUVE
Human Umbilical Vein, End.
Uni-ZAP XR
LP013



remake




HTHB HTHC HTHD
Human Thymus
Uni-ZAP XR
LP013


HSTA HSTB HSTC HSTD
Human Skin Tumor
Uni-ZAP XR
LP013


HTAA HTAB HTAC HTAD
Human Activated T-cells
Uni-ZAP XR
LP013


HTAE





HFEA HFEB HFEC
Human Fetal Epithelium
Uni-ZAP XR
LP013



(skin)




HJPA HJPB HJPC HJPD
Human Jurkat Membrane
Uni-ZAP XR
LP013



Bound Polysomes




HESA
Human Epithelioid Sarcoma
Uni-ZAP XR
LP013


HALS
Human Adult Liver,
Uni-ZAP XR
LP013



Subtracted




HFTA HFTB HFTC HFTD
Human Fetal Dura Mater
Uni-ZAP XR
LP013


HCAA HCAB HCAC
Cem cells, cyclohexamide
Uni-ZAP XR
LP013



treated




HRGA HRGB HRGC HRGD
Raji Cells, cyclohexamide
Uni-ZAP XR
LP013



treated




HE9A HE9B HE9C HE9D
Nine Week Old Early Stage
Uni-ZAP XR
LP013


HE9E
Human




HSFA
Human Fibrosarcoma
Uni-ZAP XR
LP013


HATA HATB HATC HATD
Human Adrenal Gland Tumor
Uni-ZAP XR
LP013


HATE





HTRA
Human Trachea Tumor
Uni-ZAP XR
LP013


HE2A HE2D HE2E HE2H
12 Week Old Early Stage
Uni-ZAP XR
LP013


HE2I
Human




HE2B HE2C HE2F HE2G
12 Week Old Early Stage
Uni-ZAP XR
LP013


HE2P
Human, II




HNEA HNEB HNEC HNED
Human Neutrophil
Uni-ZAP XR
LP013


HNEE





HBGA
Human Primary Breast
Uni-ZAP XR
LP013



Cancer




HPTS HPTT HPTU
Human Pituitary, subtracted
Uni-ZAP XR
LP013


HMQA HMQB HMQC
Human Activated Monocytes
Uni-ZAP XR
LP013


HMQD





HOAA HOAB HOAC
Human Osteosarcoma
Uni-ZAP XR
LP013


HTOA HTOD HTOE HTOF
human tonsils
Uni-ZAP XR
LP013


HTOG





HMGB
Human OB MG63 control
Uni-ZAP XR
LP013



fraction I




HOPB
Human OB HOS control
Uni-ZAP XR
LP013



fraction I




HOQB
Human OB HOS treated (1 nM
Uni-ZAP XR
LP013



E2) fraction I




HAUA HAUB HAUC
Amniotic Cells - TNF
Uni-ZAP XR
LP013



induced




HAQA HAQB HAQC HAQD
Amniotic Cells - Primary
Uni-ZAP XR
LP013



Culture




HROA HROC
HUMAN STOMACH
Uni-ZAP XR
LP013


HBJA HBJB HBJC HBJD
HUMAN B CELL
Uni-ZAP XR
LP013


HBJE
LYMPHOMA




HODA HODB HODC HODD
human ovarian cancer
Uni-ZAP XR
LP013


HCPA
Corpus Callosum
Uni-ZAP XR
LP013


HSOA
stomach cancer (human)
Uni-ZAP XR
LP013


HERA
SKIN
Uni-ZAP XR
LP013


HMDA
Brain-medulloblastoma
Uni-ZAP XR
LP013


HGLA HGLB HGLD
Glioblastoma
Uni-ZAP XR
LP013


HWTA HWTB HWTC
wilm's tumor
Uni-ZAP XR
LP013


HEAA
H. Atrophic Endometrium
Uni-ZAP XR
LP013


HAPN HAPO HAPP HAPQ
Human Adult Pulmonary; re-
Uni-ZAP XR
LP013


HAPR
excision




HLTG HLTH
Human T-cell lymphoma; re-
Uni-ZAP XR
LP013



excision




HAHC HAHD HAHE
Human Adult Heart; re-
Uni-ZAP XR
LP013



excision




HAGA HAGB HAGC HAGD
Human Amygdala
Uni-ZAP XR
LP013


HAGE





HSJA HSJB HSJC
Smooth muscle-ILb induced
Uni-ZAP XR
LP013


HSHA HSHB HSHC
Smooth muscle, IL1b induced
Uni-ZAP XR
LP013


HPWA HPWB HPWC
Prostate BPH
Uni-ZAP XR
LP013


HPWD HPWE





HPIA HPIB HPIC
LNCAP prostate cell line
Uni-ZAP XR
LP013


HPJA HPJB HPJC
PC3 Prostate cell line
Uni-ZAP XR
LP013


HBTA
Bone Marrow Stroma,
Uni-ZAP XR
LP013



TNF&LPS ind




HMCF HMCG HMCH HMCI
Macrophage-oxLDL; re-
Uni-ZAP XR
LP013


HMCJ
excision




HAGG HAGH HAGI
Human Amygdala; re-excision
Uni-ZAP XR
LP013


HACA
H. Adipose Tissue
Uni-ZAP XR
LP013


HKFB
K562 + PMA (36 hrs), re-
ZAP Express
LP013



excision




HCWT HCWU HCWV
CD34 positive cells (cord
ZAP Express
LP013



blood), re-ex




HBWA
Whole brain
ZAP Express
LP013


HBXA HBXB HBXC HBXD
Human Whole Brain #2 -
ZAP Express
LP013



Oligo dT >1.5 Kb




HAVM
Temporal cortex-Alzheizmer
pT-Adv
LP014


HAVT
Hippocampus, Alzheimer
pT-Adv
LP014



Subtracted




HHAS
CHME Cell Line
Uni-ZAP XR
LP014


HAJR
Larynx normal
pSport 1
LP014


HWLE HWLF HWLG
Colon Normal
pSport 1
LP014


HWLH





HCRM HCRN HCRO
Colon Carcinoma
pSport 1
LP014


HWLI HWLJ HWLK
Colon Normal
pSport 1
LP014


HWLQ HWLR HWLS
Colon Tumor
pSport 1
LP014


HWLT





HBFM
Gastrocnemius Muscle
pSport 1
LP014


HBOD HBOE
Quadriceps Muscle
pSport 1
LP014


HBKD HBKE
Soleus Muscle
pSport 1
LP014


HCCM
Pancreatic Langerhans
pSport 1
LP014


HWGA
Larynx carcinoma
pSport 1
LP014


HWGM HWGN
Larynx carcinoma
pSport 1
LP014


HWLA HWLB HWLC
Normal colon
pSport 1
LP014


HWLM HWLN
Colon Tumor
pSport 1
LP014


HVAM HVAN HVAO
Pancreas Tumor
pSport 1
LP014


HWGQ
Larynx carcinoma
pSport 1
LP014


HAQM HAQN
Salivary Gland
pSport 1
LP014


HASM
Stomach; normal
pSport 1
LP014


HBCM
Uterus; normal
pSport 1
LP014


HCDM
Testis; normal
pSport 1
LP014


HDJM
Brain; normal
pSport 1
LP014


HEFM
Adrenal Gland, normal
pSport 1
LP014


HBAA
Rectum normal
pSport 1
LP014


HFDM
Rectum tumour
pSport 1
LP014


HGAM
Colon, normal
pSport 1
LP014


HHMM
Colon, tumour
pSport 1
LP014


HCLB HCLC
Human Lung Cancer
Lambda Zap II
LP015


HRLA
L1 Cell line
ZAP Express
LP015


HHAM
Hypothalamus, Alzheimer's
pCMVSport 3.0
LP015


HKBA
Ku 812F Basophils Line
pSport 1
LP015


HS2S
Saos2, Dexamethosome
pSport 1
LP016



Treated




HA5A
Lung Carcinoma A549
pSport 1
LP016



TNFalpha activated




HTFM
TF-1 Cell Line GM-CSF
pSport 1
LP016



Treated




HYAS
Thyroid Tumour
pSport 1
LP016


HUTS
Larynx Normal
pSport 1
LP016


HXOA
Larynx Tumor
pSport 1
LP016


HEAH
Ea.hy.926 cell line
pSport 1
LP016


HINA
Adenocarcinoma Human
pSport 1
LP016


HRMA
Lung Mesothelium
pSport 1
LP016


HLCL
Human Pre-Differentiated
Uni-Zap XR
LP017



Adipocytes




HS2A
Saos2 Cells
pSport 1
LP020


HS2I
Saos2 Cells; Vitamin D3
pSport 1
LP020



Treated




HUCM
CHME Cell Line, untreated
pSport 1
LP020


HEPN
Aryepiglottis Normal
pSport 1
LP020


HPSN
Sinus Piniformis Tumour
pSport 1
LP020


HNSA
Stomach Normal
pSport 1
LP020


HNSM
Stomach Tumour
pSport 1
LP020


HNLA
Liver Normal Met5No
pSport 1
LP020


HUTA
Liver Tumour Met 5 Tu
pSport 1
LP020


HOCN
Colon Normal
pSport 1
LP020


HOCT
Colon Tumor
pSport 1
LP020


HTNT
Tongue Tumour
pSport 1
LP020


HLXN
Larynx Normal
pSport 1
LP020


HLXT
Larynx Tumour
pSport 1
LP020


HTYN
Thymus
pSport 1
LP020


HPLN
Placenta
pSport 1
LP020


HTNG
Tongue Normal
pSport 1
LP020


HZAA
Thyroid Normal (SDCA2 No)
pSport 1
LP020


HWES
Thyroid Thyroiditis
pSport 1
LP020


HFHD
FICOLL TM ed Human Stromal
pTrip1Ex2
LP021



Cells, 5Fu treated




HFHM, HFHN
FICOLL TM ed Human Stromal
pTrip1Ex2
LP021



Cells, Untreated




HPCI
Hep G2 Cells, lambda library
lambda Zap-CMV
LP021




XR



HBCA, HBCB, HBCC
H. Lymph node breast Cancer
Uni-ZAP XR
LP021


HCOK
Chondrocytes
pSPORT1
LP022


HDCA, HDCB, HDCC
Dendritic Cells From CD34
pSPORT1
LP022



Cells




HDMA, HDMB
CD40 activated monocyte
pSPORT1
LP022



dendritic cells




HDDM, HDDN, HDDO
LPS activated derived
pSPORT1
LP022



dendritic cells




HPCR
Hep G2 Cells, PCR library
lambda Zap-CMV
LP022




XR



HAAA, HAAB, HAAC
Lung, Cancer (4005313A3):
pSPORT1
LP022



Invasive Poorly Differentiated





Lung Adenocarcinoma




HIPA, HIPB, HIPC
Lung, Cancer (4005163 B7):
pSPORT1
LP022



Invasive, Poorly Diff.





Adenocarcinoma, Metastatic




HOOH, HOOI
Ovary, Cancer: (4004562 B6)
pSPORT1
LP022



Papillary Serous Cystic





Neoplasm, Low Malignant





Pot




HIDA
Lung, Normal: (4005313 B1)
pSPORT1
LP022


HUJA, HUJB, HUJC, HUJD, HUJE
B-Cells
pCMVSport 3.0
LP022


HNOA, HNOB, HNOC, HNOD
Ovary, Normal: (9805C040R)
pSPORT1
LP022


HNLM
Lung, Normal: (4005313 B1)
pSPORT1
LP022


HSCL
Stromal Cells
pSPORT1
LP022


HAAX
Lung, Cancer: (4005313 A3)
pSPORT1
LP022



Invasive Poorly-differentiated





Metastatic lung





adenocarcinoma




HUUA, HUUB, HUUC, HUUD
B-cells (unstimulated)
pTrip1Ex2
LP022


HWWA, HWWB, HWWC, HWWD,
B-cells (stimulated)
pSPORT1
LP022


HWWE, HWWF, HWWG





HCCC
Colon, Cancer: (9808C064R)
pCMVSport 3.0
LP023


HPDO HPDP HPDQ HPDR
Ovary, Cancer (9809C332):
pSport 1
LP023


HPD
Poorly differentiated





adenocarcinoma




HPCO HPCP HPCQ HPCT
Ovary, Cancer (15395A1F):
pSport 1
LP023



Grade II Papillary Carcinoma




HOCM HOCO HOCP HOCQ
Ovary, Cancer: (15799A1F)
pSport 1
LP023



Poorly differentiated





carcinoma




HCBM HCBN HCBO
Breast, Cancer: (4004943 A5)
pSport 1
LP023


HNBT HNBU HNBV
Breast, Normal: (4005522B2)
pSport 1
LP023


HBCP HBCQ
Breast, Cancer: (4005522 A2)
pSport 1
LP023


HBCJ
Breast, Cancer: (9806C012R)
pSport 1
LP023


HSAM HSAN
Stromal cells 3.88
pSport 1
LP023


HVCA HVCB HVCC HVCD
Ovary, Cancer: (4004332 A2)
pSport 1
LP023


HSCK HSEN HSEO
Stromal cells (HBM3.18)
pSport 1
LP023


HSCP HSCQ
stromal cell clone 2.5
pSport 1
LP023


HUXA
Breast Cancer: (4005385 A2)
pSport 1
LP023


HCOM HCON HCOO HCOP
Ovary, Cancer (4004650 A3):
pSport 1
LP023


HCOQ
Well-Differentiated





Micropapillary Serous





Carcinoma




HBNM
Breast, Cancer: (9802C020E)
pSport 1
LP023


HVVA HVVB HVVC HVVD
Human Bone Marrow, treated
pSport 1
LP023


HVVE









Two nonlimiting examples are provided below for isolating a particular clone from the deposited sample of plasmid cDNAs cited for that clone in Table 1A. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to the nucleotide sequence of SEQ ID NO:X.
Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with 32P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (STRATAGENETM)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the nucleotide sequence of SEQ ID NO:X are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 μl of reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.
Several methods are available for the identification of the 5′ or 3′ non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols which are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993)).
Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.
This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene.
Example 2
Isolation of Genomic Clones Corresponding to a Polynucleotide
A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR using primers selected for the sequence corresponding to SEQ ID NO:X according to the method described in Example 1. (See also, Sambrook.)
Example 3
Tissue Specific Expression Analysis
Tissue distribution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, described by, among others, Sambrook et al. For example, a cDNA probe produced by the method described in Example 1 is labeled with P32 using the REDIPRIMETM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using CHROMA SPN-100TM column (CLONTECHTM Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for mRNA expression.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) (CLONTECHTM) are examined with the labeled probe using EXPRESSHYBTM hybridization solution (CLONTECHTM) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at −70° C. overnight, and the films developed according to standard procedures.
The Human Genome Sciences, Inc. (HGS) database is derived from sequencing tissue and/or disease specific cDNA libraries. Libraries generated from a particular tissue are selected and the specific tissue expression pattern of EST groups or assembled contigs within these libraries is determined by comparison of the expression patterns of those groups or contigs within the entire database. ESTs and assembled contigs which show tissue specific expression are selected.
The original clone from which the specific EST sequence was generated, or in the case of an assembled contig, the clone from which the 5′ most EST sequence was generated, is obtained from the catalogued library of clones and the insert amplified by PCR using methods known in the art. The PCR product is denatured and then transferred in 96 or 384 well format to a nylon membrane (Schleicher and Scheull) generating an array filter of tissue specific clones. Housekeeping genes, maize genes, and known tissue specific genes are included on the filters. These targets can be used in signal normalization and to validate assay sensitivity. Additional targets are included to monitor probe length and specificity of hybridization.
Radioactively labeled hybridization probes are generated by first strand cDNA synthesis per the manufacturer's instructions (LIFE TECHNOLOGIESTM) from mRNA/RNA samples prepared from the specific tissue being analyzed (e.g., prostate, prostate cancer, ovarian, ovarian cancer, etc.). The hybridization probes are purified by gel exclusion chromatography, quantitated, and hybridized with the array filters in hybridization bottles at 65° C. overnight. The filters are washed under stringent conditions and signals are captured using a Fuji phosphorimager.
Data is extracted using AIS software and following background subtraction, signal normalization is performed. This includes a normalization of filter-wide expression levels between different experimental runs. Genes that are differentially expressed in the tissue of interest are identified.
Example 4
Chromosomal Mapping of the Polynucleotides
An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95° C.; 1 minute, 56° C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5 minute cycle at 70° C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions are analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.
Example 5
Bacterial Expression of a Polypeptide
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 μg/ml) and Kan (25 μg/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 min at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4° C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4° C. or frozen at −80° C.
In addition to the above expression vector, the present invention further includes an expression vector, called pHE4a (ATCCTM Accession Number 209645, deposited on Feb. 25, 1998) which contains phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCCTM Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter and operator sequences are made synthetically.
DNA can be inserted into the pHE4a by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.
Example 6
Purification of a Polypeptide from an Inclusion Body
The following alternative method can be used to purify a polypeptide expressed in E. coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10° C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10° C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4° C. overnight to allow further GuHCl extraction.
Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4° C. without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 μm membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 7
Cloning and Expression of a Polypeptide in a Baculovirus Expression System
In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon, is amplified using the PCR protocol described in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“GENECLEANTM,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“GENECLEANTM” BIO 101 Inc., La Jolla, Calif.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (STRATAGENETM Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
Five μg of a plasmid containing the polynucleotide is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BACULOGOLDTM baculovirus DNA, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BACULOGOLDTM virus DNA and 5 μg of the plasmid are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (LIFE TECHNOLOGIESTM Inc., Gaithersburg, Md.). Afterwards, 10 μl LIPOFECTINTM plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCCTM CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27° C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27° C. for four days.
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (LIFE TECHNOLOGIESTM Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by LIFE TECHNOLOGIESTM Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4° C.
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from LIFE TECHNOLOGIESTM Inc., Rockville, Md.). After 42 hours, 5 μCi of 35S-methionine and 5 μCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.
Example 8
Expression of a Polypeptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (PHARMACIATM, Uppsala, Sweden), pRSVcat (ATCCTM 37152), pSV2dhfr (ATCCTM 37146), pBC12MI (ATCCTM 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991)). Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
Derivatives of the plasmid pSV2-dhfr (ATCCTM Accession No. 37146), the expression vectors pC4 (ATCCTM Accession No. 209646) and pC6 (ATCCTM Accession No. 209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the vector does not need a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“GENECLEANTM,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. Five μg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using LIPOFECTINTM (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 9
Protein Fusions
The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EPA 394,827; Traunecker, et al., Nature 331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.
For example, if pC4 (ATCCTM Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
If the naturally occurring signal sequence is used to produce the polypeptide of the present invention, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)







Human IgG Fc region:


(SEQ ID NO: 236)


GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC


 


CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA


 


ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG


 


TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG


 


GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA


 


CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT


 


GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA


 


ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC


 


ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG


 


TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG


 


GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC


 


CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG


 


ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT


 


GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG


 


TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT






Example 10
Production of an Antibody from a Polypeptide
a) Hybridoma Technology
The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of a polypeptide of the present invention is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for a polypeptide of the present invention are prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with a polypeptide of the present invention or, more preferably, with a secreted polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCCTM. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide of the present invention.
Alternatively, additional antibodies capable of binding to a polypeptide of the present invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the polypeptide-specific antibody can be blocked by said polypeptide. Such antibodies comprise anti-idiotypic antibodies to the polypeptide-specific antibody and are used to immunize an animal to induce formation of further polypeptide-specific antibodies.
It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, secreted protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)).
b) Isolation of Antibody Fragments Directed Against a Polypeptide of the Present Invention from a Library of scFvs
Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against a polypeptide of the present invention to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).
Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in International Publication No. WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking Five ml of this culture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see International Publication No. WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and grown overnight. Phage are prepared as described in International Publication No. WO 92/01047.
M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).
Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., International Publication No. WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.
Example 11
Method of Determining Alterations in a Gene Corresponding to a Polynucleotide
RNA from entire families or individual patients presenting with a phenotype of interest (such as a disease or disorder, e.g., an immune, cardiovascular, cancer, or other proliferative disease or disorder) is isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X; and/or the nucleotide sequence of the cDNA contained in ATCCTM Deposit No:Z. Suggested PCR conditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 seconds at 52-58° C.; and 60-120 seconds at 70° C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).
PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase (Epicentre Technologies). The intron-exon boundaries of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations are then cloned and sequenced to validate the results of the direct sequencing.
PCR products are cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (BOEHRINGERTM Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991)). Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.
Example 12
Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample
A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 μg/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.
The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbound polypeptide.
Next, 50 μl of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbound conjugate.
Add 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.
Example 13
Formulation
The invention also provides methods of preventing, treating and/or ameliorating a disease or disorder (such as, for example, any one or more of the diseases or disorders disclosed herein, e.g., an immune, cardiovascular, cancer, or other proliferative disease or disorder) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).
The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
Therapeutics can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).
In a preferred embodiment, polypeptide, polynucleotide, and antibody compositions of the invention are formulated in a biodegradable, polymeric drug delivery system, for example as described in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519; 5,340,849; and 5,487,897 and in International Publication Numbers WO01/35929, WO00/24374, and WO00/06117 which are hereby incorporated by reference in their entirety. In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the ATRIGEL® Biodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colo.).
Examples of biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions, include but are not limited to, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials. The preferred polymers are those that have a lower degree of crystallization and are more hydrophobic. These polymers and copolymers are more soluble in the biocompatible solvents than the highly crystalline polymers such as polyglycolide and chitin which also have a high degree of hydrogen-bonding. Preferred materials with the desired solubility parameters are the polylactides, polycaprolactones, and copolymers of these with glycolide in which there are more amorphous regions to enhance solubility. In specific preferred embodiments, the biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions are poly(lactide-co-glycolides). Polymer properties such as molecular weight, hydrophobicity, and lactide/glycolide ratio may be modified to obtain the desired polypeptide, polynucleotide, or antibody release profile (See, e.g., Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732-741 (2000), which is hereby incorporated by reference in its entirety).
It is also preferred that the solvent for the biodegradable polymer be non-toxic, water miscible, and otherwise biocompatible. Examples of such solvents include, but are not limited to, N-methyl-2-pyrrolidone, 2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alcohols, dils, triols, and tetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15 alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone; C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate; alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such as dimethylformamide, dimethylacetamide and caprolactam; C3 to C20 ethers such as tetrahydrofuran, or solketal; tweens, triacetin, propylene carbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid, 1-dodecylazacycloheptan-2-one, Other preferred solvents are benzyl alcohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate, glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleic acid, polyethylene glycol, propylene carbonate, and triethyl citrate. The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin, and propylene carbonate because of the solvating ability and their compatibility.
Additionally, formulations comprising polypeptide, polynucleotide, and antibody compositions and a biodegradable polymer may also include release-rate modification agents and/or pore-forming agents. Examples of release-rate modification agents include, but are not limited to, fatty acids, triglycerides, other like hydrophobic compounds, organic solvents, plasticizing compounds and hydrophilic compounds. Suitable release rate modification agents include, for example, esters of mono-, di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate, dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate, glycerol triacetate, di(n-butyl)sebecate, and the like; polyhydroxy alcohols, such as propylene glycol, polyethylene glycol, glycerin, sorbitol, and the like; fatty acids; triesters of glycerol, such as triglycerides, epoxidized soybean oil, and other epoxidized vegetable oils; sterols, such as cholesterol; alcohols, such as C.sub.6-C.sub.12 alkanols, 2-ethoxyethanol. The release rate modification agent may be used singly or in combination with other such agents. Suitable combinations of release rate modification agents include, but are not limited to, glycerin/propylene glycol, sorbitol/glycerine, ethylene oxide/propylene oxide, butylene glycol/adipic acid, and the like. Preferred release rate modification agents include, but are not limited to, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, and hexanediol. Suitable pore-forming agents that may be used in the polymer composition include, but are not limited to, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, polymers such as hydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone. Solid crystals that will provide a defined pore size, such as salt or sugar, are preferred.
In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the BEMATM BioErodible Mucoadhesive System, MCATM MucoCutaneous Absorption System, SMPTM Solvent MicroParticle System, or BCPTM BioCompatible Polymer System of Atrix Laboratories, Inc. (Fort Collins, Colo.).
Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.
In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
The Therapeutic is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.
The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (GENENTECHTM, Inc.), BCG (e.g., THERACYS®), MPL and nonviable preparations of Corynebacterium parvum. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diphtheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but are not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines, growth factors, and/or therapeutic treatments described below. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
In one embodiment, the Therapeutics of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the compositions of the invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADIN®), dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADONTM), acenocoumarol (e.g., nicoumalone, SINTHROMETM), indan-1,3-dione, phenprocoumon (e.g., MARCUMARTM), ethyl biscoumacetate (e.g., TROMEXANTM), and aspirin. In a specific embodiment, compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin.
In another embodiment, the Therapeutics of the invention are administered in combination with thrombolytic drugs. Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g., KABIKINASETM), antiresplace (e.g., EMINASETM), tissue plasminogen activator (t-PA, altevase, ACTIVASETM), urokinase (e.g., ABBOKINASETM), sauruplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICARTM). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.
In another embodiment, the Therapeutics of the invention are administered in combination with antiplatelet drugs. Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINETM), and ticlopidine (e.g., TICLIDTM).
In specific embodiments, the use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the prevention of occlusion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the therapeutics of the invention, alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular cannulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
Therapeutics of the invention may also be administered in combination with additional cardiovascular agents, such as, for example, beta-adrenergic blockers, calcium channel blockers, ACE inhibitors, angiotensin II blockers, alpha adrenergic blockers, hypotensive agents, antilipemic agents, and vasodilating agents.
Non-limiting examples of beta-adrenergic blockers includes TENORMINTM (atenolol), BREVIBLOCTM (esmolol), NORMODYNETM (labetalol), TRANDATETM, LOPRESSORTM (metoprolol), INDERALTM (propranolol), and BETApp96TM (sotalol). Calcium channel blockers includes, for example, NORVASCTM (amlodipine), CARDIZEMTM (diltiazem), PLENDILTM (felodipine), DYNACRICTM (isradipine), CARDENETM (nicardipine), ADALATTM (nifedipine), and CALANTM (verapamil). ACE inhibitors includes, for example, LOTENSINTM (benazepril), CAPOTENTM (captopril), VASOTECTM (enalapril), MONOPRILTM (fosinopril), PRINIVILTM (lisinopril), ACCUPRILTM (quinapril), and ALTACETM (ramipril). Non-limiting examples of angiotensin II blockers includes AVAPROTM (irbesartan), COZAARTM (losartan), and DIOVANTM (valsartan). Alpha adrenergic blockers include, for example, CARDURATM (doxazosin), MINIPRESSTM (prazosin), FLOMAXTM (tamsulosin), and terazosin. Hypotensive agents include, for example, CATAPRESTM (clonidine), APRESOLINETM (hydralazine), ALDOMETTM (methyldopa), LONITENTM (minoxidil), NIPRIDETM (nitroprusside) and reserpine. Antilipemic agents include, for example, LIPITORTM (atorvastatin), QUESTRANTM (cholestyramine), LOLESTIDTM (colestipol), TRICORTM (fenofibrate), LOPIDTM (gemfibrate), MEVACORTM (lovstatin), PRAVACHOLTM (pravastatin), and ZOCORTM (simvastatin). Non-limiting examples of vasodilating agents include alprostadil, amyl nitrite, PERSANTINTM (dipyridamole), FLONANTM (epoprostenol), ISORDILTM (isosorbide dinitrate), IMDURTM (isosorbide mononitrate), NIMOTOPTM (nimodipine), INOmaxTM (nitric oxide gas), nitroglycerin, papaverine, and PRISCOLINETM (tolazoline).
In certain embodiments, therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddI), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM (zidovudine/lamivudine). NNRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir), INVIRASETM (saquinavir), and VIRACEPTTM (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.
Additional NRTIs include LODENOSINETM (F-ddA; an acid-stable adenosine NRTI; Triangle/ABBOTTTM; COVIRACILTM (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro; Triangle/ABBOTTTM); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is PMEA-pp); TENOFOVIRTM (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/ABBOTTTM); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGENTM (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′ azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of 13-L-FD4C and 13-L-FddC (WO 98/17281).
Additional NNRTIs include COACTINONTM (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/ABBOTTTM); CAPRAVIRINETM (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; AGOURONTM); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; PHARMACIATM & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DUPONTTM); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (WO 99/49830).
Additional protease inhibitors include LOPINAVIRTM (ABT378/r; ABBOTTTM Laboratories); BMS-232632 (an azapeptide; BRISTOL-MYERS SQUIBBTM); TIPRANAVIRTM (PNU-140690, a non-peptic dihydropyrone; PHARIVIACIATM & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; BRISTOL-MYERS SQUIBBTM); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DUPONTTM); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; AGOURONTM); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Wellcome); CGP61755 (Ciba); and AGENERASETM (amprenavir; Glaxo Wellcome Inc.).
Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor; Trimeris).
Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibit fusion.
Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIRTM (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.
Additional antiretroviral agents include hydroxyurea-like compounds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOXTM (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors such as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; ROCHETM).
Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.
Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKINTM (aldesleukin/L2-7001; CHIRONTM), IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as RemuneTM (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targeted to the ER to block surface expression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).
In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, ATOVAQUONETM, ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM, AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM, ITRACONAZOLETM, KETOCONAZOLETM, ACYCLOVIRTM, FAMCICOLVIRTM, PYRIMETHAMINETM, LEUCOVORINTM, NEUPOGENTM (filgrastim/G-CSF), and LEUKINETM (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, and/or ATOVAQUONETM to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, and/or ETHAMBUTOLTM to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTINTM, CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLETM, ITRACONAZOLETM, and/or KETOCONAZOLETM to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINETM and/or LEUCOVORINTM to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat or prevent an opportunistic bacterial infection.
In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.
In other embodiments, the Therapeutics of the invention are administered in combination with immunostimulants. Immunostimulants that may be administered in combination with the Therapeutics of the invention include, but are not limited to, levamisole (e.g., ERGAMISOLTM), isoprinosine (e.g. INOSIPLEXTM), interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).
In other embodiments, Therapeutics of the invention are administered in combination with immunosuppressive agents. Immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININTM), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNETM, NEORALTM, SANGDYATM (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURANTM (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONETM (prednisone) and HYDELTRASOLTM (prednisolone), FOLEXTM and MEXATETM (methotrxate), OXSORALEN-ULTRATM (methoxsalen) and RAPAMUNETM (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S/DTM, ATGAMTM (antithymocyte glubulin), and GAMIMUNETM. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
In certain embodiments, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (ENTREMEDTM, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (TAXOLTM), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262 (4): 1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.
Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (CELGENETM, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard et al., J Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXIGENETM, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (TAP PHARMACEUTICALSTM, Deerfield, Ill.); ZD-0101 ASTRAZENECATM (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (NOLVADEXTM); Tazarotene; Tetrathiomolybdate; XELODATM (Capecitabine); and 5-Fluorouracil.
Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositions of the invention include, but are not limited to, AG-3340 (AGOURONTM, La Jolla, Calif.), BAY-12-9566 (BAYERTM, West Haven, Conn.), BMS-275291 (BRISTOL-MYERS SQUIBBTM, Princeton, N.J.), CGS-27032A (NOVARTISTM, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and METASTATTM (AETERNATM, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositions of the invention include, but are not limited to, EMD-121974 (MERCKTM KcgaA Darmstadt, Germany) and VITAXINTM (IXSYSTM, La Jolla, Calif./MEDIMMUNETM, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositions of the invention include, but are not limited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (GENENTECHTM, S. San Francisco, Calif.), PTK-787/ZK-225846 (NOVARTISTM, Basel, Switzerland), SU-101 (SUGENTM, S. San Francisco, Calif.), SU-5416 (SUGENTM/PHARMACIATM Upjohn, Bridgewater, N.J.), and SU-6668 (SUGENTM). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositions of the invention include, but are not limited to, IM-862 (CYTRANTM, Kirkland, Wash.), Interferon-alpha, IL-12 (ROCHETM, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).
In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.
In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.
In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to alkylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for example, Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin (2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted ureas (for example, Hydroxyurea), methylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine; MIH), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing hormone analogs (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone, estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, glutamic acid, and mitotane).
In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as RemicadeTM Centocor, Inc.), Trocade (ROCHETM, RO-32-3555), Leflunomide (also known as AravaTM from HOECHST MARION ROUSSELTM), KineretTM (an IL-1 Receptor antagonist also known as Anakinra from AMGENTM, Inc.)
In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.
In another specific embodiment, the compositions of the invention are administered in combination ZEVALINTM. In a further embodiment, compositions of the invention are administered with ZEVALINTM and CHOP, or ZEVALINTM and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. ZEVALINTM may be associated with one or more radioisotopes. Particularly preferred isotopes are 90Y and 111In.
In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.
In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are herein incorporated by reference in their entireties.
In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINETM, PROKINETM), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGENTM), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGENTM, PROCRITTM), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any one or more of IL-1 through IL-12, interferon-gamma, or thrombopoietin.
In certain embodiments, Therapeutics of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, osmolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.
In another embodiment, the Therapeutics of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amiodarone, bretylium, digitalis, digoxin, digitoxin, diliazem, diisopyramide, esmolol, flecaimide, lidocaine, mexiletine, moricizine, phenyloin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocamide, and verapamil).
In another embodiment, the Therapeutics of the invention are administered in combination with diuretic agents, such as carbonic anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibit Na+—K+-2Cl− symport (e.g., furosemide, bumetamide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylclothiazide, polythiazide, trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor antagonists (e.g., spironolactone, canrenone, and potassium canrenoate).
In one embodiment, the Therapeutics of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, 127I, radioactive isotopes of iodine such as 131I and 123I; recombinant growth hormone, such as HUMATROPETM (recombinant somatropin); growth hormone analogs such as PROTROPINTM (somatrem); dopamine agonists such as PARLODELTM (bromocriptine); somatostatin analogs such as SANDOSTATINTM (octreotide); gonadotropin preparations such as PREGNYLTM, A.P.L.TM and PROFASITM (chorionic gonadotropin (CG)), PERGONALTM (menotropins), and METRODINTM (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTRELTM and LUTREPULSETM (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRONTM (leuprolide acetate), SUPPRELINTM (histrelin acetate), SYNARELTM (nafarelin acetate), and ZOLADEXTM (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRHTM and THYPINONETM (protirelin); recombinant human TSH such as THYROGENTM; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T4TM, SYNTHROIDTM and LEVOTHROIDTM (levothyroxine sodium), L-T3TM, CYTOMELTM and TRIOSTATTM (liothyroine sodium), and THYROLARTM (liotrix); antithyroid compounds such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLETM (methimazole), NEO-MERCAZOLETM (carbimazole); beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca2+ channel blockers; dexamethasone and iodinated radiological contrast agents such as TELEPAQUETM (iopanoic acid) and ORAGRAFINTM (sodium ipodate).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or conjugated estrogens such as ESTRACETM (estradiol), ESTINYLTM (ethinyl estradiol), PREMARINTM, ESTRATABTM, ORTHO-ESTTM, OGENTM and estropipate (estrone), ESTROVISTM (quinestrol), ESTRADERMTM (estradiol), DELESTROGENTM and VALERGENTM (estradiol valerate), DEPO-ESTRADIOL CYPIONATETM and ESTROJECT LATM (estradiol cypionate); antiestrogens such as NOLVADEXTM (tamoxifen), SEROPHENETM and CLOMIDTM (clomiphene); progestins such as DURALUTINTM (hydroxyprogesterone caproate), MPATM and DEPO-PROVERATM (medroxyprogesterone acetate), PROVERATM and CYCRINTM (MPA), MEGACETM (megestrol acetate), NORLUTINTM (norethindrone), and NORLUTATETM and AYGESTINTM (norethindrone acetate); progesterone implants such as NORPLANT SYSTEMTM (subdermal implants of norgestrel); antiprogestins such as RU 486TM (mifepristone); hormonal contraceptives such as ENOVIDTM (norethynodrel plus mestranol), PROGESTASERTTM (intrauterine device that releases progesterone), LOESTRINTM, BREVICONTM, MODICONTM, GENORATM, NELONATM, NORINYLTM, OVACON-35TM and OVACON-50TM (ethinyl estradiol/norethindrone), LEVLENTM, NORDETTETM, TRI-LEVLENTM and TRIPHASIL-21TM (ethinyl estradiol/levonorgestrel) LO/OVRALTM and OVRALTM (ethinyl estradiol/norgestrel), DEMULENTM (ethinyl estradiol/ethynodiol diacetate), NORINYLTM, ORTHO-NOVUMTM, NORETHINTM, GENORATM, and NELOVATM (norethindrone/mestranol), DESOGENTM and ORTHO-CEPTTM (ethinyl estradiol/desogestrel), ORTHO-CYCLENTM and ORTHO-TRICYCLENTM (ethinyl estradiol/norgestimate), MICRONORTM and NOR-QDTM (norethindrone), and OVRETTETM (norgestrel).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50TM (testosterone), TESTEXTM (testosterone propionate), DELATESTRYLTM (testosterone enanthate), DEPO-TESTOSTERONETM (testosterone cypionate), DANOCRINETM (danazol), HALOTESTINTM (fluoxymesterone), ORETON METHYLTM, TESTREDTM and VIRILONTM (methyltestosterone), and OXANDRINTM (oxandrolone); testosterone transdermal systems such as TESTODERMTM; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCURTM (cyproterone acetate), EULEXINTM (flutamide), and PROSCARTM (finasteride); adrenocorticotropic hormone preparations such as CORTROSYNTM (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATETM (alclometasone dipropionate), CYCLOCORTTM (amcinonide), BECLOVENTTM and VANCERILTM (beclomethasone dipropionate), CELESTONETM (betamethasone), BENISONETM and UTICORTTM (betamethasone benzoate), DIPROSONETM (betamethasone dipropionate), CELESTONE PHOSPHATETM (betamethasone sodium phosphate), CELESTONE SOLUSPANTM (betamethasone sodium phosphate and acetate), BETA-VALTM and VALISONETM (betamethasone valerate), TEMOVATETM (clobetasol propionate), CLODERMTM (clocortolone pivalate), CORTEFTM and HYDROCORTONETM (cortisol (hydrocortisone)), HYDROCORTONE ACETATETM (cortisol (hydrocortisone) acetate), LOCOIDTM (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATETM (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORTTM and SOLU CORTEFTM (cortisol (hydrocortisone) sodium succinate), WESTCORTTM (cortisol (hydrocortisone) valerate), CORTISONE ACETATETM (cortisone acetate), DESOWENTM and TRIDESILONTM (desonide), TOPICORTTM (desoximetasone), DECADRONTM (dexamethasone), DECADRON LATM (dexamethasone acetate), DECADRON PHOSPHATETM and HEXADROL PHOSPHATETM (dexamethasone sodium phosphate), FLORONETM and MAXIFLORTM (diflorasone diacetate), FLORINEF ACETATETM (fludrocortisone acetate), AEROBIDTM and NASALIDETM (flunisolide), FLUONIDTM and SYNALARTM (fluocinolone acetonide), LIDEXTM (fluocinonide), FLUOROPTM and FMLTM (fluorometholone), CORDRANTM (flurandrenolide), HALOGTM (halcinonide), HMS LIZUIFILMTM (medrysone), MEDROLTM (methylprednisolone), DEPO-MEDROLTM and MEDROL ACETATETM (methylprednisone acetate), A-METHAPREDTM and SOLUMEDROLTM (methylprednisolone sodium succinate), ELOCONTM (mometasone furoate), HALDRONETM (paramethasone acetate), DELTA-CORTEFTM (prednisolone), ECONOPREDTM (prednisolone acetate), HYDELTRASOLTM (prednisolone sodium phosphate), HYDELTRA-T.B.ATM (prednisolone tebutate), DELTASONETM (prednisone), ARISTOCORTTM and KENACORTTM (triamcinolone), KENALOGTM (triamcinolone acetonide), ARISTOCORTTM and KENACORT DIACETATETM (triamcinolone diacetate), and ARISTOSPANTM (triamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADRENTM (aminoglutethimide), NIZORALTM (ketoconazole), MODRASTANETM (trilostane), and METOPIRONETM (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULINTM and NOVOLINTM; oral hypoglycemic agents such as ORAMIDETM and ORINASETM (tolbutamide), DIABINESETM (chlorpropamide), TOLAMIDETM and TOLINASETM (tolazamide), DYMELORTM (acetohexamide), glibenclamide, MICRONASETM, DIBETATM and GLYNASETM (glyburide), GLUCOTROLTM (glipizide), and DIAMICRONTM (gliclazide), GLUCOPHAGETM (metformin), PRECOSETM (acarbose), AMARYLTM (glimepiride), and ciglitazone; thiazolidinediones (TZDs) such as rosiglitazone, AVANDIATM (rosiglitazone maleate), ACTOSTM (piogliatazone), troglitazone, ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATINTM (octreotide); and diazoxides such as PROGLYCEMTM (diazoxide). In still other embodiments, Therapeutics of the invention are administered in combination with one or more of the following: a biguanide antidiabetic agent, a glitazone antidiabetic agent, and a sulfonylurea antidiabetic agent.
In one embodiment, the Therapeutics of the invention are administered in combination with treatments for uterine motility disorders. Treatments for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens (e.g., PREMARIN® and ESTRATAB®), estradiols (e.g., CLIMARA® and ALORA®), estropipate, and chlorotrianisene; progestin drugs (e.g., AMEN® (medroxyprogesterone), MICRONOR® (norethidrone acetate), PROMETRIUM® progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogens/medroxyprogesterone (e.g., PREMPROTM and PREMPHASE®) and norethindrone acetate/ethinyl estsradiol (e.g., FEMHRTTM).
In an additional embodiment, the Therapeutics of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but not limited to, ferrous sulfate (iron sulfate, FEOSOLTM), ferrous fumarate (e.g., FEOSTATTM), ferrous gluconate (e.g., FERGONTM), polysaccharide-iron complex (e.g., NIFEREXTM), iron dextran injection (e.g., INFEDTM), cupric sulfate, pyroxidine, riboflavin, Vitamin B12, cyancobalamin injection (e.g., REDISOLTM, RUBRAMIN PCTM), hydroxocobalamin, folic acid (e.g., FOLVITETM), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.
In certain embodiments, the Therapeutics of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the Therapeutics of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).
In other embodiments, the Therapeutics of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the Therapeutics of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenyloin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantadine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).
In another embodiment, Therapeutics of the invention are administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the Therapeutics of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enelaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., amyl nitrite, isosorbide dinitrate, isosorbide mononitrate, nitric oxide gas, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.
Other vasodilating agents that may be administered with the Therapeutics of the invention include, but are not limited to, epoprostenol and alprostadil.
In certain embodiments, the Therapeutics of the invention are administered in combination with treatments for cardiovascular disorders. Treatments for cardiovascular disorders that may be administered with the Therapeutic of the invention include, but are not limited to, angiotensin II blockers (e.g., irbesartan, losartan, and valsartan), alpha adrenergic blockers (e.g., doxazosin, prazosin, tamsulosin, and terazosin), hypotensive agents (e.g., clonidine, hydralazine, methyldopa, minoxidil, nitroprusside, and reserpine) and antilipemic agents (e.g., atorvastatin, cholestyramine, colestipol, fenofibrate, gemfibrate, lovstatin, pravastatin, and simvastatin).
In certain embodiments, the Therapeutics of the invention are administered in combination with treatments for gastrointestinal disorders. Treatments for gastrointestinal disorders that may be administered with the Therapeutic of the invention include, but are not limited to, H2 histamine receptor antagonists (e.g., TAGAMETTM (cimetidine), ZANTACTM (ranitidine), PEPCIDTM (famotidine), and AXIDTM (nizatidine)); inhibitors of H+, K+ ATPase (e.g., PREVACIDTM (lansoprazole) and PRILOSECTM (omeprazole)); Bismuth compounds (e.g., PEPTO-BISMOLTM (bismuth subsalicylate) and DE-NOLTM (bismuth subcitrate)); various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTECTM (misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g., surfactant laxatives, stimulant laxatives, saline and osmotic laxatives); antidiarrheal agents (e.g., LOMOTILTM (diphenoxylate), MOTOFENTM (diphenoxin), and IMODIUMTM (loperamide hydrochloride)), synthetic analogs of somatostatin such as SANDOSTATINTM (octreotide), antiemetic agents (e.g., ZOFRANTM (ondansetron), KYTRILTM (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, proclorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide and chlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholic acid; and pancreatic enzyme preparations such as pancreatin and pancrelipase.
In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
Example 14
Method of Treating Decreased Levels of the Polypeptide
The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of polypeptides (including agonists thereto), and/or antibodies of the invention. Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a polypeptide of the present invention in an individual may be treated by administering agonists of said polypeptide. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the agonist (including polypeptides and antibodies of the present invention) to increase the activity level of the polypeptide in such an individual.
For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 μg/kg of the agonist for six consecutive days. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 13.
Example 15
Method of Treating Increased Levels of the Polypeptide
The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).
In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, due to a variety of etiologies, such as cancer.
For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The antisense polynucleotides of the present invention can be formulated using techniques and formulations described herein (e.g. see Example 13), or otherwise known in the art.
Example 16
Method of Treatment Using Gene Therapy—Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.
The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
Example 17
Gene Therapy Using Endogenous Genes Corresponding to Polynucleotides of the Invention
Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.
The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel, then purified by phenol extraction and ethanol precipitation.
In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.
Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×106 cells/ml. Electroporation should be performed immediately following resuspension.
Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′ end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′ end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′ end and a HindIII site at the 3′ end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2—BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×106 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.
Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.
Example 18
Method of Treatment Using Gene Therapy—In Vivo
Another aspect of the present invention is using in vivo gene therapy methods to prevent, treat, and/or ameliorate diseases, disorders, and conditions (such as immune, cardiovascular, cancer, and other proliferative diseases, disorders, and conditions). The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to (i.e., associated with) a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622, U.S. Pat. No. 5,705,151, U.S. Pat. No. 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).
The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, LIPOFECTINTM or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of tissues within an animal, including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% AvertinTM. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 μm cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be used to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.
Example 19
Transgenic Animals
The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 20
Knock-Out Animals
Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (e.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.
In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form that, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 21
Assays Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation
Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.
One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.
In Vitro Assay—Agonists or antagonists of the invention can be assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the agonists or antagonists of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).
Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10−5M 2ME, 100 U/ml penicillin, 10 μg/ml streptomycin, and 10−5 dilution of SAC) in a total volume of 150 μl. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.
In vivo Assay—BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of agonists or antagonists of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with agonists or antagonists of the invention identify the results of the activity of the agonists or antagonists on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.
Flow cytometric analyses of the spleens from mice treated with agonist or antagonist are used to indicate whether the agonists or antagonists specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.
Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and agonists or antagonists-treated mice.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 22
T Cell Proliferation Assay
Proliferation Assay for Resting PBLs
A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4° C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of agonists or antagonists of the invention (total volume 200 μl). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37° C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatant is removed and stored at −20° C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 μl of medium containing 0.5 μCi of 3H-thymidine and cultured at 37° C. for 18-24 hr. Wells are harvested and incorporation of 3H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative control for the effects of agonists or antagonists of the invention.
Alternatively, a proliferation assay on resting PBL (peripheral blood lymphocytes) is measured by the up-take of 3H-thymidine. The assay is performed as follows. PBMC are isolated by FICOLLTM (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non-adherent cells are collected, washed and used in the proliferation assay. The assay is performed in a 96 well plate using 2×104 cells/well in a final volume of 200 μl. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 μl are added to 140 μl of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2 (*), IFNγ, TNFα, IL-10 and TR2. In addition to the control supernatants, recombinant human IL-2 (R & D Systems, Minneapolis, Minn.) at a final concentration of 100 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 μCi of 3H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of 3H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error. (*) The amount of the control cytokines IL-2, IFNγ, TNFα, and IL-10 produced in each transfection varies between 300 pg to 5 ng/ml.
Costimulation Assay.
A costimulation assay on resting PBL (peripheral blood lymphocytes) is performed in the presence of immobilized antibodies to CD3 and CD28. The use of antibodies specific for the invariant regions of CD3 mimic the induction of T cell activation that would occur through stimulation of the T cell receptor by an antigen. Cross-linking of the TCR (first signal) in the absence of a costimulatory signal (second signal) causes very low induction of proliferation and will eventually result in a state of “anergy”, which is characterized by the absence of growth and inability to produce cytokines. The addition of a costimulatory signal such as an antibody to CD28 mimics the action of the costimulatory molecule. B7-1 expressed on activated APCs, results in enhancement of T cell responses including cell survival and production of IL-2. Therefore this type of assay allows to detect both positive and negative effects caused by addition of supernatants expressing the proteins of interest on T cell proliferation.
The assay is performed as follows. Ninety-six well plates are coated with 100 ng/ml anti-CD3 and 5 μg/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 100 ul and incubated overnight at 4° C. Plates are washed twice with PBS before use. PBMC are isolated by FICOLLTM (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% FCS (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non adherent cells are collected, washed and used in the proliferation assay. The assay is performed in a 96 well plate using 2×104 cells/well in a final volume of 200 μl. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 ul are added to 140 μl of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector only (negative control), IL-2, IFNγ, TNFα, IL-10 and TR2. In addition to the control supernatants recombinant human IL-2 (R & D Systems, Minneapolis, Minn.) at a final concentration of 10 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 μCi of 3H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of 3H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error.
Costimulation Assay: IFN γ and IL-2 ELISA.
The assay is performed as follows. Twenty-four well plates are coated with either 300 ng/ml or 600 ng/ml anti-CD3 and 5 μg/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 500 ul and incubated overnight at 4° C. Plates are washed twice with PBS before use. PBMC are isolated by FICOLLTM (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% FCS (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non adherent cells are collected, washed and used in the costimulation assay. The assay is performed in the pre-coated twenty-four well plate using 1×105 cells/well in a final volume of 900 μl. The supernatants (293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 300 μl are added to 600 μl of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector only (negative control), IL-2, IFNγ, IL-12 and IL-18. In addition to the control supernatants recombinant human IL-2 (all cytokines were purchased from R & D Systems, Minneapolis, Minn.) at a final concentration of 10 ng/ml, IL-12 at a final concentration of 1 ng/ml and IL-18 at a final concentration of 50 ng/ml are also used. Controls and unknown samples are tested in duplicate. Supernatant samples (250 μl) are collected 2 days and 5 days after the beginning of the assay. ELISAs to test for IFNγ and IL-2 secretion are performed using kits purchased from R & D Systems, (Minneapolis, Minn.). Results are expressed as an average of duplicate samples plus or minus standard error.
Proliferation Assay for Preactivated-Resting T Cells.
A proliferation assay on preactivated-resting T cells is performed on cells that are previously activated with the lectin phytohemagglutinin (PHA). Lectins are polymeric plant proteins that can bind to residues on T cell surface glycoproteins including the TCR and act as polyclonal activators. PBLs treated with PHA and then cultured in the presence of low doses of IL-2 resemble effector T cells. These cells are generally more sensitive to further activation induced by growth factors such as IL-2. This is due to the expression of high affinity IL-2 receptors that allows this population to respond to amounts of IL-2 that are 100 fold lower than what would have an effect on a naïve T cell. Therefore the use of this type of cells might enable to detect the effect of very low doses of an unknown growth factor, that would not be sufficient to induce proliferation on resting (naïve) T cells.
The assay is performed as follows. PBMC are isolated by F/H gradient centrifugation from human peripheral blood, and are cultured in 10% FCS (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.) in the presence of 2 ug/ml PHA (SIGMATM, Saint Louis, Mo.) for three days. The cells are then washed in PBS and cultured in 10% FCS/RPMI in the presence of 5 ng/ml of human recombinant IL-2 (R & D Systems, Minneapolis, Minn.) for 3 days. The cells are washed and rested in starvation medium (1% FCS/RPMI) for 16 hours prior to the beginning of the proliferation assay. An aliquot of the cells is analyzed by FACS to determine the percentage of T cells (CD3 positive cells) present; this usually ranges between 93-97% depending on the donor. The assay is performed in a 96 well plate using 2×104 cells/well in a final volume of 200 μl. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 μl are added to 140 μl of in 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2, IFNγ, TNFα, IL-10 and TR2. In addition to the control supernatants recombinant human IL-2 at a final concentration of 10 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 μCi of 3H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of 3H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 23
Effect of Agonists or Antagonists of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells
Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.
FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of agonist or antagonist of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Th1 helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (106/ml) are treated with increasing concentrations of agonists or antagonists of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.
Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increased expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.
FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of agonists or antagonists of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Agonists or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a HISTOPAQUETM gradient (SIGMATM). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.
Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated processes (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×106/ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.
Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×105 cells/ml with increasing concentrations of agonists or antagonists of the invention and under the same conditions, but in the absence of agonists or antagonists. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in the presence of agonist or antagonist of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.
Oxidative burst. Purified monocytes are plated in 96-well plate at 2-1×105 cell/well. Increasing concentrations of agonists or antagonists of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37° C. for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H2O2 produced by the macrophages, a standard curve of a H2O2 solution of known molarity is performed for each experiment.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 24
Biological Effects of Agonists or Antagonists of the Invention Astrocyte and Neuronal Assays
Agonists or antagonists of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate an agonist or antagonist of the invention's activity on these cells.
Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of an agonist or antagonist of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.
Fibroblast and Endothelial Cell Assays.
Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. ALAMAR BLUETM (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CYTOFLUORTM fluorescence reader. For the PGE2 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or agonists or antagonists of the invention with or without IL-1α for 24 hours. The supernatants are collected and assayed for PGE2 by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without agonists or antagonists of the invention IL-1α for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).
Human lung fibroblasts are cultured with FGF-2 or agonists or antagonists of the invention for 3 days in basal medium before the addition of ALAMAR BLUETM to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with agonists or antagonists of the invention.
Parkinson Models.
The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released. Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.
It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).
Based on the data with FGF-2, agonists or antagonists of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of an agonist or antagonist of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.
Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if an agonist or antagonist of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the agonist or antagonist may be involved in Parkinson's Disease.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 25
The Effect of Agonists or Antagonists of the Invention on the Growth of Vascular Endothelial Cells
On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. An agonist or antagonist of the invention, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.
An increase in the number of HUVEC cells indicates that the compound of the invention may proliferate vascular endothelial cells, while a decrease in the number of HUVEC cells indicates that the compound of the invention inhibits vascular endothelial cells.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 26
Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells
For evaluation of mitogenic activity of growth factors, the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, PROMEGATM). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 27
Inhibition of PDGF-induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect
HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number. In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271(36):21985-21992 (1996).
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 28
Stimulation of Endothelial Migration
This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.
Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, Md.; Falk, W., et al., J. Immunological Methods 1980; 33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 μM (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5×105 cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37° C. in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified by counting cells of three random high-power fields (40×) in each well, and all groups are performed in quadruplicate.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 29
Stimulation of Nitric Oxide Production by Endothelial Cells
Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.
Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC.
Briefly, NO release from cultured HUVEC monolayer is measured with a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation:

2KNO2+2KI+2H2SO462NO+I2+2H2O+2K2SO4 

The standard calibration curve is obtained by adding graded concentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing K1 and H2SO4. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37° C. The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per 1×106 endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).
The studies described in this example tested activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 30
Effect of Polypeptides of the Invention on Cord Formation in Angiogenesis
Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.
CADMEC (microvascular endothelial cells) are purchased from Cell Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell Applications' CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor Medium (200 ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth Medium is then replaced with 300 mg Cell Applications' Chord Formation Medium containing control buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr. The numbers and lengths of the capillary-like chords are quantitated through use of the Boeckeler VIA-170 video image analyzer. All assays are done in triplicate.
Commercial (R&D) VEGF (50 ng/ml) is used as a positive control. b-estradiol (1 ng/ml) is used as a negative control. The appropriate buffer (without protein) is also utilized as a control.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 31
Angiogenic Effect on Chick Chorioallantoic Membrane
Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined.
Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese quail (Coturnix coturnix) are incubated at 37.8° C. and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old quail embryos is studied with the following methods.
On Day 4 of development, a window is made into the egg shell of chick eggs. The embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13. THERMANOXTM coverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and about 3.3 mg/5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 32
Angiogenesis Assay Using a MATRIGELTM Implant in Mouse
In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (MATRIGELTM). The protein is mixed with the liquid MATRIGELTM at 4 degree C. and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid “plug” of MATRIGELTM is removed and examined for the presence of new blood vessels. MATRIGELTM is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.
When thawed at 4 degree C. the MATRIGELTM material is a liquid. The MATRIGELTM is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with the mixture of MATRIGELTM and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the MATRIGELTM plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 ng/ml). MATRIGELTM alone is used to determine basal levels of angiogenesis.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 33
Rescue of Ischemia in Rabbit Lower Limb Model
To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model is created by surgical removal of one femoral artery as described previously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshita et al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter. On day 30, various parameters are measured in these rabbits: (a) BP ratio—The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL:resting FL; (c) Angiographic Score—This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density—The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.
The studies described in this example tested activity of polynucleotides and polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the agonists, and/or antagonists of the invention.
Example 34
Effect of Polypeptides of the Invention on Vasodilation
Since dilation of vascular endothelium is important in reducing blood pressure, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as the mean+/−SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 35
Rat Ischemic Skin Flap Model
The evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization.
The study in this model is divided into three parts as follows:
Ischemic skin
Ischemic skin wounds
Normal wounds
The experimental protocol includes:

    
    
        Raising a 3×4 cm, single pedicle full-thickness random skin flap (myocutaneous flap over the lower back of the animal).
        An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap).
        Topical treatment with a polypeptide of the invention of the excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: 1 mg to 100 mg.
        Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding for histological, immunohistochemical, and in situ studies.
    
    


The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 36
Peripheral Arterial Disease Model
Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes:
One side of the femoral artery is ligated to create ischemic muscle of the hindlimb, the other side of hindlimb serves as a control.
A polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.
The ischemic muscle tissue is collected after ligation of the femoral artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology. Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 37
Ischemic Myocardial Disease Model
A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes:
The heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed.
A polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4 weeks.
Thirty days after the surgery, the heart is removed and cross-sectioned for morphometric and in situ analyzes.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 38
Rat Corneal Wound Healing Model
This animal model shows the effect of an agonist or antagonist of the invention on neovascularization. The experimental protocol includes:
a) Making a 1-1.5 mm long incision from the center of cornea into the stromal layer.
b) Inserting a spatula below the lip of the incision facing the outer corner of the eye.
c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).
d) Positioning a pellet, containing 50 ng-5 μg of an agonist or antagonist of the invention, within the pocket.
e) Treatment with an agonist or antagonist of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 39
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
Diabetic db+/db+ Mouse Model.
To demonstrate that an agonist or antagonist of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).
The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).
The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).
Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of AvertinTM (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
An agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with an agonist or antagonist of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.
Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.
Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue is used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
Steroid Impaired Rat Model
The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahl et al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impair wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
To demonstrate that an agonist or antagonist of the invention can accelerate the healing process, the effects of multiple topical applications of the agonist or antagonist on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.
Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
The agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with an agonist or antagonist of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 40
Lymphadema Animal Model
The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of an agonist or antagonist of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.
Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately  ̃350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.
Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated or suture ligated.
Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.
Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of  ̃0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.
To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect of plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.
Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people and those 2 readings are averaged. Readings are taken from both control and edematous limbs.
Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), and both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into the instrument to each marked level then measured by Buxco edema software (Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.
Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2+ comparison.
Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.
Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at −80 EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 41
Suppression of TNF Alpha-Induced Adhesion Molecule Expression by an Agonist or Antagonist of the Invention
The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.
Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.
The potential of an agonist or antagonist of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.
To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO2. HUVECs are seeded in 96-well plates at concentrations of 1×104 cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.
Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 μl of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.
Fixative is then removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. The wells should not be allowed to dry. 10 μl of diluted primary antibody is then added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.
Then add 20 μl of diluted Extravidin®-Alkaline Phosphotase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the Extravidin®-Alkaline Phosphotase in glycine buffer: 1:5,000 (100)>10−0.5>10−1>10−1.5. 5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.
The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 42
Production of Polypeptide of the Invention for High-Throughput Screening Assays
The following protocol produces a supernatant containing polypeptide of the present invention to be tested. This supernatant can then be used in the Screening Assays described in Examples 44-53.
First, dilute Poly-D-Lysine (644 587 BOEHRINGERTM Mannheim) stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml. Add 200 μl of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.
Plate 293T cells (do not carry cells past P+20) at 2×105 cells/well in 0.5 ml DMEM (Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS (14-503F Biowhittaker)/1× Penstrep (17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 μl LIPOFECTAMINETM (18324-012 Gibco/BRL) and 5 ml OPTI-MEMTM I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2 μg of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8-10, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50 μl of the LIPOFECTAMINETM/OPTI-MEMTM I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multi-channel pipetter to add 150 μl OPTI-MEMTM I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.
Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, and person B, using a12-channel pipetter with tips on every other channel, adds the 200 μl of DNA/LIPOFECTAMINETM/OPTI-MEMTM I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degree C. for 6 hours.
While cells are incubating, prepare appropriate media, either 1% BSA in DMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO4-5H2O; 0.050 mg/L of Fe(NO3)3-9H2O; 0.417 mg/L of FeSO4-7H2O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl2; 48.84 mg/L of MgSO4; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO3; 62.50 mg/L of NaH2PO4—H20; 71.02 mg/L of Na2HPO4; 0.4320 mg/L of ZnSO4-7H2O; 0.002 mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H2O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H20; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H20; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalanine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryosine-2Na-2H20; and 99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin B12; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 μM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamine and 1× penstrep. (BSA (81-068-3 BAYERTM) 100 gm dissolved in 1 L DMEM for a 10% BSA stock solution). Filter the media and collect 50 μl for endotoxin assay in 15 ml polystyrene conical.
The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5 ml appropriate media to each well. Incubate at 37 degree C. for 45 or 72 hours depending on the media used: 1% BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300 μl multichannel pipetter, aliquot 600 μl in one 1 ml deep well plate and the remaining supernatant into a 2 ml deep well. The supernatants from each well can then be used in the assays described in Examples 44-51.
It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide of the present invention directly (e.g., as a secreted protein) or by polypeptide of the present invention inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.
Example 43
Construction of GAS Reporter Construct
One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.
GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.
The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995)). A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO:237)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (See Table below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.














JAKs














Ligand
tyk2
Jak1
Jak2
Jak3
STATS
GAS(elements) or ISRE





IFN family








IFN-a/B
+
+
−
−
1, 2, 3
ISRE


IFN-g

+
+
−
1
GAS (IRF1 > Lys6 > IFP)


Il-10
+
?
?
−
1, 3


gp130 family


IL-6 (Pleiotropic)
+
+
+
?
1, 3
GAS (IRF1 > Lys6 > IFP)


Il-11 (Pleiotropic)
?
+
?
?
1, 3


OnM (Pleiotropic)
?
+
+
?
1, 3


LIF (Pleiotropic)
?
+
+
?
1, 3


CNTF (Pleiotropic)
−/+
+
+
?
1, 3


G-CSF (Pleiotropic)
?
+
?
?
1, 3


IL-12 (Pleiotropic)
+
−
+
+
1, 3


g-C family


IL-2 (lymphocytes)
−
+
−
+
1, 3, 5
GAS


IL-4 (lymph/myeloid)
−
+
−
+
6
GAS (IRF1 = IFP >> Ly6)(IgH)


IL-7 (lymphocytes)
−
+
−
+
5
GAS


IL-9 (lymphocytes)
−
+
−
+
5
GAS


IL-13 (lymphocyte)
−
+
?
?
6
GAS


IL-15
?
+
?
+
5
GAS


gp140 family


IL-3 (myeloid)
−
−
+
−
5
GAS (IRF1 > IFP >> Ly6)


IL-5 (myeloid)
−
−
+
−
5
GAS


GM-CSF (myeloid)
−
−
+
−
5
GAS


Growth hormone family


GH
?
−
+
−
5


PRL
?
+/−
+
−
1, 3, 5


EPO
?
−
+
−
5
GAS(B-CAS > IRF1 = IFP >> Ly6)


Receptor Tyrosine Kinases


EGF
?
+
+
−
1, 3
GAS (IRF1)


PDGF
?
+
+
−
1, 3


CSF-1
?
+
+
−
1, 3
GAS (not IRF1)









To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 44-45, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18 bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is:







(SEQ ID NO: 238)


5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTC


 


CCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′






The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:239)
PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from CLONTECHTM. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (STRATAGENETM.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:







(SEQ ID NO: 240)


5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCG


 


AAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGT


 


CCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCC


 


ATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG


 


GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGG


 


AGGCCTAGGCTTTTGCAAAAAGCTT:3′






With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.
The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from CLONTECHTM using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (CLONTECHTM), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 44-45.
Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing EGR and NF-KB promoter sequences are described in Examples 46 and 47. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 44
High-Throughput Screening Assay for T-Cell Activity
The following protocol is used to assess T-cell activity by identifying factors, and determining whether supernatant containing a polypeptide of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 43. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCCTM Accession No. TIB-152), although Molt-3 cells (ATCCTM Accession No. CRL-1552) and Molt-4 cells (ATCCTM Accession No. CRL-1582) cells can also be used.
Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (LIFE TECHNOLOGIESTM) (transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml geneticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 μl of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEMTM (LIFE TECHNOLOGIESTM) with 10 μg of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEMTM containing 50 μl of DMRIE-C and incubate at room temperature for 15-45 mins.
During the incubation period, count cell concentration, spin down the required number of cells (107 per transfection), and resuspend in OPTI-MEMTM to a final concentration of 107 cells/ml. Then add 1 ml of 1×107 cells in OPTI-MEMTM to T25 flask and incubate at 37 degree C. for 6 hrs. After the incubation, add 10 ml of RPMI+15% serum.
The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Geneticin, and 1% Pen-Strep. These cells are treated with supernatants containing polypeptide of the present invention or polypeptide of the present invention induced polypeptides as produced by the protocol described in Example 42.
On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI+10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.
Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 μl of cells into each well (therefore adding 100,000 cells per well).
After all the plates have been seeded, 50 μl of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10, and H11 to serve as additional positive controls for the assay.
The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 μl samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at −20 degree C. until SEAP assays are performed according to Example 48. The plates containing the remaining treated cells are placed at 4 degree C. and serve as a source of material for repeating the assay on a specific well if desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.
The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.
Example 45
High-Throughput Screening Assay Identifying Myeloid Activity
The following protocol is used to assess myeloid activity of polypeptide of the present invention by determining whether polypeptide of the present invention proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 43. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 43, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×107 U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.
Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 μg GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 μM Na2HPO4.7H2O, 1 mM MgCl2, and 675 μM CaCl2. Incubate at 37 degrees C. for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degree C. for 36 hr.
The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 μg/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 μg/ml G418 for couple of passages.
These cells are tested by harvesting 1×108 cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5×105 cells/ml. Plate 200 μl cells per well in the 96-well plate (or 1×105 cells/well).
Add 50 μl of the supernatant prepared by the protocol described in Example 42. Incubate at 37 degree C. for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example 48.
Example 46
High-Throughput Screening Assay Identifying Neuronal Activity
When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, activation of cells can be assessed by polypeptide of the present invention.
Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat pheochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells by polypeptide of the present invention can be assessed.
The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (−633 to +1) (Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:








5′ GCGCTCGAGGGATGACAGCGATAGAACCCCG
(SEQ ID NO: 241)


G-3′



 


5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCT
(SEQ ID NO: 242)


C-3′







Using the GAS:SEAP/Neo vector produced in Example 43, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.
To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.
PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.
Transfect the EGR/SEAP/Neo construct into PC12 using the LIPOFECTAMINETM protocol described in Example 42. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 μg/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 μg/ml G418 for couple of passages.
To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5×105 cells/ml.
Add 200 μl of the cell suspension to each well of 96-well plate (equivalent to 1×105 cells/well). Add 50 μl supernatant produced by Example 42, 37 degree C. for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/μl of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 48.
Example 47
High-Throughput Screening Assay for T-Cell Activity
NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF-KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.
In non-stimulated conditions, NF-KB is retained in the cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.
Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 42. Activators or inhibitors of NF-KB would be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.
To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:243), 18 by of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site:







(SEQ ID NO: 244)


5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGG


 


GACTTTCCATCCTGCCATCTCAATTAG:3′






The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a Hind III site:







(SEQ ID NO: 239)


5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′






PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from CLONTECHTM. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2-. (STRATAGENETM) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence:







(SEQ ID NO: 245)


5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTT


 


TCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCC


 


GCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATG


 


GCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCT


 


GAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTG


 


CAAAAAGCTT:3′






Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (CLONTECHTM) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (CLONTECHTM), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.
Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 44. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 44. As a positive control, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.
Example 48
Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 44-47, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 μl of 2.5× dilution buffer into Optiplates containing 35 μl of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C. for 30 min. Separate the Optiplates to avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the Table below). Add 50 μl Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on a luminometer, thus one should treat 5 plates at each time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation:












# of plates
Rxn buffer diluent (ml)
CSPD (ml)

















10
60
3


11
65
3.25


12
70
3.5


13
75
3.75


14
80
4


15
85
4.25


16
90
4.5


17
95
4.75


18
100
5


19
105
5.25


20
110
5.5


21
115
5.75


22
120
6


23
125
6.25


24
130
6.5


25
135
6.75


26
140
7


27
145
7.25


28
150
7.5


29
155
7.75


30
160
8


31
165
8.25


32
170
8.5


33
175
8.75


34
180
9


35
185
9.25


36
190
9.5


37
195
9.75


38
200
10


39
205
10.25


40
210
10.5


41
215
10.75


42
220
11


43
225
11.25


44
230
11.5


45
235
11.75


46
240
12


47
245
12.25


48
250
12.5


49
255
12.75


50
260
13









Example 49
High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.
The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.
For adherent cells, seed the cells at 10,000-20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO2 incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 μl of HBSS (Hank's Balanced Salt Solution) leaving 100 μl of buffer after the final wash.
A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 μl of 12 μg/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C. in a CO2 incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 μl of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5×106 cells/ml with HBSS in a 50-ml conical tube. 4 μl of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×106 cells/ml, and dispensed into a microplate, 100 μl/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley Cell Wash with 200 μl, followed by an aspiration step to 100 μl final volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The supernatant is added to the well, and a change in fluorescence is detected.
To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 μl. Increased emission at 530 nm indicates an extracellular signaling event caused by the a molecule, either polypeptide of the present invention or a molecule induced by polypeptide of the present invention, which has resulted in an increase in the intracellular Ca++ concentration.
Example 50
High-Throughput Screening Assay Identifying Tyrosine Kinase Activity
The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
Because of the wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether polypeptide of the present invention or a molecule induced by polypeptide of the present invention is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.
Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well LOPRODYNETM Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from SIGMATM Chemicals (St. Louis, Mo.) or 10% MATRIGELTM purchased from Becton Dickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of ALAMAR BLUETM as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford, Mass.) are used to cover the LOPRODYNETM Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.
To prepare extracts, A431 cells are seeded onto the nylon membranes of LOPRODYNETM plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 μl of the supernatant produced in Example 42, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 αmM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease inhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.)) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4° C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C. at 16,000×g.
Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.
Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from BOEHRINGERTM Mannheim.
The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 μl of 5 uM Biotinylated Peptide, then 10 μl ATP/Mg2+ (5 mM ATP/50 mM MgCl2), then 10 μl of 5× Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl2, 5 mM MnCl2 0.5 mg/ml BSA), then 5 μl of Sodium Vanadate (1 mM), and then 5 μl of water. Mix the components gently and preincubate the reaction mix at 30 degree C. for 2 min. Initial the reaction by adding 10 μl of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 μl of 120 mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 μl aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C. for 20 min. This allows the streptavidin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 μl of anti-phosphotyrosine antibody conjugated to horse radish peroxidase (anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37 degree C. for one hour. Wash the well as above.
Next add 100 μl of peroxidase substrate solution (BOEHRINGERTM Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.
Example 51
High-Throughput Screening Assay Identifying Phosphorylation Activity
As a potential alternative and/or complement to the assay of protein tyrosine kinase activity described in Example 50, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.
Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degree C. until use.
A431 cells are seeded at 20,000/well in a 96-well LOPRODYNETM filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 μl of the supernatants obtained in Example 42 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation by polypeptide of the present invention or a molecule induced by polypeptide of the present invention.
Example 52
Assay for the Stimulation of Bone Marrow CD34+ Cell Proliferation
This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of isolated polypeptides expressed in mammalian cells to stimulate proliferation of CD34+ cells.
It has been previously shown that most mature precursors will respond to only a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of polypeptides on hematopoietic activity of a wide range of progenitor cells, the assay contains a given polypeptide in the presence or absence of other hematopoietic growth factors. Isolated cells are cultured for 5 days in the presence of Stem Cell Factor (SCF) in combination with tested sample. SCF alone has a very limited effect on the proliferation of bone marrow (BM) cells, acting in such conditions only as a “survival” factor. However, combined with any factor exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore, if the tested polypeptide has a stimulatory effect on hematopoietic progenitors, such activity can be easily detected. Since normal BM cells have a low level of cycling cells, it is likely that any inhibitory effect of a given polypeptide, or agonists or antagonists thereof, might not be detected. Accordingly, assays for an inhibitory effect on progenitors is preferably tested in cells that are first subjected to in vitro stimulation with SCF+IL+3, and then contacted with the compound that is being evaluated for inhibition of such induced proliferation.
Briefly, CD34+ cells are isolated using methods known in the art. The cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md. Cat#160-204-101). After several gentle centrifugation steps at 200×g, cells are allowed to rest for one hour. The cell count is adjusted to 2.5×105 cells/ml. During this time, 100 μl of sterile water is added to the peripheral wells of a 96-well plate. The cytokines that can be tested with a given polypeptide in this assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat#255-SC) at 50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat#203-ML) at 30 ng/ml. After one hour, 10 μl of prepared cytokines, 50 μl of the supernatants prepared in Example 42 (supernatants at 1:2 dilution=50 μl) and 20 μl of diluted cells are added to the media which is already present in the wells to allow for a final total volume of 100 μl. The plates are then placed in a 37° C./5% CO2 incubator for five days.
Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H] Thymidine is added in a 10 μl volume to each well to determine the proliferation rate. The experiment is terminated by harvesting the cells from each 96-well plate to a filtermat using the Tomtec Harvester 96. After harvesting, the filtermats are dried, trimmed and placed into OMNIFILTERTM assemblies consisting of one OMNIFILTERTM plate and one OMNIFILTERTM Tray. 60 μl MICROSCINTTM is added to each well and the plate sealed with TopSeal-A press-on sealing film. A bar code 15 sticker is affixed to the first plate for counting. The sealed plates are then loaded and the level of radioactivity determined via the Packard Top Count and the printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.
The studies described in this example test the activity of a given polypeptide to stimulate bone marrow CD34+ cell proliferation. One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof. As a nonlimiting example, potential antagonists tested in this assay would be expected to inhibit cell proliferation in the presence of cytokines and/or to increase the inhibition of cell proliferation in the presence of cytokines and a given polypeptide. In contrast, potential agonists tested in this assay would be expected to enhance cell proliferation and/or to decrease the inhibition of cell proliferation in the presence of cytokines and a given polypeptide.
The ability of a gene to stimulate the proliferation of bone marrow CD34+ cells indicates that polynucleotides and polypeptides corresponding to the gene are useful for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein.
Example 53
Assay for Extracellular Matrix Enhanced Cell Response (EMECR)
The objective of the Extracellular Matrix Enhanced Cell Response (EMECR) assay is to identify gene products (e.g., isolated polypeptides) that act on the hematopoietic stem cells in the context of the extracellular matrix (ECM) induced signal.
Cells respond to the regulatory factors in the context of signal(s) received from the surrounding microenvironment. For example, fibroblasts, and endothelial and epithelial stem cells fail to replicate in the absence of signals from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the ECM protein fibronectin (fn). Adhesion of cells to fn is mediated by the α5.β1 and α4.β1 integrin receptors, which are expressed by human and mouse hematopoietic stem cells. The factor(s) which integrate with the ECM environment and are responsible for stimulating stem cell self-renewal have not yet been identified. Discovery of such factors should be of great interest in gene therapy and bone marrow transplant applications
Briefly, polystyrene, non tissue culture treated, 96-well plates are coated with fn fragment at a coating concentration of 0.2 μg/cm2. Mouse bone marrow cells are plated (1,000 cells/well) in 0.2 ml of serum-free medium. Cells cultured in the presence of IL-3 (5 ng/ml)+SCF (50 ng/ml) would serve as the positive control, conditions under which little self-renewal but pronounced differentiation of the stem cells is to be expected. Gene products of the invention (e.g., including, but not limited to, polynucleotides and polypeptides of the present invention, and supernatants produced in Example 42), are tested with appropriate negative controls in the presence and absence of SCF (5.0 ng/ml), where test factor supernatants represent 10% of the total assay volume. The plated cells are then allowed to grow by incubating in a low oxygen environment (5% CO2, 7% O2, and 88% N2) tissue culture incubator for 7 days. The number of proliferating cells within the wells is then quantitated by measuring thymidine incorporation into cellular DNA. Verification of the positive hits in the assay will require phenotypic characterization of the cells, which can be accomplished by scaling up of the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.
If a particular polypeptide of the present invention is found to be a stimulator of hematopoietic progenitors, polynucleotides and polypeptides corresponding to the gene encoding said polypeptide may be useful for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein. The gene product may also be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
Additionally, the polynucleotides and/or polypeptides of the gene of interest and/or agonists and/or antagonists thereof, may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.
Moreover, polynucleotides and polypeptides corresponding to the gene of interest may also be useful for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of hematopoietic related disorders such as, for example, anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
Example 54
Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation
The polypeptide of interest is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the polypeptide of interest on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflammatory or immunomodulatory outcome. Assays are run with and without co-TNFa stimulation, in order to check for costimulatory or inhibitory activity.
Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media. NHDF culture media contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5% FBS. After incubation at 37° C. for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 0.4% FBS. Incubate at 37° C. until day 2.
On day 2, serial dilutions and templates of the polypeptide of interest are designed such that they always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Add 1⁄3 vol media containing controls or polypeptides of the present invention and incubate at 37° C./5% CO2 until day 5.
Transfer 60 μl from each well to another labeled 96-well plate, cover with a plate-sealer, and store at 4° C. until Day 6 (for IL6 ELISA). To the remaining 100 μl in the cell culture plate, aseptically add ALAMAR BLUETM in an amount equal to 10% of the culture volume (10 μl). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530 nm and emission at 590 nm using the CYTOFLUORTM. This yields the growth stimulation/inhibition data.
On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 μl/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.
On day 6, empty the plates into the sink and blot on paper towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 μl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then add 50 μl/well of diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.
Plates are washed with wash buffer and blotted on paper towels. Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 μl/well. Cover the plate and incubate 1 h at RT. Plates are again washed with wash buffer and blotted on paper towels.
Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.
A positive result in this assay suggests AoSMC cell proliferation and that the polypeptide of the present invention may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of polypeptides, polynucleotides, agonists and/or antagonists of the polynucleotide/polypeptide of the present invention which gives a positive result. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, polypeptides of the present invention and polynucleotides of the present invention may be used in wound healing and dermal regeneration, as well as the promotion of vasculogenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, antagonists of polypeptides and polynucleotides of the invention may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular agent (e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, antagonists of polypeptides and polynucleotides of the invention may be useful in treating anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.
Example 55
Cellular Adhesion Molecule (CAM) Expression on Endothelial Cells
The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.
Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are then incubated at 37° C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min. Fixative is removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed three times with PBS (+Ca,Mg)+0.5% BSA. 20 μl of diluted Extravidin®-Alkaline Phosphatase (1:5,000 dilution, referred to herein as the working dilution) are added to each well and incubated at 37° C. for 30 min. Wells are washed three times with PBS (+Ca, Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin®-Alkaline Phosphotase in glycine buffer: 1:5,000 (100)>10−0.5>10−1>10−1.5. 5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.
Example 56
ALAMAR BLUETM Endothelial Cells Proliferation Assay
This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard ALAMAR BLUETM Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration. Dilutions of the protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.
Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37° C. overnight. After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of the protein of interest or control protein sample(s) (prepared in SFM) in triplicate wells with additional bFGF to a concentration of 10 ng/ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37° C. incubator for three days. After three days 10 ml of stock ALAMAR BLUETM (Biosource Cat# DAL1100) is added to each well and the plate(s) is/are placed back in the 37° C. incubator for four hours. The plate(s) are then read at 530 nm excitation and 590 nm emission using the CYTOFLUORTM fluorescence reader. Direct output is recorded in relative fluorescence units.
ALAMAR BLUETM is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) form to reduced (fluorescent red) form (i.e., stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity). The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (bFGF in growth medium) and protein dilutions.
Example 57
Detection of Inhibition of a Mixed Lymphocyte Reaction
This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated polypeptides). Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. Multiple cells may be targeted by these polypeptides since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.
Polypeptides of interest found to inhibit the MLR may find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.
Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from two donors are adjusted to 2×106 cells/ml in RPMI-1640 (LIFE TECHNOLOGIESTM, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor are adjusted to 2×105 cells/ml. Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of test materials (50 μl) are added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number 202-IL) is added to a final concentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg/ml. Cells are cultured for 7-8 days at 37° C. in 5% CO2, and 1 μC of [3H] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount. Data is expressed as the mean and standard deviation of triplicate determinations.
Samples of the protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, IL-2 (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.
Example 58
Assays for Protease Activity
The following assay may be used to assess protease activity of the polypeptides of the invention.
Gelatin and casein zymography are performed essentially as described (Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson et al., Journal of Urology, 149:653-658 (1993)). Samples are run on 10% polyacrylamide/0.1% SDS gels containing 1% gelatin orcasein, soaked in 2.5% triton at room temperature for 1 hour, and in 0.1M glycine, pH 8.3 at 37° C. 5 to 16 hours. After staining in amido black areas of proteolysis appear as clear areas against the blue-black background. Trypsin (SIGMATM T8642) is used as a positive control.
Protease activity is also determined by monitoring the cleavage of n-a-benzoyl-L-arginine ethyl ester (BAEE) (SIGMATM B-4500). Reactions are set up in (25 mMNaPO4, 1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples are added and the change in absorbance at 260 nm is monitored on the Beckman DU-6 spectrophotometer in the time-drive mode. Trypsin is used as a positive control.
Additional assays based upon the release of acid-soluble peptides from casein or hemoglobin measured as absorbance at 280 nm or colorimetrically using the Folin method are performed as described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assays involve the solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).
Example 59
Identifying Serine Protease Substrate Specificity
Methods known in the art or described herein may be used to determine the substrate specificity of the polypeptides of the present invention having serine protease activity. A preferred method of determining substrate specificity is by the use of positional scanning synthetic combinatorial libraries as described in GB 2 324 529 (incorporated herein in its entirety).
Example 60
Ligand Binding Assays
The following assay may be used to assess ligand binding activity of the polypeptides of the invention.
Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The purified ligand for a polypeptide is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its polypeptide. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, specific polypeptide binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
Example 61
Functional Assay in Xenopus Oocytes
Capped RNA transcripts from linearized plasmid templates encoding the polypeptides of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response polypeptides and polypeptide agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.
Example 62
Microphysiometric Assays
Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process. The pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of detecting the activation of polypeptide that is coupled to an energy utilizing intracellular signaling pathway.
Example 63
Extract/Cell Supernatant Screening
A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligands banks as identified to date. Accordingly, the polypeptides of the invention can also be functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify its natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
Example 64
Calcium and cAMP Functional Assays
Seven transmembrane receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition. Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day >150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.
Example 65
ATP-Binding Assay
The following assay may be used to assess ATP-binding activity of polypeptides of the invention.
ATP-binding activity of the polypeptides of the invention may be detected using the ATP-binding assay described in U.S. Pat. No. 5,858,719, which is herein incorporated by reference in its entirety. Briefly, ATP-binding to polypeptides of the invention is measured via photoaffinity labeling with 8-azido-ATP in a competition assay. Reaction mixtures containing 1 mg/ml of the ABC transport protein of the present invention are incubated with varying concentrations of ATP, or the non-hydrolyzable ATP analog adenyl-5′-imidodiphosphate for 10 minutes at 4° C. A mixture of 8-azido-ATP (SIGMATM Chem. Corp., St. Louis, Mo.) plus 8-azido-ATP (32P-ATP) (5 mCi/μmol, ICN, Irvine Calif.) is added to a final concentration of 100 μM and 0.5 ml aliquots are placed in the wells of a porcelain spot plate on ice. The plate is irradiated using a short wave 254 nm UV lamp at a distance of 2.5 cm from the plate for two one-minute intervals with a one-minute cooling interval in between. The reaction is stopped by addition of dithiothreitol to a final concentration of 2 mM. The incubations are subjected to SDS-PAGE electrophoresis, dried, and autoradiographed. Protein bands corresponding to the particular polypeptides of the invention are excised, and the radioactivity quantified. A decrease in radioactivity with increasing ATP or adenyl-5′-imidodiphosphate provides a measure of ATP affinity to the polypeptides.
Example 66
Small Molecule Screening
This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and polypeptide of the invention.
Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the invention. These methods comprise contacting such an agent with a polypeptide of the invention or fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the invention.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is herein incorporated by reference in its entirety. Briefly stated, large numbers of different small molecule test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with polypeptides of the invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide that shares one or more antigenic epitopes with a polypeptide of the invention.
Example 67
Phosphorylation Assay
In order to assay for phosphorylation activity of the polypeptides of the invention, a phosphorylation assay as described in U.S. Pat. No. 5,958,405 (which is herein incorporated by reference) is utilized. Briefly, phosphorylation activity may be measured by phosphorylation of a protein substrate using gamma-labeled 32P-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. The polypeptides of the invention are incubated with the protein substrate, 32P-ATP, and a kinase buffer. The 32P incorporated into the substrate is then separated from free 32P-ATP by electrophoresis, and the incorporated 32P is counted and compared to a negative control. Radioactivity counts above the negative control are indicative of phosphorylation activity of the polypeptides of the invention.
Example 68
Detection of Phosphorylation Activity (Activation) of the Polypeptides of the Invention in the Presence of Polypeptide Ligands
Methods known in the art or described herein may be used to determine the phosphorylation activity of the polypeptides of the invention. A preferred method of determining phosphorylation activity is by the use of the tyrosine phosphorylation assay as described in U.S. Pat. No. 5,817,471 (incorporated herein by reference).
Example 69
Identification of Signal Transduction Proteins that Interact with Polypeptides of the Present Invention
The purified polypeptides of the invention are research tools for the identification, characterization and purification of additional signal transduction pathway proteins or receptor proteins. Briefly, labeled polypeptides of the invention are useful as reagents for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptides of the invention are covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as carcinoma tissues, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The protein complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 70
IL-6 Bioassay
To test the proliferative effects of the polypeptides of the invention, the IL-6 Bioassay as described by Marz et al. is utilized (Proc. Natl. Acad. Sci., U.S.A., 95:3251-56 (1998), which is herein incorporated by reference). Briefly, IL-6 dependent B9 murine cells are washed three times in IL-6 free medium and plated at a concentration of 5,000 cells per well in 50 μl, and 50 μl of the IL-6-like polypeptide is added. After 68 hrs. at 37° C., the number of viable cells is measured by adding the tetrazolium salt thiazolyl blue (MTT) and incubating for a further 4 hrs. at 37° C. B9 cells are lysed by SDS and optical density is measured at 570 nm. Controls containing IL-6 (positive) and no cytokine (negative) are utilized. Enhanced proliferation in the test sample(s) relative to the negative control is indicative of proliferative effects mediated by polypeptides of the invention.
Example 71
Support of Chicken Embryo Neuron Survival
To test whether sympathetic neuronal cell viability is supported by polypeptides of the invention, the chicken embryo neuronal survival assay of Senaldi et al is utilized (Proc. Natl. Acad. Sci., U.S.A., 96:11458-63 (1998), which is herein incorporated by reference). Briefly, motor and sympathetic neurons are isolated from chicken embryos, resuspended in L15 medium (with 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; LIFE TECHNOLOGIESTM, Rockville, Md.) and Dulbecco's modified Eagles medium [with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH 7.2); LIFE TECHNOLOGIESTM, Rockville, Md.], respectively, and incubated at 37° C. in 5% CO2 in the presence of different concentrations of the purified IL-6-like polypeptide, as well as a negative control lacking any cytokine. After 3 days, neuron survival is determined by evaluation of cellular morphology, and through the use of the colorimetric assay of Mosmann (Mosmann, T., J. Immunol. Methods, 65:55-63 (1983)). Enhanced neuronal cell viability as compared to the controls lacking cytokine is indicative of the ability of the inventive purified IL-6-like polypeptide(s) to enhance the survival of neuronal cells.
Example 72
Assay for Phosphatase Activity
The following assay may be used to assess serine/threonine phosphatase (PTPase) activity of the polypeptides of the invention.
In order to assay for serine/threonine phosphatase (PTPase) activity, assays can be utilized which are widely known to those skilled in the art. For example, the serine/threonine phosphatase (PSPase) activity is measured using a PSPase assay kit from New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate for PSPase, is phosphorylated on serine and threonine residues with cAMP-dependent Protein Kinase in the presence of [32P]ATP. Protein serine/threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from 32P-labeled MyBP.
Example 73
Interaction of Serine/Threonine Phosphatases with Other Proteins
The polypeptides of the invention with serine/threonine phosphatase activity as determined in Example 72 are research tools for the identification, characterization and purification of additional interacting proteins or receptor proteins, or other signal transduction pathway proteins. Briefly, labeled polypeptide(s) of the invention is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptide of the invention is covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as neural or liver cells, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The polypeptides of the invention—complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 74
Assaying for Heparanase Activity
In order to assay for heparanase activity of the polypeptides of the invention, the heparanase assay described by Vlodaysky et al is utilized (Vlodaysky, I., et al., Nat. Med., 5:793-802 (1999)). Briefly, cell lysates, conditioned media or intact cells (1×106 cells per 35-mm dish) are incubated for 18 hrs at 37° C., pH 6.2-6.6, with 35S-labeled ECM or soluble ECM derived peak I proteoglycans. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration on a Sepharose CL-6B column (0.9×30 cm). Fractions are eluted with PBS and their radioactivity is measured. Degradation fragments of heparan sulfate side chains are eluted from Sepharose 6B at 0.5<Kav<0.8 (peak II). Each experiment is done at least three times. Degradation fragments corresponding to “peak II,” as described by Vlodaysky et al., is indicative of the activity of the polypeptides of the invention in cleaving heparan sulfate.
Example 75
Immobilization of Biomolecules
This example provides a method for the stabilization of polypeptides of the invention in non-host cell lipid bilayer constructs (see, e.g., Bieri et al., Nature Biotech 17:1105-1108 (1999), hereby incorporated by reference in its entirety herein) that can be adapted for the study of polypeptides of the invention in the various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation is used to confine a biotin tag to the extracellular domain of the polypeptides of the invention, thus allowing uniform orientation upon immobilization. A 50 uM solution of polypeptides of the invention in washed membranes is incubated with 20 mM NaIO4 and 1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr at room temperature (reaction volume, 150 ul). Then the sample is dialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford Ill.) at 4° C. first for 5 h, exchanging the buffer after each hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodium phosphate, pH7). Just before addition into a cuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mM octylglucoside).
Example 76
TAQMAN®
Quantitative PCR (QPCR). Total RNA from cells in culture are extracted by TRIZOLTM separation as recommended by the supplier (LifeTechnologies). (Total RNA is treated with DNase I (LIFE TECHNOLOGIESTM) to remove any contaminating genomic DNA before reverse transcription.) Total RNA (50 ng) is used in a one-step, 50 ul, RT-QPCR, consisting of TaqMan® Buffer A (Perkin-Elmer; 50 mM KCl/10 mM Tris, pH 8.3), 5.5 mM MgCl2, 240 μM each dNTP, 0.4 units RNase inhibitor (PROMEGATM), 8% glycerol, 0.012% Tween-20, 0.05% gelatin, 0.3 μM primers, 0.1 μM probe, 0.025 units Amplitaq Gold® (Perkin-Elmer) and 2.5 units Superscript II reverse transcriptase (LIFE TECHNOLOGIESTM). As a control for genomic contamination, parallel reactions are setup without reverse transcriptase. The relative abundance of (unknown) and 18S RNAs are assessed by using the Applied Biosystems Prism 7700 Sequence Detection System (Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W. & Deetz, K. (1995) PCR Methods Appl. 4, 357-362). Reactions are carried out at 48° C. for 30 min, 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s, 60° C. for 1 min. Reactions are performed in triplicate.
Primers (f & r) and FRET probes sets are designed using Primer Express Software (Perkin-Elmer). Probes are labeled at the 5′-end with the reporter dye 6-FAM and on the 3′-end with the quencher dye TAMRA (Biosource International, Camarillo, Calif. or Perkin-Elmer).
Example 77
Assays for Metalloproteinase Activity
Metalloproteinases (EC 3.4.24.-) are peptide hydrolases which use metal ions, such as Zn2+, as the catalytic mechanism. Metalloproteinase activity of polypeptides of the present invention can be assayed according to the following methods.
Proteolysis of Alpha-2-Macroglobulin
To confirm protease activity, purified polypeptides of the invention are mixed with the substrate alpha-2-macroglobulin (0.2 unit/ml; BOEHRINGERTM Mannheim, Germany) in 1× assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl2, 25 μM ZnCl2 and 0.05% Brij-35) and incubated at 37° C. for 1-5 days. Trypsin is used as positive control. Negative controls contain only alpha-2-macroglobulin in assay buffer. The samples are collected and boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide gel. After electrophoresis the proteins are visualized by silver staining Proteolysis is evident by the appearance of lower molecular weight bands as compared to the negative control.
Inhibition of Alpha-2-Macroglobulin Proteolysis by Inhibitors of Metalloproteinases
Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA, AND HgCl2), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2), and commercial small molecule MMP inhibitors) are used to characterize the proteolytic activity of polypeptides of the invention. The three synthetic MMP inhibitors used are: MMP inhibitor I, [IC50=1.0 μM against MMP-1 and MMP-8; IC50=30 μM against MMP-9; IC50=150 μM against MMP-3]; MMP-3 (stromelysin-1) inhibitor I [IC50=5 μM against MMP-3], and MMP-3 inhibitor II [Ki=130 nM against MMP-3]; inhibitors available through Calbiochem, catalog #444250, 444218, and 444225, respectively). Briefly, different concentrations of the small molecule MMP inhibitors are mixed with purified polypeptides of the invention (50 μg/ml) in 22.9 μl of 1×HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl2, 25 μM ZnCl2 and 0.05% Brij-35) and incubated at room temperature (24° C.) for 2-hr, then 7.1 μl of substrate alpha-2-macroglobulin (0.2 unit/ml) is added and incubated at 37° C. for 20-hr. The reactions are stopped by adding 4× sample buffer and boiled immediately for 5 minutes. After SDS-PAGE, the protein bands are visualized by silver stain.
Synthetic Fluorogenic Peptide Substrates Cleavage Assay
The substrate specificity for polypeptides of the invention with demonstrated metalloproteinase activity can be determined using synthetic fluorogenic peptide substrates (purchased from BACHEM Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105, M-2110, and M-2255. The first four are MMP substrates and the last one is a substrate of tumor necrosis factor-α (TNF-α) converting enzyme (TACE). All the substrates are prepared in 1:1 dimethyl sulfoxide (DMSO) and water. The stock solutions are 50-500 μM. Fluorescent assays are performed by using a Perkin Elmer LS 50B luminescence spectrometer equipped with a constant temperature water bath. The excitation λ is 328 nm and the emission λ is 393 nm. Briefly, the assay is carried out by incubating 176 μl 1×HEPES buffer (0.2 M NaCl, 10 mM CaCl2, 0.05% Brij-35 and 50 mM HEPES, pH 7.5) with 4 μl of substrate solution (50 μM) at 25° C. for 15 minutes, and then adding 20 μl of a purified polypeptide of the invention into the assay cuvett. The final concentration of substrate is 1 μM. Initial hydrolysis rates are monitored for 30-min.
Example 78
Characterization of the cDNA Contained in a Deposited Plasmid
The size of the cDNA insert contained in a deposited plasmid may be routinely determined using techniques known in the art, such as PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the cDNA sequence. For example, two primers of 17-30 nucleotides derived from each end of the cDNA (i.e., hybridizable to the absolute 5′ nucleotide or the 3′ nucleotide end of the sequence of SEQ ID NO:X, respectively) are synthesized and used to amplify the cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 μl of reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
Example 79
Cloning, Sequence Analysis and Chromosomal Localization of the Novel Human Integrin Alpha 11 Subunit
The integrins are a large family of cell adhesion molecules consisting of noncovalently associated αβ heterodimers. We have cloned and sequenced a novel human integrin α-subunit cDNA, designated α11. The α11 cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120 residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced α11 protein shows the typical structural features of integrin α-subunits and is similar to a distinct group of α-subunits from collagen-binding integrins. However, it differs from most integrin α-chains by an incompletely preserved cytoplasmic GFFKR motif. The human ITGA11 gene was located to bands q22.3-23 on chromosome 15, and its transcripts were found predominantly in bone, cartilage as well as in cardiac and skeletal muscle. Expression of the 5.5 kilobase α11 mRNA was also detectable in ovary and small intestine.
Introduction
All vertebrate cells express members of the integrin family of cell adhesion molecules, which mediate cellular adhesion to other cells and extracellular subtratum, cell migration and participate in important physiologic processes from signal transduction to cell proliferation and differentiation (Hynes, 92; Springer, 92). Integrins are structurally homologous heterodimeric type-I membrane glycoproteins formed by the noncovalent association of one of eight β-subunits with one of the 17 different α-subunits described to date, resulting in at least 22 different αβ complexes. Their binding specificities for cellular and extracellular ligands are determined by both subunits and are dynamically regulated in a cell-type-specific mode by the cellular environment as well as by the developmental and activation state of the cell (Diamond and Springer, 94). In integrin α-subunits, the aminoterminal region of the large extracellular domain consists of a seven-fold repeated structure which is predicted to fold into a β-propeller domain (Corbi et al., 1987; Springer, 1997). The three or four C-terminal repeats contain putative divalent cation binding motifs that are thought to be important for ligand binding and subunit association (Diamond and Springer, 94). The α1, α2, α10, αD, αE, αL, αM and αX-subunits contain an approximately 200 amino acid I-domain inserted between the second and third repeat that is not present in other α-chains (Larson et al., 1989). Several isolated I-domains have been shown to independently bind the ligands of the parent integrin heterodimer (Kamata and Takada, 1994; Randi and Hogg, 1994). The α3, α5-8, αIIb and αV-subunits are proteolytically processed at a conserved site into disulphide-linked heavy and light chains, while the α4-subunit is cleaved at a more aminoterminal site into two fragments that remain noncovalently associated (Hemler et al., 90). Additional α-subunit variants are generated by alternative splicing of primary transcripts (Ziober et al., 93; Delwel et al., 95; Leung et al., 98). The extracellular domains of α-integrin subunits are connected by a single spanning transmembrane domain to short, diverse cytoplasmic domains whose only conserved feature is a membrane-proximal KXGFF(K/R)R motif (Sastry and Horwitz, 1993). The cytoplasmic domains have been implicated in the cell-type-specific modulation of integrin affinity states (Williams et al., 1994).
Here we report the cDNA cloning, sequence analysis, expression and chromosomal localization of the human α-integrin subunit.
Materials and Methods
Library Screening and DNA Sequencing.
A human fetal heart cDNA library in λgt10 (Clontech Laboratories, Inc., Palo Alto, Calif., USA) was screened with 32P-labelled (REDIPRIMETM, Amersham New Zealand Ltd., Auckland, New Zealand) probes corresponding to the regions 473 to 749 and 2394 to 3189 of the α11 cDNA using standard procedures. Inserts were subcloned from λgt10 into pUC21 and sequenced on both strands according to a successive specific primer strategy on an automated sequencer (Applied Biosystems 373A, The Centre for Gene Technology, School of Biological Sciences, The University of Auckland).
Northern Blot Analysis and Tissue Distribution.
A 1341 bp PCR fragment corresponding to the region 351-1692 of the α cDNA was 32P-labelled (REDIPRIMETM) and hybridized with human multiple tissue Northern blots (MTN I and MTN II, CLONTECHTM) for 16 h at 60∞ C in EXPRESSHYBTM solution (CLONTECHTM). Filters were washed twice with 0.1×SSC/1% SDS at 50° C. for 30 min, and autoradiographed. Human DNA from 63 tissue-specific cDNA libraries (Express-CheckTM, American Type Culture Collection, Manassas, Va., USA) was amplified using primers KL120 (5′-GCAGGGATGCCACCTGCC) and KL119 (5′-GATGAAGACTGTGGTGTCGAAGG) according to the manufacturers instructions. PCR-products were resolved by agarose gel electrophoresis and transferred to Hybond C+ (Amersham). Filters were hybridized by standard procedures (Ausubel et al., 98) with a 502 bp 32P-labelled (REDIPRIMETM) probe fragment obtained from the cloned “11 cDNA with the same oligonucleotides.
Chromosomal Assignment.
500 ng genomic DNA prepared from a panel of 21 human-rodent somatic cell hybrids or from human, mouse and hamster cells (Kelsell et al., 95) was amplified with oligonucleotides KL175 (5′-GGTGCCAGACCTACATGGAC) and KL189 (5′-CGTGCAAATTCAATGCCAAATGCC) in a standard PCR reaction of 30 cycles (94° C. for 1 min, 55° C. for 1 min, 72° C. for 2 min). All PCR reactions were resolved in a 2% agarose gel. Southern hybridization was performed as detailed above, except that the probe fragment was obtained from clone HOHBY69 with oligonucleotides KL175 and KL189. For fluorescent in situ hybridization, metaphase spreads were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes of a 46,XY male donor using standard cytogenetic procedures. A purified 3.7 kB fragment representing the entire coding region of clone HOHBY69 was labelled with biotin-16-dUTP using the High Prime labelling kit (Roche Molecular Biochemicals, Auckland, NZ). Conditions for hybridization and immunofluorescent detection were essentially as described (Morris et al., 93), except that C0t−1 suppression was not required, slides were washed to a stringency of 0.1×SSC/60° C. after hybridization, and an additional amplification step was needed because of the small size of the probe. For precise chromosome band localization, DAPI and FITC images were captured using a Photometrics KAF1400 CCD camera and QUIPS Smartcapture FISH software version 1.3 (Vysis Inc., Downers Grove, Ill., USA). QUIPS CGH/Karyotyping software (version 3.0.2) assisted karyotype analysis.
Results
Cloning of a Novel Human α-Integrin Subunit cDNA:
A protein homology search (Altschul et al., 90) of the human expressed sequence tag (EST) databases of Human Genome Sciences, Inc. (Ni et al., 97) and The Institute for Genomic Research (Kirkness and Kerlavage, 97) identified the clones HRDAF83 and HOEAM34 as candidate novel integrin α-subunit cDNAs. Clone HRDAF83 was isolated from a human rhabdomyosarcoma cDNA library and sequenced on both strands. The 1223 bp insert contains largely incompletely processed hnRNA and a 277 bp region that showed homology to the aminoterminal half of the α1-integrin I-domain. The 2517 bp insert of clone HOEAM34 was derived from a human osteoblast cDNA library. It is homologous to the C-terminal part of the human α1-subunit and contains 1324 nucleotides of 3′-untranslated region. In order to isolate the full-length cDNAs for these integrin α-subunits, a cDNA library prepared from human fetal heart in λgt10 was screened with the 277 bp fragment from clone HRDAF83 homologous to the α1-I-domain. Two clones, λ831 and λ832, were isolated and both strands of their inserts sequenced. Clone λ832 contains the entire 5′ half of a novel α-subunit cDNA, while clone λ831 covers the same region, but is 358 bp and 173 bp shorter than λ831 at its 5′- and 3′-ends, respectively. A screening of the same library with a 795 bp fragment from the extreme 5′-terminus of clone HOEAM34 identified clone λ342, which contained essentially the same region as clone HOEAM34 but has a 317 bp shorter 3′-untranslated region. Rescreening the EST databases with the sequences derived from the human fetal heart library led to the identification of clone HOHBY69, which was isolated from a osteoblast cDNA library. Both strands of the 4681 bp insert of clone HOHBY69 were sequenced. The 5′-region of HOHBY69 was identical to the HRDAF83/λ832/λ831-group, while the 3′-region of HOHBY69 was largely identical to HOEAM34 and λ342, thereby demonstrating that the two groups of partial cDNAs represent the 5′- and 3′-portions of the same cDNA. One major difference between the HOHBY69 and HOEAM34/λ342 is the presence of an additional GTA-triplet at position 3088 in HOHBY69. From the overlapping clones, a total of 4986 bp of cDNA was assembled to the composite sequence shown in FIG. 19A-F and has been submitted to GenBankTM with accession number AF109681. This cDNA encodes a previously unidentified human integrin α-subunit that was designated α11.
Structure of the Human α11-Subunit.
The all cDNA contains a 5′-untranslated region of 72 nucleotides and a single open reading frame extending from a predicted translation initiation codon at position +1 to a TGA termination codon at position 3570. This is followed by 1324 nucleotides of 3′ untranslated region which contains an AATTAAA polyadenylation signal (Wahle and Keller, 1996) 12 nucleotides upstream of a poly(A) stretch. The deduced amino acid sequence contains a 22 residue N-terminal region with the characteristics of a cleaved signal peptide (von Heijne, 83; Nielsen et al., 97), a large extracellular domain of 1120 amino acids followed by a 23 amino acid hydrophobic stretch that resembles a transmembrane domain, and a short 24 residue cytoplasmic domain. The molecular weight of the mature 1167 amino acid all-subunit is predicted to be 131 kDa, but the addition of carbohydrate side chains to any of the 15 potential N-glycosylation sequons [NX(S/T)] within the extracellular domain is likely to increase the molecular weight of the native protein. An I-domain of 207 amino acids is inserted between the second and third repeat. Consistent with the structure of an typical I-domain-containing integrin α-subunit, it lacks a potential dibasic protease cleavage site in the C-terminal region of the extracellular domain.
The α11-subunit is most closely related to the recently discovered α10-subunit (Camper et al., 98, Lehnert et al., in preparation) and the α1- and α2-subunits. Overall, the mature α11-protein is 45% identical to the α10 chain, while the homologies to the α1- and α2-subunits are 41% and 39%, respectively. Even greater homology exists between the I-domains of the α10- and α11-subunits which are 60% identical to each other. The high degree of homology seen in the extracellular domains of the subunits is in contrast to the low similarity of their cytoplasmic domains. Interestingly, the KXGFF(K/R)R motif that is absolutely conserved in all other α-subunit cytoplasmic domains is only partially preserved in both subunits. The sequence in α11 is KLGFFRS, while the α10-subunit contains a KLGFFAH motif. A graphical comparison of the similarity between all integrin α-subunits is shown in FIG. 3 (Lehnert et al.). Together with the α-subunits from the collagen-binding integrins α1β1, α2β1 and α10β1, the α11-subunit forms a group distinct from the other I-domain-containing integrin subunits.
Tissue Distribution and Expression of the Integrin α11-Subunit.
The tissue distribution of the α11 mRNA was assessed by screening multiple human tissue Northern blots with a probe corresponding to the region 351-1692 of the α11 cDNA. A single transcript of approximately 5.5 kb was found weakly expressed only in ovary and small intestine. Integrin α11-subunit expression was further analyzed by amplification and Southern hybridization of a 502 bp fragment corresponding to the region 1988-2490 in the α11 cDNA from tissue-specific human cDNA libraries. α11 cDNA was detected in five different cDNA libraries prepared from fetal heart (day 57-75), in two fetal brain libraries, and in a cDNA library from large intestine (not shown). An analysis of the Human Genome Sciences Database revealed eight different α11-related ESTs in human osteoblast libraries, three EST in a human chondrosarcoma cDNA library and two EST in a human stromal osteoclastoma library.
Chromosomal Localization of the Integrin α11-Subunit.
Genomic DNA from a collection of 21 human-rodent somatic cell hybrids (Kelsell et al., 95) was amplified by PCR using oligonucleotide primers directed the region 473 to 749 of the human α11 cDNA. In Southern hybridization, a signal corresponding to a 1.4 kb fragment was detectable only with DNA from a hybrid cell line that contains human chromosome 15. A fragment of the same size was also amplified from human genomic DNA, but not from mouse or hamster DNA (FIG. 5C (Lehnert et al.)). Cloning and sequence of the PCR product from chromosome 15 revealed the presence of a 1154 bp intron inserted after cDNA-position 600, thus resulting in a PCR-product of 1431 bp. The ITGA11 gene was also localized by fluorescent in situ hybridization of metaphase chromosomes with the entire coding region from clone HOHBY69. All of 20 metaphase cells analyzed showed fluorescent signal on both chromosomes 15, specifically across bands q22.3-q23. No additional signals were detected on any other chromosome (FIG. 5A (Lehnert et al.)).
Discussion:
We have cloned and sequenced a novel cDNA encoding a protein that shares extensive structural homology with integrin α-chains. The aminoterminal 22 amino acids of the deduced protein sequence show the characteristic features of a hydrophobic leader peptide, including a signal peptidase recognition motif at positions −3 and −1 (von Heijne 83). Proteolytic cleavage of the precursor protein at this position would result in an aminoterminal sequence for the mature α11-chain of FNMD, which is similar to the consensus sequence[(F/Y)N(L/V)D] of all other integrin α11-subunits (Tuckwell et al., 94). The N-terminal half of the large extracellular region of α11 is composed of seven repeats that each contain FG-GAP-GxxY consensus motifs (FG-GAP repeats). These repeats can be found in all integrin α1-subunits and are predicted to fold into a β-propeller domain (Springer, 97). Inserted between the second and third FG-GAP repeats is a 207 amino acid I-domain spanning from glutamine138 to methionine344. It contains a divalent cation coordination motif that has been shown to directly bind Mg2+ ions in the αM subunit (Michishita et al., 96). The noncontiguous amino acid side chains involved in the coordination of magnesium or manganese ions have been identified by mutagenesis analysis and from crystal structures of the isolated α2, αL and αM-subunit I-domains (Emsley et al., 97; Qu and Leahy, 95; Lee et al., 95). All residues required for the coordination of the divalent cations in these subunits are preserved in the α11-I-domain. These are the asparagines at positions 148 and 249, the serine residues at position 150 and 152, and the threonine at position 218.
The crystal structure of the α2-subunit has revealed a small “-helix that is not present in the I-domains of the β2-associated α-subunits. Together with the MIDAS sphere, amino acid residues from this C-helix and the adjacent turn region have been proposed to make physical contacts to a collagen triple helix (Emsley et al., 97). Interestingly, the small C-helix is structurally conserved in the α-subunits of the collagen-binding integrins α1β1, α2β1 and α10β1, and is also present in the α11 I-domain (G279YYNR283). In addition, asparagine154 and histidine258 of the α2-I-domain were predicted to contact the collagen triple helix, and both are preserved in the α1, α10 and α11-I-domains, but not in other integrin all-subunits. The conservation of structural motifs required for collagen binding suggests that collagen may be a ligand for the α11 integrin. Each of the repeats 5-7 of the α11-subunit accommodates the sequence Dx(D/N)xDxxxD. Three or four copies of these putative divalent cation binding sites are conserved in all integrin α-subunits and their presence is consistent with the divalent cation requirement for the adhesive function of integrins (Larson et al., 89; Fujimura and Phillips, 83; Hynes, 92). The extracellular domain of the integrin all-subunit contains 20 cysteine residues. Only the intramolecular disulfide bonds in the “IIb subunit have been biochemically characterized (Calvete et al., 89), but the location of many cysteines is conserved in integrin α-subunits. In the α11-subunit, the cysteine residues 637 and 646, 652 and 707, 759 and 765, and 859 and 871 are homologous to the residues that form the four carboxyterminal disulfide bonds in the heavy chain of “IIb (Calvete et al., 89). Based on the proposed structure of the integrin α-subunit propeller domain (Springer et al., 97), additional disulfide bonds within the α11 subunit can be predicted between cysteine residues 54 and 61, 99 and 117, and between 107 and 137. Two additional cysteine residues are found within a short segment (residues 783 to 798) that is unique to the all-subunit.
The integrin cytoplasmic domains play central roles in integrin affinity modulation and in cellular signal transmission. The membrane-proximal sequence KxGFF(K/R)R is strictly conserved among integrin α-subunit cytoplasmic domains (Williams et al., 94). Within this motif, both phenylalanine residues and the last arginine have been implicated in maintaining the default low affinity state of integrins αLβ2 and αIIbβ3, as their substitution or deletion resulted in constitutively activated ligand binding (O'Toole et al., 94, Lu and Spriner 97). Interestingly, the last arginine residue is replaced by a serine in the α11 cytoplasmic domain and with a histidine in the α10 subunit, suggesting that both integrins might be in a default “high” affinity state. It will be interesting to analyze whether substitution of these residues with a conserved arginine will affect their affinity status.
We have isolated α11 cDNAs from osteoclast, osteoblast, myosarcoma and fetal heart libraries. Amongst the HGS EST databases, integrin α11 transcripts were predominantly found in libraries prepared from osteoblast, osteoclast and chondrosarcoma cells. A search for further “11-related sequences in the EST division of the GenBank database revealed two clones (accession numbers Z50157 and Z50167) from primary human myoblasts (Genini et al., 96), two clones from human trabecular bone cells (AA852614 and AA852615), as well as clones from fibroblast cells (W45078), pancreatic tumor (U53091) and breast tissue (H16112). In contrast, Northern blot analysis detected α11-expression only in ovary and small intestine. only fetal heart, fetal brain and large intestine. Of the tissues represented in the tissue-specific cDNA-library panel, only fetal heart, fetal brain and large intestine showed detectable α11-expression. However, bone- and muscle-derived tissues were not included in the Northern blot, and cDNA libraries prepared from these tissues were also not represented in the tissue-specific cDNA panel.
The ITGA11 gene was localized to chromosome 15, bands q22.3-23, by FISH and PCR analysis of human-rodent somatic cell hybrids. This segment is overrepresented in squamous cell carcinomas (Wolff et al., 1998), but appears to only infrequently affected in other cancers. Genes at this region encode neogenin, a protein expressed ubiquitously expressed in human tissues (Meyerhardt et al., 97); tropomyosin 1, expressed in cardiac and skeletal muscle tissues (Tiso et al., 97); and the human homologue of the metalloprotease-disintegrin kuzbanian, which is overexpressed in tumors of sympathoadrenal origin (Yavari et al., 98). In addition, the region 15q22.3-q23 is linked to Bardet-Biedl syndrome 4, a heterogeneous autosomal disorder characterized by obesity and associated with cardiovascular anomalities (Carmi et al., 95).
In conclusion, we have cloned and sequenced the cDNA for the novel integrin α11-subunit which is closely related to the “-subunits of the collagen-binding integrins β1β1, α2β1 and α10β1. The high degree of homology of α11 to these subunits suggests that it associates with the integrin β1-subunit, and may function as an additional collagen receptor.
All references referred to above and presented below are hereby incorporated herein by reference:

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    Camper L., Hellman, U., and Lundgren-Akerlund, E. (1998). Isolation, cloning and sequence analysis of the integrin subunit α10, a β1-associated collagen binding integrin expressed on chondrocytes. Journal of Biological Chemistry 273: 20383-20389.
    Carmi R., Rokhlina, T., Kwitek-Black, A. E., Elbedour, K., Nishimura, D., Stone, E. M., and Sheffield, V. C. (1995). Use of a DNA pooling strategy to identify a human obesity syndrome locus on chromosome 15. Human Molecular Genetics 4: 9-13.
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    Ziober B. L., Vu, M. P., Waleh, N., Crawford, J., Lin, C. S., Kramer, and R H (1993). Alternative extracellular and cytoplasmic domains of the integrin alpha 7 subunit are differentially expressed during development. Journal of Biological Chemistry 268: 26773-83.
























TABLE 8





Res
Position
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV






























Met
1
A
.
.
.
.
.
.
1.30
−0.61
.
.
.
0.95
2.10


Arg
2
.
.
B
.
.
.
.
0.88
−1.04
*
.
.
0.95
3.21


Lys
3
.
.
B
B
.
.
.
0.98
−0.79
*
.
.
0.75
2.07


Thr
4
A
.
.
B
.
.
.
1.02
−0.30
.
.
.
0.45
2.20


Arg
5
.
.
B
B
.
.
.
0.60
−0.49
.
.
F
0.60
1.11


Leu
6
A
.
.
B
.
.
.
0.39
0.20
*
.
.
−0.30
0.46


Trp
7
.
.
B
B
.
.
.
−0.01
0.89
*
.
.
−0.60
0.26


Gly
8
A
.
.
B
.
.
.
−0.66
1.31
.
*
.
−0.60
0.14


Leu
9
A
.
.
B
.
.
.
−1.16
1.93
*
.
.
−0.60
0.17


Leu
10
.
.
B
B
.
.
.
−1.97
1.93
*
*
.
−0.60
0.13


Trp
11
.
.
B
B
.
.
.
−2.01
1.80
.
.
.
−0.60
0.12


Met
12
.
.
B
B
.
.
.
−2.02
2.01
.
.
.
−0.60
0.10


Leu
13
A
.
.
B
.
.
.
−1.68
1.71
.
.
.
−0.60
0.17


Phe
14
A
.
.
B
.
.
.
−1.68
1.03
*
*
.
−0.60
0.28


Val
15
A
.
.
B
.
.
.
−0.76
0.80
*
*
.
−0.60
0.23


Ser
16
A
A
.
.
.
.
.
−1.06
0.19
*
*
.
−0.30
0.55


Glu
17
A
A
.
.
.
.
.
−1.04
0.00
.
*
.
−0.30
0.64


Leu
18
A
A
.
.
.
.
.
−0.54
−0.29
*
*
.
0.30
0.88


Arg
19
A
A
.
.
.
.
.
0.20
−0.44
*
*
.
0.30
0.94


Ala
20
A
A
.
.
.
.
.
0.24
−0.83
*
*
.
0.75
1.09


Ala
21
A
A
.
.
.
.
.
0.23
−0.14
*
*
.
0.45
1.09


Thr
22
A
A
.
.
.
.
.
0.23
−0.34
*
*
F
0.45
0.80


Lys
23
A
A
.
.
.
.
.
1.04
−0.34
*
*
F
0.60
1.38


Leu
24
A
A
.
.
.
.
.
0.98
−0.84
*
*
F
0.90
2.36


Thr
25
A
A
.
.
.
.
.
1.32
−1.34
.
.
F
0.90
3.27


Glu
26
A
A
.
.
.
.
.
1.91
−1.07
.
.
F
0.90
2.56


Glu
27
A
A
.
.
.
.
.
1.41
−1.07
.
*
F
0.90
5.38


Lys
28
A
A
.
.
.
.
.
1.41
−1.07
.
*
F
0.90
3.08


Tyr
29
A
A
.
.
.
.
.
2.22
−1.56
.
*
F
0.90
3.55


Glu
30
A
A
.
.
.
.
.
2.19
−1.56
.
*
F
0.90
3.55


Leu
31
A
A
.
.
.
.
.
2.19
−1.13
.
*
F
0.90
1.76


Lys
32
A
A
.
.
.
.
.
1.88
−0.73
*
*
F
0.90
1.94


Glu
33
A
A
.
.
.
.
.
1.02
−1.00
*
*
F
0.90
1.62


Gly
34
A
A
.
.
.
.
.
1.27
−0.31
.
*
F
0.60
1.62


Gln
35
A
A
.
.
.
.
.
0.41
−1.00
.
*
F
0.90
1.35


Thr
36
A
A
.
.
.
.
.
1.27
−0.36
.
*
F
0.45
0.58


Leu
37
A
A
.
.
.
.
.
0.56
−0.36
.
*
F
0.60
1.17


Asp
38
.
A
B
.
.
.
.
0.56
−0.21
.
*
.
0.30
0.36


Val
39
.
A
B
.
.
.
.
0.66
−0.61
.
*
.
0.60
0.42


Lys
40
.
A
B
.
.
.
.
0.34
−0.34
.
*
.
0.30
0.80


Cys
41
.
.
B
.
.
T
.
−0.16
−0.54
.
*
.
1.00
0.69


Asp
42
A
.
.
.
.
T
.
0.66
0.14
.
*
.
0.10
0.77


Tyr
43
A
.
.
.
.
T
.
0.70
−0.50
.
*
.
0.70
0.66


Thr
44
A
.
.
.
.
T
.
0.86
−0.50
*
*
.
0.85
2.47


Leu
45
A
A
.
.
.
.
.
0.22
−0.29
*
*
.
0.45
1.28


Glu
46
A
A
.
.
.
.
.
0.59
0.21
*
.
.
−0.30
0.83


Lys
47
A
A
.
.
.
.
.
0.29
−0.16
.
.
.
0.30
0.77


Phe
48
A
A
.
.
.
.
.
0.53
−0.26
*
.
F
0.60
1.25


Ala
49
A
.
.
.
.
T
.
0.89
−0.54
.
.
F
1.30
1.25


Ser
50
A
.
.
.
.
T
.
1.11
−0.54
*
.
F
1.30
1.25


Ser
51
A
.
.
.
.
T
.
0.82
−0.04
.
.
F
1.00
1.46


Gln
52
A
.
.
.
.
T
.
0.78
0.09
.
*
F
0.40
1.51


Lys
53
A
.
.
.
.
.
.
0.59
−0.01
*
.
F
0.80
1.96


Ala
54
A
.
.
B
.
.
.
0.29
0.29
*
.
.
−0.15
1.02


Trp
55
A
.
.
B
.
.
.
0.70
0.59
*
.
.
−0.60
0.41


Gln
56
.
.
B
B
.
.
.
1.00
0.19
*
.
.
−0.30
0.41


Ile
57
.
.
B
B
.
.
.
0.66
0.19
*
.
.
0.04
0.67


Ile
58
.
.
B
.
.
T
.
0.61
0.11
*
.
.
0.78
0.63


Arg
59
.
.
B
.
.
T
.
0.60
−0.80
*
.
.
2.02
0.63


Asp
60
.
.
.
.
T
T
.
0.68
−0.59
*
.
F
2.91
0.89


Gly
61
.
.
.
.
T
T
.
0.72
−0.84
*
.
F
3.40
1.97


Glu
62
.
.
.
.
.
.
C
1.30
−1.53
*
.
F
2.66
2.01


Met
63
.
.
.
.
.
T
C
1.38
−1.04
.
.
F
2.52
1.74


Pro
64
.
.
.
.
T
T
.
0.68
−0.36
*
.
F
2.08
1.45


Lys
65
.
.
.
.
T
T
.
0.01
−0.29
.
.
F
1.59
0.84


Thr
66
A
.
.
.
.
T
.
0.04
0.29
.
.
F
0.25
0.46


Leu
67
A
A
.
.
.
.
.
0.04
0.16
*
.
.
−0.30
0.43


Ala
68
A
A
.
.
.
.
.
0.76
−0.27
*
.
.
0.30
0.37


Cys
69
A
A
.
.
.
.
.
0.76
−0.27
*
.
.
0.30
0.50


Thr
70
A
A
.
.
.
.
.
0.41
−0.33
*
.
.
0.64
0.94


Glu
71
A
.
.
.
.
.
.
0.77
−0.63
*
.
F
1.78
1.25


Arg
72
A
.
.
.
.
.
.
1.58
−1.13
*
.
F
2.12
4.65


Pro
73
.
.
.
.
T
.
.
1.87
−1.30
.
.
F
2.86
5.18


Ser
74
.
.
.
.
T
T
.
2.50
−1.40
*
.
F
3.40
4.01


Lys
75
.
.
.
.
T
T
.
2.60
−0.90
*
.
F
3.06
2.79


Asn
76
.
.
.
.
T
T
.
1.74
−0.47
*
.
F
2.42
2.79


Ser
77
.
.
.
.
.
T
C
1.63
−0.26
.
*
F
1.88
1.54


His
78
.
.
B
B
.
.
.
0.99
−0.24
.
.
F
0.94
1.34


Pro
79
.
.
B
B
.
.
.
0.94
0.40
.
.
.
−0.60
0.62


Val
80
.
.
B
B
.
.
.
1.01
0.43
.
*
.
−0.60
0.46


Gln
81
.
.
B
B
.
.
.
0.12
0.04
*
.
.
−0.30
0.66


Val
82
.
.
B
B
.
.
.
−0.47
0.23
*
.
.
−0.30
0.30


Gly
83
.
.
B
B
.
.
.
−1.24
0.49
*
*
.
−0.60
0.28


Arg
84
.
.
B
B
.
.
.
−1.03
0.53
*
.
.
−0.60
0.13


Ile
85
.
.
B
B
.
.
.
−0.18
0.13
*
.
.
−0.30
0.31


Ile
86
.
.
B
B
.
.
.
−0.42
−0.51
*
.
.
0.60
0.53


Leu
87
.
.
B
B
.
.
.
0.40
−0.19
*
*
.
0.30
0.42


Glu
88
.
.
B
B
.
.
.
0.74
0.31
*
*
.
−0.30
0.82


Asp
89
A
.
.
.
.
.
.
0.60
−0.37
*
*
.
0.65
1.95


Tyr
90
A
.
.
.
.
.
.
1.14
−0.56
.
.
.
0.95
3.21


His
91
A
.
.
.
.
T
.
1.22
−0.81
.
.
.
1.15
1.84


Asp
92
A
.
.
.
.
T
.
1.22
−0.13
.
*
.
0.70
0.91


His
93
A
.
.
.
.
T
.
1.33
0.56
*
*
.
−0.20
0.48


Gly
94
A
.
.
.
.
T
.
0.48
−0.20
.
*
.
0.70
0.69


Leu
95
A
.
.
B
.
.
.
0.83
−0.06
.
*
.
0.30
0.31


Leu
96
A
.
.
B
.
.
.
0.27
−0.06
.
*
.
0.30
0.44


Arg
97
.
.
B
B
.
.
.
−0.59
0.06
.
*
.
−0.30
0.44


Val
98
.
.
B
B
.
.
.
−0.56
0.27
.
*
.
−0.30
0.40


Arg
99
.
.
B
B
.
.
.
−1.02
−0.01
.
*
.
0.30
0.77


Met
100
.
.
B
B
.
.
.
−0.21
−0.01
.
*
.
0.30
0.32


Val
101
.
.
B
B
.
.
.
−0.26
0.39
.
*
.
−0.30
0.76


Asn
102
.
.
B
B
.
.
.
−0.37
0.39
.
*
.
−0.30
0.29


Leu
103
.
.
B
B
.
.
.
0.49
0.39
*
*
.
−0.30
0.50


Gln
104
.
.
B
B
.
.
.
0.08
−0.23
*
*
.
0.73
1.13


Val
105
.
.
B
B
.
.
.
0.33
−0.49
.
*
.
0.86
0.94


Glu
106
.
.
B
B
.
.
.
0.38
−0.46
.
*
F
1.44
1.13


Asp
107
.
.
.
.
T
T
.
0.13
−0.46
.
*
F
2.37
0.54


Ser
108
.
.
.
.
T
T
.
0.94
−0.10
.
*
F
2.80
1.14


Gly
109
.
.
.
.
T
T
.
0.28
−0.34
.
.
F
2.52
1.14


Leu
110
.
.
.
.
T
T
.
0.28
0.23
.
.
.
1.34
0.36


Tyr
111
.
.
B
B
.
.
.
−0.61
0.87
.
.
.
−0.04
0.20


Gln
112
.
.
B
B
.
.
.
−0.86
1.17
.
.
.
−0.32
0.14


Cys
113
.
.
B
B
.
.
.
−0.56
1.50
.
.
.
−0.60
0.27


Val
114
.
.
B
B
.
.
.
−0.42
1.21
.
.
.
−0.60
0.30


Ile
115
.
.
B
B
.
.
.
0.18
0.89
*
.
.
−0.26
0.27


Tyr
116
.
.
B
.
.
.
.
0.47
0.91
*
.
.
0.28
0.77


Gln
117
.
.
.
.
.
.
C
0.47
0.34
*
.
.
1.27
2.09


Pro
118
.
.
.
.
.
T
C
0.92
−0.30
*
.
F
2.56
5.15


Pro
119
.
.
.
.
T
T
.
1.74
−0.56
*
.
F
3.40
5.08


Lys
120
.
.
.
.
.
T
C
2.03
−0.81
*
.
F
2.86
3.99


Glu
121
.
.
.
.
.
T
C
1.47
−0.60
*
.
F
2.52
2.56


Pro
122
A
.
.
.
.
.
.
0.77
−0.34
*
.
F
1.48
1.36


His
123
A
.
.
B
.
.
.
0.98
0.01
*
.
.
0.04
0.59


Met
124
A
.
.
B
.
.
.
1.30
0.01
*
.
.
−0.30
0.57


Leu
125
A
.
.
B
.
.
.
0.37
0.01
*
*
.
−0.30
0.72


Phe
126
A
.
.
B
.
.
.
0.48
0.27
*
*
.
−0.30
0.37


Asp
127
A
.
.
B
.
.
.
−0.12
−0.23
*
*
.
0.30
0.74


Arg
128
A
.
.
B
.
.
.
−0.94
−0.16
*
*
.
0.30
0.74


Ile
129
A
.
.
B
.
.
.
−1.20
−0.20
*
*
.
0.30
0.63


Arg
130
.
.
B
B
.
.
.
−0.70
−0.34
*
*
.
0.30
0.28


Leu
131
.
.
B
B
.
.
.
0.04
0.14
*
*
.
−0.30
0.21


Val
132
.
.
B
B
.
.
.
−0.30
0.14
*
*
.
−0.30
0.59


Val
133
.
.
B
B
.
.
.
−1.11
−0.11
*
*
.
0.30
0.30


Thr
134
.
.
B
B
.
.
.
−0.52
0.67
*
*
F
−0.45
0.31


Lys
135
.
.
B
B
.
.
.
−0.98
0.37
.
*
F
−0.15
0.56


Gly
136
.
.
B
.
.
T
.
−0.48
0.16
.
.
F
0.25
0.75


Phe
137
.
.
B
.
.
T
.
0.17
0.00
.
.
F
0.25
0.75


Ser
138
.
.
B
.
.
T
.
0.68
−0.06
.
.
F
0.85
0.58


Gly
139
.
.
.
.
.
T
C
0.69
0.37
.
.
F
0.45
0.58


Thr
140
.
.
.
.
.
T
C
0.64
0.33
.
.
F
0.45
0.90


Pro
141
.
.
.
.
.
T
C
0.99
−0.06
.
.
F
1.20
1.08


Gly
142
.
.
.
.
.
T
C
1.69
−0.44
.
.
F
1.54
1.89


Ser
143
.
.
.
.
.
T
C
1.69
−0.47
.
.
F
1.88
2.11


Asn
144
.
.
.
.
.
T
C
1.72
−0.57
.
.
F
2.52
1.82


Glu
145
.
.
.
.
.
T
C
2.03
−0.51
.
.
F
2.86
2.66


Asn
146
.
.
.
.
T
T
.
2.24
−0.54
.
.
F
3.40
3.44


Ser
147
.
.
.
.
T
T
.
1.73
−0.53
.
.
F
3.06
3.44


Thr
148
.
.
.
B
T
.
.
1.79
−0.29
*
.
F
2.02
1.47


Gln
149
.
.
B
B
.
.
.
1.83
0.47
*
.
F
0.38
1.44


Asn
150
.
.
B
B
.
.
.
0.94
0.07
*
.
F
0.34
2.14


Val
151
.
.
B
B
.
.
.
0.73
0.37
*
.
F
0.00
1.04


Tyr
152
.
.
B
B
.
.
.
0.82
0.31
*
.
.
−0.30
0.93


Lys
153
.
.
B
B
.
.
.
0.82
0.34
*
.
F
−0.15
0.89


Ile
154
.
.
B
B
.
.
.
0.51
0.43
*
.
F
−0.30
1.74


Pro
155
.
.
B
.
.
T
.
0.20
0.27
*
.
F
0.40
1.60


Pro
156
.
.
.
.
T
T
.
1.10
0.00
*
.
F
0.80
1.15


Thr
157
.
.
.
.
T
T
.
0.76
0.00
*
.
F
0.80
3.29


Thr
158
.
.
B
.
.
T
.
−0.10
−0.19
*
.
F
1.00
2.15


Thr
159
.
A
B
.
.
.
.
0.12
0.07
*
.
F
0.00
1.15


Lys
160
.
A
B
.
.
.
.
0.12
0.21
*
.
F
−0.15
0.43


Ala
161
.
A
B
.
.
.
.
−0.48
0.16
*
.
.
−0.30
0.46


Leu
162
.
A
B
.
.
.
.
−0.41
0.36
*
.
.
−0.30
0.26


Cys
163
.
.
B
.
.
T
.
−0.41
0.63
*
.
.
−0.20
0.20


Pro
164
.
.
B
.
.
T
.
−0.40
1.11
*
.
.
−0.20
0.29


Leu
165
.
.
B
.
.
T
.
−0.66
1.00
*
*
.
−0.20
0.47


Tyr
166
.
.
B
.
.
T
.
0.04
0.74
*
*
.
−0.05
1.37


Thr
167
.
.
B
.
.
.
.
0.54
0.17
*
.
F
0.20
1.74


Ser
168
.
.
.
.
.
T
C
0.36
0.23
*
.
F
0.60
3.04


Pro
169
.
.
B
.
.
T
.
0.26
0.19
*
.
F
0.40
1.44


Arg
170
.
.
B
.
.
T
.
1.07
−0.09
*
*
F
1.00
1.44


Thr
171
.
.
B
.
.
T
.
0.72
−0.17
*
.
F
1.00
1.86


Val
172
.
.
B
B
.
.
.
0.82
−0.06
*
.
F
0.60
1.21


Thr
173
.
.
B
B
.
.
.
0.91
−0.06
*
*
F
0.73
0.96


Gln
174
.
.
B
B
.
.
.
1.17
0.37
*
*
F
0.56
1.03


Ala
175
.
.
B
B
.
.
.
0.76
−0.11
*
*
F
1.44
2.77


Pro
176
.
.
.
.
.
T
C
0.76
−0.37
.
.
F
2.32
2.57


Pro
177
.
.
.
.
T
T
.
1.02
−0.37
.
*
F
2.80
2.14


Lys
178
.
.
.
.
T
T
.
1.33
−0.27
.
*
F
2.52
2.14


Ser
179
.
.
B
.
.
T
.
0.48
−0.77
.
*
F
2.14
2.31


Thr
180
.
.
B
B
.
.
.
0.77
−0.56
.
*
F
1.46
1.11


Ala
181
.
.
B
B
.
.
.
0.67
−0.60
.
*
F
1.03
0.74


Asp
182
.
.
B
B
.
.
.
0.67
−0.11
.
*
F
0.45
0.80


Val
183
.
.
B
B
.
.
.
0.62
−0.07
.
*
F
0.79
0.86


Ser
184
.
.
B
.
.
.
.
0.62
−0.56
.
.
F
1.78
1.42


Thr
185
.
.
B
.
.
T
.
0.93
−0.67
.
*
F
2.32
1.14


Pro
186
.
.
.
.
.
T
C
0.63
−0.67
.
*
F
2.86
2.66


Asp
187
.
.
.
.
T
T
.
0.63
−0.63
.
*
F
3.40
1.39


Ser
188
.
.
B
.
.
T
.
0.68
−0.61
.
*
F
2.66
1.55


Glu
189
.
.
B
.
.
.
.
0.67
−0.41
.
*
F
1.67
0.83


Ile
190
.
.
B
.
.
.
.
0.98
−0.36
.
*
F
1.33
0.71


Asn
191
.
.
B
.
.
.
.
0.33
0.04
.
*
.
0.24
0.86


Leu
192
.
.
B
B
.
.
.
0.02
0.30
.
*
.
−0.30
0.37


Thr
193
.
.
B
B
.
.
.
0.32
0.79
.
*
F
−0.60
0.76


Asn
194
.
.
B
B
.
.
.
−0.57
0.10
*
*
F
−0.15
0.78


Val
195
.
.
B
B
.
.
.
−0.57
0.39
*
*
F
−0.15
0.67


Thr
196
.
.
B
B
.
.
.
−0.46
0.39
*
*
F
−0.15
0.32


Asp
197
.
.
B
B
.
.
.
−0.50
−0.10
*
*
.
0.30
0.39


Ile
198
.
.
B
B
.
.
.
−0.40
0.14
*
*
.
−0.30
0.39


Ile
199
.
.
B
B
.
.
.
−1.26
−0.07
*
*
.
0.30
0.42


Arg
200
.
.
B
B
.
.
.
−1.10
0.09
*
*
.
−0.30
0.19


Val
201
.
.
B
B
.
.
.
−0.79
0.87
*
.
.
−0.60
0.23


Pro
202
.
.
B
B
.
.
.
−1.68
0.59
*
.
.
−0.60
0.53


Val
203
.
.
B
B
.
.
.
−1.64
0.59
.
*
.
−0.60
0.19


Phe
204
.
.
B
B
.
.
.
−1.64
1.23
.
*
.
−0.60
0.19


Asn
205
.
.
B
B
.
.
.
−2.57
1.27
.
*
.
−0.60
0.09


Ile
206
.
.
B
B
.
.
.
−2.52
1.53
.
.
.
−0.60
0.10


Val
207
.
.
B
B
.
.
.
−2.90
1.57
.
.
.
−0.60
0.09


Ile
208
.
.
B
B
.
.
.
−2.39
1.29
.
.
.
−0.60
0.06


Leu
209
.
.
B
B
.
.
.
−2.03
1.31
.
*
.
−0.60
0.08


Leu
210
.
.
B
.
.
T
.
−2.73
1.06
.
.
.
−0.20
0.11


Ala
211
.
.
B
.
.
T
.
−2.66
1.20
.
.
.
−0.20
0.13


Gly
212
A
.
.
.
.
T
.
−2.10
1.20
.
.
.
−0.20
0.13


Gly
213
A
.
.
.
.
T
.
−1.17
0.90
.
.
.
−0.20
0.22


Phe
214
A
.
.
.
.
.
.
−0.66
0.21
.
.
.
−0.10
0.43


Leu
215
A
.
.
.
.
.
.
−0.66
0.10
.
.
F
0.05
0.58


Ser
216
.
.
B
.
.
T
.
−0.92
0.36
.
.
F
0.25
0.48


Lys
217
.
.
B
.
.
T
.
−1.28
0.57
.
.
F
−0.05
0.42


Ser
218
.
.
B
.
.
T
.
−1.23
0.57
.
.
F
−0.05
0.44


Leu
219
.
.
B
.
.
T
.
−1.39
0.27
.
.
.
0.10
0.44


Val
220
.
.
B
B
.
.
.
−1.39
0.53
.
.
.
−0.60
0.16


Phe
221
.
.
B
B
.
.
.
−1.79
1.21
.
.
.
−0.60
0.10


Ser
222
.
.
B
B
.
.
.
−2.42
1.61
.
.
.
−0.60
0.10


Val
223
.
.
B
B
.
.
.
−2.98
1.43
.
.
.
−0.60
0.14


Leu
224
.
.
B
B
.
.
.
−2.48
1.43
.
*
.
−0.60
0.12


Phe
225
A
.
.
B
.
.
.
−2.43
1.13
*
*
.
−0.60
0.13


Ala
226
A
.
.
B
.
.
.
−1.62
1.43
*
*
.
−0.60
0.15


Val
227
A
.
.
B
.
.
.
−1.62
0.79
*
*
.
−0.60
0.35


Thr
228
.
.
B
B
.
.
.
−1.47
0.49
.
*
.
−0.60
0.54


Leu
229
.
.
B
B
.
.
.
−1.51
0.49
*
*
.
−0.60
0.46


Arg
230
.
.
B
B
.
.
.
−1.02
0.63
*
*
.
−0.60
0.46


Ser
231
.
.
B
B
.
.
.
−0.82
0.41
.
*
.
−0.60
0.49


Phe
232
.
.
B
B
.
.
.
−0.36
0.36
.
*
.
−0.30
0.76


Val
233
.
.
B
B
.
.
.
−0.43
0.10
*
*
.
−0.30
0.50


Pro
234
.
.
B
B
.
.
.
−0.01
0.53
*
*
.
−0.60
0.48































TABLE 9





Res
Position
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV






























Met
1
A
A
.
.
.
.
.
0.61
−0.60
.
.
.
0.75
1.47


Lys
2
A
A
.
.
.
.
.
0.41
−0.64
.
.
.
0.96
1.54


Arg
3
.
A
B
.
.
.
.
0.46
−0.57
*
.
.
1.17
1.22


Ala
4
A
A
.
.
.
.
.
0.50
−0.57
*
.
.
1.38
1.22


Ser
5
A
.
.
.
.
T
.
0.59
−0.76
*
.
F
1.99
0.60


Ala
6
.
.
.
.
.
T
C
1.30
−0.37
*
.
F
2.10
0.41


Gly
7
.
.
.
.
.
T
C
0.44
−0.37
*
.
F
1.89
0.80


Gly
8
.
.
.
.
.
T
C
−0.48
−0.19
*
*
F
1.68
0.49


Ser
9
.
A
.
.
.
.
C
−0.48
0.11
.
.
F
0.47
0.40


Arg
10
.
A
B
.
.
.
.
−0.47
0.11
*
.
F
0.06
0.41


Leu
11
.
A
B
.
.
.
.
−0.73
0.60
*
.
.
−0.60
0.43


Leu
12
.
A
B
.
.
.
.
−1.20
0.81
*
.
.
−0.60
0.24


Ala
13
.
A
B
.
.
.
.
−1.14
1.11
*
.
.
−0.60
0.10


Trp
14
.
A
B
.
.
.
.
−1.66
2.03
.
*
.
−0.60
0.13


Val
15
.
A
B
.
.
.
.
−1.77
2.03
.
*
.
−0.60
0.13


Leu
16
A
A
.
.
.
.
.
−1.54
1.74
.
.
.
−0.60
0.22


Trp
17
A
A
.
.
.
.
.
−1.02
1.74
.
.
.
−0.60
0.21


Leu
18
A
A
.
.
.
.
.
−0.43
1.74
.
.
.
−0.60
0.30


Gln
19
A
A
.
.
.
.
.
−1.00
1.50
.
.
.
−0.60
0.63


Ala
20
.
A
.
.
T
.
.
−0.73
1.46
.
.
.
−0.20
0.45


Trp
21
.
A
.
.
T
.
.
−0.51
1.04
.
.
.
−0.20
0.55


Gln
22
.
A
B
.
.
.
.
−0.43
0.86
.
.
.
−0.60
0.32


Val
23
.
A
B
.
.
.
.
−0.29
0.89
.
.
.
−0.60
0.49


Ala
24
.
A
B
.
.
.
.
−0.50
0.96
.
.
.
−0.60
0.25


Ala
25
.
A
B
.
.
.
.
−0.26
0.47
.
.
.
−0.60
0.22


Pro
26
.
A
.
.
T
.
.
−0.56
0.50
.
*
.
−0.20
0.30


Cys
27
.
.
.
.
T
T
.
−1.22
0.36
.
.
.
0.50
0.30


Pro
28
.
.
.
.
T
T
.
−1.22
0.43
.
.
.
0.20
0.16


Gly
29
.
.
.
.
T
T
.
−1.30
0.57
.
.
.
0.20
0.08


Ala
30
.
.
B
.
.
T
.
−0.96
0.71
.
.
.
−0.20
0.08


Cys
31
.
.
B
.
.
.
.
−0.74
0.90
.
.
.
−0.40
0.08


Val
32
.
.
B
.
.
.
.
−0.08
0.87
.
.
.
−0.40
0.12


Cys
33
.
.
B
.
.
T
.
−0.08
0.44
.
.
.
−0.20
0.21


Tyr
34
.
.
B
.
.
T
.
0.31
0.37
.
.
.
0.36
0.62


Asn
35
.
.
B
.
.
T
.
0.04
−0.20
.
.
F
1.52
1.66


Glu
36
.
.
B
.
.
T
.
0.40
−0.20
.
.
F
1.78
2.30


Pro
37
.
.
.
B
T
.
.
0.94
−0.29
.
.
F
2.04
2.11


Lys
38
.
.
.
B
T
.
.
1.31
−0.56
.
*
F
2.60
1.90


Val
39
.
.
.
B
T
.
.
0.89
−0.57
.
*
F
2.34
1.47


Thr
40
.
.
B
B
.
.
.
0.68
0.00
.
*
F
0.63
0.51


Thr
41
.
.
B
B
.
.
.
0.68
0.00
.
*
F
0.37
0.39


Ser
42
.
.
B
B
.
.
.
0.89
0.40
*
*
F
−0.19
0.92


Cys
43
.
.
B
.
.
T
.
0.50
0.16
*
*
F
0.40
1.10


Pro
44
.
.
.
.
T
T
.
0.54
0.10
.
.
F
0.65
0.76


Gln
45
.
.
.
.
T
T
.
0.86
0.30
.
.
F
0.65
0.47


Gln
46
.
.
B
.
.
T
.
0.58
0.31
*
.
F
0.40
1.50


Gly
47
.
A
B
.
.
.
.
0.02
0.24
*
.
F
−0.15
0.98


Leu
48
.
A
B
B
.
.
.
0.48
0.46
.
.
.
−0.60
0.42


Gln
49
.
A
B
B
.
.
.
−0.17
0.49
.
.
.
−0.60
0.38


Ala
50
.
A
B
B
.
.
.
−0.51
0.73
*
.
.
−0.60
0.28


Val
51
.
A
B
B
.
.
.
−1.40
0.73
*
.
.
−0.60
0.34


Pro
52
.
.
B
B
.
.
.
−1.27
0.73
.
.
.
−0.60
0.14


Val
53
.
.
B
B
.
.
.
−1.04
0.76
.
.
.
−0.60
0.21


Gly
54
.
.
B
.
.
.
.
−1.63
0.76
.
.
.
−0.40
0.29


Ile
55
.
.
B
.
.
.
.
−1.34
0.61
.
.
.
−0.40
0.19


Pro
56
.
.
B
.
.
.
.
−0.49
0.57
.
*
.
−0.40
0.34


Ala
57
A
.
.
.
.
.
.
−0.17
0.33
.
*
.
−0.10
0.59


Ala
58
A
.
.
.
.
.
.
−0.20
−0.10
*
.
.
0.65
1.64


Ser
59
A
.
.
B
.
.
.
−0.56
−0.10
*
*
F
0.45
0.75


Gln
60
.
.
B
B
.
.
.
−0.48
0.26
*
*
F
−0.15
0.64


Arg
61
.
.
B
B
.
.
.
−0.30
0.44
.
*
F
−0.45
0.52


Ile
62
.
.
B
B
.
.
.
−0.06
0.44
.
*
.
−0.60
0.53


Phe
63
.
.
B
B
.
.
.
0.53
0.49
*
*
.
−0.60
0.30


Leu
64
.
.
B
B
.
.
.
0.94
0.49
*
*
.
−0.60
0.25


His
65
.
.
.
.
T
T
.
0.06
0.49
*
*
.
0.26
0.69


Gly
66
.
.
.
.
T
T
.
−0.36
0.49
*
*
F
0.47
0.56


Asn
67
.
.
.
.
T
T
.
0.50
0.09
*
.
F
0.83
0.91


Arg
68
.
.
.
.
T
T
.
0.34
−0.10
.
.
F
1.49
0.91


Ile
69
.
.
.
.
T
.
.
0.94
0.04
.
.
.
0.60
0.69


Ser
70
.
.
B
.
.
.
.
0.39
0.04
*
.
.
0.14
0.66


His
71
.
.
B
.
.
.
.
0.14
0.14
*
.
.
0.08
0.34


Val
72
.
.
B
.
.
.
.
−0.16
0.64
*
.
.
−0.28
0.49


Pro
73
.
.
B
.
.
.
.
−0.97
0.34
*
*
.
−0.04
0.49


Ala
74
.
.
.
.
T
.
.
0.03
0.74
*
*
.
0.00
0.31


Ala
75
A
.
.
.
.
.
.
−0.26
0.24
.
*
.
−0.10
0.82


Ser
76
A
.
.
.
.
.
.
−0.89
0.10
*
.
.
−0.10
0.54


Phe
77
A
.
.
.
.
.
.
0.08
0.24
*
.
.
−0.10
0.29


Arg
78
A
.
.
.
.
.
.
0.29
−0.26
*
*
.
0.60
0.55


Ala
79
A
.
.
.
.
.
.
0.07
−0.36
*
*
.
0.70
0.66


Cys
80
A
.
.
.
.
T
.
0.34
−0.06
*
*
.
1.00
0.63


Arg
81
.
.
.
.
T
T
.
−0.24
−0.36
*
*
.
1.50
0.47


Asn
82
.
.
.
.
T
T
.
−0.36
0.33
*
*
.
1.00
0.32


Leu
83
.
.
B
.
.
T
.
−0.76
0.51
*
.
.
0.20
0.50


Thr
84
.
.
B
B
.
.
.
−0.98
0.86
*
.
.
−0.30
0.27


Ile
85
.
.
B
B
.
.
.
−0.34
1.54
.
.
.
−0.40
0.14


Leu
86
.
.
B
B
.
.
.
−0.76
1.64
.
.
.
−0.50
0.23


Trp
87
.
.
B
B
.
.
.
−0.76
1.34
.
*
.
−0.60
0.21


Leu
88
.
.
B
B
.
.
.
−0.80
1.26
.
.
.
−0.60
0.48


His
89
.
.
B
.
.
T
.
−1.30
1.21
.
.
.
−0.20
0.43


Ser
90
.
.
.
.
.
T
C
−1.00
1.21
*
*
.
0.00
0.34


Asn
91
.
.
.
.
.
T
C
−0.08
0.80
*
*
.
0.00
0.42


Val
92
A
.
.
.
.
T
.
−0.68
0.11
.
*
.
0.10
0.60


Leu
93
A
A
.
.
.
.
.
0.13
0.30
.
*
.
−0.30
0.31


Ala
94
A
A
.
.
.
.
.
−0.42
−0.09
.
*
.
0.30
0.33


Arg
95
A
A
.
.
.
.
.
−0.71
0.01
*
*
.
−0.30
0.44


Ile
96
A
A
.
.
.
.
.
−1.30
−0.13
*
*
.
0.30
0.54


Asp
97
A
A
.
.
.
.
.
−1.14
−0.31
.
*
.
0.30
0.54


Ala
98
A
A
.
.
.
.
.
−0.64
−0.03
*
*
.
0.30
0.24


Ala
99
A
A
.
.
.
.
.
−0.40
0.46
*
*
.
−0.60
0.49


Ala
100
A
A
.
.
.
.
.
−1.32
0.20
.
*
.
−0.30
0.29


Phe
101
A
A
.
.
.
.
.
−1.02
0.89
.
.
.
−0.60
0.24


Thr
102
A
A
.
.
.
.
.
−1.83
0.89
.
.
.
−0.60
0.24


Gly
103
A
A
.
.
.
.
.
−2.06
1.07
.
.
.
−0.60
0.19


Leu
104
A
A
.
.
.
.
.
−1.47
1.26
.
.
.
−0.60
0.19


Ala
105
A
A
.
.
.
.
.
−0.88
0.47
.
.
.
−0.60
0.22


Leu
106
A
A
.
.
.
.
.
−0.99
0.39
.
.
.
−0.30
0.39


Leu
107
A
A
.
.
.
.
.
−0.68
0.64
.
*
.
−0.60
0.39


Glu
108
A
A
.
.
.
.
.
−1.14
−0.04
.
*
.
0.30
0.64


Gln
109
A
A
.
.
.
.
.
−0.63
0.14
.
*
.
−0.30
0.64


Leu
110
A
A
.
.
.
.
.
−0.04
−0.16
.
*
.
0.45
1.05


Asp
111
A
A
.
.
.
.
.
0.77
−0.84
.
*
.
0.75
1.01


Leu
112
A
A
.
.
.
.
.
0.99
−0.44
.
*
.
0.30
0.94


Ser
113
A
.
.
.
.
T
.
0.99
−0.34
.
*
F
1.00
1.15


Asp
114
A
.
.
.
.
T
.
0.18
−0.63
.
*
F
1.30
1.19


Asn
115
A
.
.
.
.
T
.
1.10
0.06
.
*
F
0.40
1.19


Ala
116
A
.
.
.
.
T
.
0.80
−0.63
.
*
.
1.15
1.74


Gln
117
.
A
B
.
.
.
.
0.76
−0.63
.
*
.
0.75
1.40


Leu
118
.
A
B
.
.
.
.
1.06
0.01
.
*
.
−0.30
0.64


Arg
119
.
A
B
.
.
.
.
0.84
−0.39
*
*
F
0.60
1.07


Ser
120
.
A
B
.
.
.
.
0.26
−0.46
*
*
F
0.53
0.95


Val
121
.
.
B
.
.
.
.
0.53
−0.36
.
*
F
0.96
1.17


Asp
122
.
.
B
.
.
T
.
−0.17
−0.56
.
*
F
1.39
0.86


Pro
123
.
.
B
.
.
T
.
0.61
0.23
*
*
F
0.57
0.55


Ala
124
.
.
B
.
.
T
.
0.16
0.34
*
.
F
0.80
1.02


Thr
125
.
.
B
.
.
T
.
−0.36
0.13
.
.
.
0.42
0.60


Phe
126
.
.
B
.
.
.
.
0.16
0.81
.
*
.
−0.16
0.32


His
127
A
.
.
.
.
.
.
0.27
0.81
*
*
.
−0.24
0.31


Gly
128
.
.
.
.
.
.
C
−0.33
0.31
*
*
.
0.18
0.43


Leu
129
.
A
.
.
.
.
C
0.22
0.51
*
*
.
−0.40
0.41


Gly
130
.
A
.
.
.
.
C
0.22
0.23
*
*
.
−0.10
0.41


Arg
131
A
A
.
.
.
.
.
0.11
0.21
*
*
.
−0.30
0.59


Leu
132
A
A
.
.
.
.
.
0.11
0.47
.
*
.
−0.60
0.59


His
133
.
A
B
.
.
.
.
−0.36
0.29
*
*
.
−0.30
0.82


Thr
134
.
A
B
.
.
.
.
0.46
0.54
*
.
.
−0.60
0.34


Leu
135
.
A
B
.
.
.
.
0.91
0.54
*
*
.
−0.38
0.70


His
136
.
A
B
.
.
.
.
0.13
−0.14
*
.
.
0.89
1.00


Leu
137
.
A
B
.
.
.
.
0.60
−0.07
.
.
.
0.96
0.37


Asp
138
.
.
.
.
T
T
.
−0.18
−0.13
*
.
.
1.98
0.45


Arg
139
.
.
.
.
T
T
.
0.13
−0.13
.
.
.
2.20
0.27


Cys
140
.
.
.
.
T
T
.
0.94
−0.23
*
.
.
1.98
0.57


Gly
141
.
.
B
.
.
T
.
0.17
−0.91
*
.
.
1.66
0.59


Leu
142
.
A
B
.
.
.
.
0.63
−0.23
*
.
.
0.74
0.25


Gln
143
.
A
B
.
.
.
.
0.42
0.20
*
.
.
−0.08
0.46


Glu
144
.
A
B
.
.
.
.
−0.03
0.06
*
*
F
−0.15
0.72


Leu
145
.
A
B
.
.
.
.
−0.18
0.06
*
.
F
−0.15
0.86


Gly
146
.
.
.
.
.
T
C
−0.53
0.06
*
*
F
0.45
0.41


Pro
147
.
.
.
.
T
T
.
0.39
0.44
*
*
F
0.35
0.20


Gly
148
.
.
.
.
.
T
C
0.04
0.44
*
.
F
0.15
0.49


Leu
149
.
.
B
.
.
T
.
−0.77
0.19
*
*
.
0.10
0.49


Phe
150
.
A
B
.
.
.
.
−0.54
0.44
*
.
.
−0.60
0.26


Arg
151
.
A
B
.
.
.
.
−0.79
0.51
*
*
.
−0.60
0.26


Gly
152
.
A
B
.
.
.
.
−1.39
0.59
*
*
.
−0.60
0.32


Leu
153
A
A
.
.
.
.
.
−1.04
0.59
*
*
.
−0.60
0.31


Ala
154
A
A
.
.
.
.
.
−0.48
0.20
*
*
.
−0.30
0.27


Ala
155
A
A
.
.
.
.
.
−0.59
0.96
*
*
.
−0.60
0.43


Leu
156
A
A
.
.
.
.
.
−0.94
1.21
.
*
.
−0.60
0.43


Gln
157
.
A
B
.
.
.
.
−1.41
1.29
.
.
.
−0.60
0.67


Tyr
158
.
A
B
.
.
.
.
−0.60
1.47
.
.
.
−0.60
0.55


Leu
159
.
A
B
.
.
.
.
−0.01
1.37
.
.
.
−0.45
1.15


Tyr
160
.
A
B
.
.
.
.
0.58
0.69
.
.
.
−0.45
1.11


Leu
161
.
.
B
.
.
T
.
0.80
0.69
.
.
.
−0.05
1.14


Gln
162
.
.
B
.
.
T
.
−0.01
0.43
.
.
.
−0.05
1.39


Asp
163
.
.
B
.
.
T
.
0.23
0.43
.
.
.
−0.20
0.73


Asn
164
.
.
B
.
.
T
.
0.46
0.07
*
.
.
0.25
1.54


Ala
165
.
A
B
.
.
.
.
−0.11
−0.11
*
.
.
0.30
0.90


Leu
166
.
A
B
.
.
.
.
0.49
0.17
*
.
.
−0.30
0.44


Gln
167
.
A
B
.
.
.
.
0.49
0.60
*
.
.
−0.32
0.43


Ala
168
.
A
B
.
.
.
.
0.49
0.20
*
.
.
0.26
0.70


Leu
169
.
.
B
.
.
T
.
0.18
−0.30
*
.
.
1.69
1.43


Pro
170
.
.
B
.
.
T
.
0.07
−0.50
*
*
F
2.12
1.19


Asp
171
.
.
.
.
T
T
.
0.99
−0.11
*
*
F
2.80
1.02


Asp
172
.
.
B
.
.
T
.
0.99
−0.61
*
.
F
2.42
2.42


Thr
173
.
.
B
.
.
.
.
0.77
−1.30
*
.
F
1.94
2.61


Phe
174
.
.
B
.
.
.
.
1.23
−1.04
*
.
F
1.87
1.29


Arg
175
.
.
B
.
.
.
.
1.44
−0.61
*
*
F
1.65
0.76


Asp
176
A
.
.
.
.
.
.
0.63
−0.21
*
*
F
1.28
0.85


Leu
177
.
.
.
.
T
.
.
0.32
−0.01
*
*
F
1.89
0.81


Gly
178
.
.
.
.
T
.
.
0.60
−0.31
*
*
F
2.10
0.60


Asn
179
.
.
.
.
.
.
C
0.49
0.19
*
*
.
0.94
0.49


Leu
180
A
.
.
B
.
.
.
−0.32
0.87
*
.
.
0.03
0.49


Thr
181
.
.
B
B
.
.
.
−1.13
0.97
*
.
.
−0.18
0.43


His
182
.
.
B
B
.
.
.
−0.36
1.23
*
.
.
−0.39
0.22


Leu
183
.
.
B
B
.
.
.
−0.36
1.33
.
*
.
−0.60
0.36


Phe
184
.
.
B
B
.
.
.
−0.36
1.07
*
.
.
−0.60
0.25


Leu
185
.
.
B
B
.
.
.
0.57
0.99
*
.
.
−0.60
0.29


His
186
.
.
B
.
.
T
.
−0.01
0.49
*
.
.
−0.20
0.69


Gly
187
.
.
.
.
T
T
.
−0.28
0.49
*
.
F
0.35
0.56


Asn
188
.
.
.
.
T
T
.
0.23
0.09
*
.
F
0.65
0.91


Arg
189
.
.
.
.
T
T
.
0.08
−0.21
.
.
F
1.25
0.90


Ile
190
.
.
.
.
.
.
C
0.68
−0.07
.
.
F
0.85
0.67


Ser
191
.
.
.
.
.
.
C
0.71
−0.07
*
.
F
0.85
0.65


Ser
192
.
.
B
.
.
.
.
1.17
−0.47
*
*
F
0.65
0.57


Val
193
.
.
B
.
.
.
.
0.58
−0.47
*
.
F
0.80
1.60


Pro
194
.
A
B
.
.
.
.
−0.23
−0.66
*
.
F
0.90
1.21


Glu
195
.
A
B
.
.
.
.
0.77
−0.26
*
.
F
0.45
0.78


Arg
196
A
A
.
.
.
.
.
0.72
−0.64
*
.
F
0.90
2.06


Ala
197
A
A
.
.
.
.
.
0.21
−0.86
*
.
.
0.75
1.32


Phe
198
A
A
.
.
.
.
.
1.03
−0.60
*
.
.
0.60
0.63


Arg
199
A
A
.
.
.
.
.
0.94
−0.10
*
.
.
0.30
0.44


Gly
200
A
A
.
.
.
.
.
0.13
0.29
*
*
.
−0.30
0.58


Leu
201
A
A
.
.
.
.
.
0.02
0.47
*
.
.
−0.60
0.55


His
202
.
A
.
.
.
.
C
0.72
−0.31
*
*
.
0.50
0.47


Ser
203
.
A
.
.
.
.
C
0.61
−0.31
*
*
.
0.50
0.93


Leu
204
A
A
.
.
.
.
.
−0.31
−0.06
*
*
.
0.30
0.93


Asp
205
A
A
.
.
.
.
.
−0.78
−0.06
*
.
.
0.30
0.56


Arg
206
A
A
.
B
.
.
.
0.00
0.13
*
.
.
−0.30
0.35


Leu
207
A
A
.
B
.
.
.
0.03
0.24
*
.
.
−0.30
0.57


Leu
208
A
A
.
B
.
.
.
0.33
−0.04
*
.
.
0.30
0.59


Leu
209
A
A
.
B
.
.
.
1.26
0.36
*
.
.
−0.30
0.49


His
210
A
.
.
.
.
T
.
0.40
0.36
*
.
.
0.25
1.16


Gln
211
A
.
.
.
.
T
.
−0.30
0.31
*
*
F
0.40
1.04


Asn
212
A
.
.
.
.
T
.
0.48
0.13
.
.
F
0.40
1.28


Arg
213
A
.
.
.
.
T
.
0.43
−0.06
.
.
.
0.85
1.28


Val
214
A
A
.
.
.
.
.
1.21
0.09
.
.
.
−0.30
0.55


Ala
215
.
A
B
.
.
.
.
1.03
0.19
.
.
.
−0.30
0.46


His
216
.
A
B
.
.
.
.
1.00
0.21
.
*
.
−0.30
0.37


Val
217
.
A
B
.
.
.
.
0.41
0.71
*
.
.
−0.60
0.67


His
218
.
A
B
.
.
.
.
−0.40
0.57
*
.
.
−0.60
0.67


Pro
219
.
A
B
.
.
.
.
0.57
0.86
*
.
.
−0.60
0.43


His
220
A
.
.
.
.
.
.
1.16
0.36
*
.
.
0.05
1.12


Ala
221
A
.
.
.
.
.
.
0.38
−0.29
*
.
.
0.65
1.38


Phe
222
A
.
.
.
.
.
.
0.89
−0.10
*
*
.
0.50
0.74


Arg
223
A
.
.
.
.
.
.
1.03
−0.10
*
*
.
0.50
0.54


Asp
224
A
.
.
.
.
.
.
0.43
−0.60
*
*
F
1.10
1.04


Leu
225
A
.
.
.
.
.
.
−0.13
−0.41
*
*
F
0.65
0.99


Gly
226
A
.
.
.
.
.
.
0.14
−0.59
*
*
F
0.95
0.50


Arg
227
A
.
.
B
.
.
.
0.03
−0.10
*
*
.
0.30
0.43


Leu
228
.
.
B
B
.
.
.
−0.32
0.59
*
*
.
−0.60
0.43


Met
229
.
.
B
B
.
.
.
−1.13
0.66
*
.
.
−0.60
0.68


Thr
230
.
.
B
B
.
.
.
−1.02
0.91
.
.
.
−0.60
0.29


Leu
231
.
.
B
B
.
.
.
−1.27
1.70
.
*
.
−0.60
0.30


Tyr
232
.
.
B
B
.
.
.
−1.38
1.51
.
.
.
−0.60
0.31


Leu
233
.
.
B
B
.
.
.
−0.57
1.30
.
.
.
−0.60
0.34


Phe
234
A
.
.
.
.
T
.
−0.78
1.21
.
.
.
−0.20
0.67


Ala
235
A
.
.
.
.
T
.
−0.77
1.21
.
.
.
−0.20
0.35


Asn
236
A
.
.
.
.
T
.
−0.54
0.84
*
.
.
−0.20
0.57


Asn
237
.
.
.
.
.
T
C
−1.11
0.66
.
.
.
0.00
0.67


Leu
238
.
A
.
.
.
.
C
−0.51
0.56
.
.
.
−0.40
0.55


Ser
239
.
A
.
.
.
.
C
−0.12
0.49
*
.
.
−0.40
0.52


Ala
240
.
A
.
.
.
.
C
0.47
0.57
*
.
.
−0.40
0.47


Leu
241
.
A
.
.
.
.
C
−0.12
0.17
*
.
.
−0.10
0.99


Pro
242
A
A
.
.
.
.
.
−0.93
−0.01
.
.
F
0.45
0.75


Thr
243
A
A
.
.
.
.
.
−0.71
0.29
.
.
F
−0.15
0.61


Glu
244
A
A
.
.
.
.
.
−0.62
0.29
.
.
F
−0.15
0.75


Ala
245
A
A
.
.
.
.
.
−0.84
0.03
*
*
.
−0.30
0.75


Leu
246
A
A
.
.
.
.
.
0.08
0.29
*
*
.
−0.30
0.43


Ala
247
A
A
.
.
.
.
.
−0.30
−0.20
*
*
.
0.30
0.48


Pro
248
A
A
.
.
.
.
.
−0.80
0.30
*
*
.
−0.30
0.48


Leu
249
A
A
.
.
.
.
.
−0.80
0.49
*
*
.
−0.60
0.48


Arg
250
A
A
.
.
.
.
.
−0.46
0.20
*
*
.
−0.30
0.83


Ala
251
A
A
.
.
.
.
.
−0.46
0.46
*
*
.
−0.60
0.84


Leu
252
A
A
.
.
.
.
.
0.24
0.71
*
*
.
−0.60
0.84


Gln
253
.
A
B
.
.
.
.
−0.36
0.03
*
*
.
−0.30
0.84


Tyr
254
.
A
B
.
.
.
.
0.46
0.71
.
*
.
−0.60
0.68


Leu
255
.
A
B
.
.
.
.
0.34
0.61
*
*
.
−0.17
1.33


Arg
256
.
A
B
.
.
.
.
0.93
−0.07
*
*
.
1.01
1.28


Leu
257
.
A
.
.
T
.
.
1.53
−0.07
*
*
.
1.69
1.32


Asn
258
.
.
.
.
T
T
.
1.24
−0.40
*
*
F
2.52
2.47


Asp
259
.
.
.
.
T
T
.
0.63
−0.17
.
*
F
2.80
1.33


Asn
260
.
.
.
.
.
T
C
0.78
0.47
.
*
F
1.42
1.19


Pro
261
.
.
.
.
T
T
.
0.67
0.36
.
*
F
1.49
0.40


Trp
262
.
.
.
.
T
.
.
0.81
−0.04
.
*
.
1.46
0.40


Val
263
.
.
B
.
.
.
.
0.92
0.53
.
*
.
0.19
0.13


Cys
264
.
.
B
.
.
T
.
0.33
0.13
.
*
.
0.72
0.17


Asp
265
.
.
B
.
.
T
.
0.44
0.20
.
*
.
1.03
0.16


Cys
266
.
.
B
.
.
T
.
0.44
−0.71
.
*
.
2.24
0.43


Arg
267
.
.
.
.
T
T
.
−0.08
−0.93
.
*
.
3.10
1.23


Ala
268
.
.
.
.
T
.
.
0.49
−0.81
.
*
.
2.44
0.61


Arg
269
.
.
.
.
.
T
C
0.57
0.10
.
*
.
1.38
1.19


Pro
270
.
.
.
.
.
T
C
0.28
0.03
.
*
.
0.92
0.61


Leu
271
A
.
.
.
.
T
.
0.13
0.94
.
*
.
0.11
0.64


Trp
272
A
.
.
.
.
T
.
0.02
1.13
*
*
.
−0.20
0.27


Ala
273
A
A
.
.
.
.
.
0.66
1.53
*
.
.
−0.60
0.30


Trp
274
A
A
.
.
.
.
.
−0.16
1.10
*
*
.
−0.60
0.73


Leu
275
A
A
.
.
.
.
.
0.17
1.20
*
*
.
−0.60
0.60


Gln
276
.
A
B
.
.
.
.
0.63
0.29
*
*
.
−0.15
1.17


Lys
277
.
A
B
.
.
.
.
0.62
0.21
.
*
F
0.34
1.10


Phe
278
.
A
.
.
T
.
.
0.91
−0.31
.
*
F
1.68
1.79


Arg
279
.
A
.
.
T
.
.
0.90
−0.61
*
*
F
2.32
1.38


Gly
280
.
.
.
.
T
T
.
1.71
−0.63
*
*
F
2.91
0.93


Ser
281
.
.
.
.
T
T
.
0.86
−0.63
*
*
F
3.40
1.85


Ser
282
.
.
.
.
.
T
C
0.60
−0.77
.
*
F
2.71
0.70


Ser
283
.
.
.
.
T
T
.
0.63
−0.34
.
*
F
2.42
1.10


Glu
284
.
.
.
.
T
.
.
0.22
−0.20
.
*
F
1.73
0.44


Val
285
.
.
B
.
.
T
.
−0.24
−0.20
.
.
F
1.19
0.44


Pro
286
.
.
.
.
T
T
.
−0.16
0.10
.
.
.
0.50
0.27


Cys
287
.
.
.
.
T
T
.
0.14
0.14
.
*
.
0.50
0.24


Ser
288
.
.
B
.
.
T
.
0.56
0.54
*
*
.
−0.20
0.56


Leu
289
.
.
B
.
.
.
.
−0.26
−0.10
*
*
F
0.65
0.71


Pro
290
.
.
B
.
.
.
.
0.01
0.16
.
*
F
0.20
1.10


Gln
291
.
A
B
.
.
.
.
−0.12
0.09
*
*
F
−0.15
0.83


Arg
292
.
A
B
.
.
.
.
0.66
0.13
*
*
F
−0.15
0.99


Leu
293
.
A
B
.
.
.
.
0.96
−0.56
*
*
F
0.90
1.26


Ala
294
.
A
B
.
.
.
.
0.96
−0.99
*
*
F
0.90
1.21


Gly
295
A
.
.
.
.
T
.
1.21
−0.70
*
*
F
1.15
0.51


Arg
296
A
.
.
.
.
T
.
1.32
−0.70
*
*
F
1.30
1.24


Asp
297
A
.
.
.
.
T
.
0.40
−1.39
*
*
F
1.30
2.40


Leu
298
A
.
.
.
.
T
.
0.62
−1.20
*
*
F
1.30
2.00


Lys
299
A
A
.
.
.
.
.
0.62
−1.13
*
*
F
0.90
1.03


Arg
300
A
A
.
.
.
.
.
0.97
−0.63
*
.
.
0.60
0.62


Leu
301
A
A
.
.
.
.
.
0.86
−0.23
*
.
.
0.45
1.22


Ala
302
A
A
.
.
.
.
.
0.04
−0.91
*
.
.
0.75
1.01


Ala
303
A
A
.
.
.
.
.
0.86
−0.23
*
.
.
0.30
0.43


Asn
304
A
A
.
.
.
.
.
0.47
0.17
*
.
.
−0.30
0.90


Asp
305
A
A
.
.
.
.
.
−0.31
−0.09
*
*
F
0.45
0.88


Leu
306
A
.
.
.
.
T
.
−0.09
−0.01
.
.
F
0.85
0.47


Gln
307
.
.
B
.
.
T
.
−0.36
−0.01
.
.
.
0.70
0.29


Gly
308
.
.
B
.
.
T
.
−0.36
0.23
.
.
.
0.10
0.13


Cys
309
.
.
B
.
.
T
.
−0.67
0.73
.
.
.
−0.20
0.16


Ala
310
.
.
B
B
.
.
.
−1.01
0.53
.
*
.
−0.60
0.13


Val
311
.
.
B
B
.
.
.
−0.41
0.56
.
.
.
−0.60
0.13


Ala
312
.
.
B
B
.
.
.
−0.66
0.56
.
.
.
−0.60
0.38


Thr
313
.
.
B
B
.
.
.
−0.34
0.74
.
.
F
−0.45
0.59


Gly
314
.
.
B
.
.
T
.
0.11
0.74
.
.
F
0.10
1.09


Pro
315
.
.
.
.
T
T
.
−0.19
0.53
.
.
F
0.50
1.67


Tyr
316
.
.
.
.
.
T
C
0.38
0.71
.
.
.
0.00
0.81


His
317
.
.
B
.
.
T
.
0.66
1.14
.
.
.
−0.20
0.86


Pro
318
.
.
B
.
.
.
.
0.62
1.20
.
*
.
−0.40
0.80


Ile
319
.
.
B
.
.
.
.
1.08
1.20
.
*
.
−0.40
0.51


Trp
320
.
.
B
.
.
T
.
0.70
0.44
.
*
.
−0.20
0.73


Thr
321
.
.
B
.
.
T
.
0.63
0.44
.
*
F
−0.05
0.48


Gly
322
.
.
.
.
.
T
C
0.67
0.50
.
*
F
0.15
0.98


Arg
323
.
.
.
.
.
T
C
0.88
−0.19
.
*
F
1.20
1.56


Ala
324
.
A
.
.
.
.
C
1.77
−1.10
.
.
F
1.10
1.87


Thr
325
.
A
.
.
.
.
C
1.84
−1.59
.
*
F
1.41
3.28


Asp
326
.
A
.
.
.
.
C
1.34
−1.59
.
*
F
1.72
2.59


Glu
327
.
A
B
.
.
.
.
1.34
−0.90
.
*
F
1.83
2.11


Glu
328
A
.
.
.
.
T
.
0.42
−0.97
.
*
F
2.54
1.45


Pro
329
.
.
.
.
T
T
.
0.80
−0.77
.
.
F
3.10
0.72


Leu
330
.
.
.
.
T
T
.
1.16
−0.34
.
.
F
2.49
0.64


Gly
331
.
.
.
.
T
T
.
0.49
−0.34
.
.
F
2.18
0.74


Leu
332
.
.
.
.
.
.
C
−0.18
0.23
.
.
.
0.72
0.26


Pro
333
.
.
.
.
T
T
.
−0.18
0.37
.
.
.
0.81
0.17


Lys
334
.
.
.
.
T
T
.
−0.18
0.09
.
.
.
0.50
0.29


Cys
335
.
.
B
.
.
T
.
0.63
0.09
.
.
.
0.10
0.55


Cys
336
.
.
B
.
.
T
.
0.39
−0.60
.
.
.
1.00
0.59


Gln
337
.
.
B
.
.
T
.
0.61
−0.53
.
.
.
1.00
0.30


Pro
338
.
.
B
.
.
T
.
0.82
−0.03
.
.
F
0.85
0.56


Asp
339
A
.
.
.
.
T
.
0.82
−0.60
.
.
F
1.30
1.75


Ala
340
A
.
.
.
.
T
.
0.90
−1.17
.
*
F
1.30
2.02


Ala
341
A
A
.
.
.
.
.
1.27
−1.07
.
*
F
0.90
1.32


Asp
342
A
A
.
.
.
.
.
0.41
−1.11
.
*
F
0.90
1.06


Lys
343
A
A
.
.
.
.
.
−0.19
−0.47
.
.
F
0.45
0.78


Ala
344
A
A
.
.
.
.
.
−0.19
−0.29
.
.
F
0.45
0.63


Ser
345
.
A
B
.
.
.
.
0.19
−0.79
.
.
.
0.60
0.66


Val
346
.
A
B
.
.
.
.
0.43
−0.36
.
*
.
0.64
0.51


Leu
347
.
A
B
.
.
.
.
0.54
0.07
.
*
.
0.38
0.50


Glu
348
.
.
B
.
.
T
.
0.29
−0.43
*
*
F
1.87
0.73


Pro
349
.
.
.
.
T
T
.
0.29
−0.39
*
.
F
2.76
1.52


Gly
350
.
.
.
.
T
T
.
0.29
−0.53
*
.
F
3.40
1.86


Arg
351
.
.
.
.
.
T
C
0.56
−0.83
*
.
F
2.86
1.44


Pro
352
.
.
.
.
.
.
C
1.02
−0.33
.
.
F
1.87
0.94


Ala
353
A
.
.
.
.
.
.
1.02
−0.33
.
.
F
1.33
0.94


Ser
354
A
.
.
.
.
T
.
0.64
−0.36
.
.
F
1.19
0.77


Ala
355
A
.
.
.
.
T
.
0.18
0.14
.
*
F
0.25
0.50


Gly
356
A
.
.
.
.
T
.
0.11
0.40
.
*
F
0.25
0.41


Asn
357
.
.
B
.
.
T
.
−0.02
−0.10
.
*
.
0.70
0.61


Ala
358
.
.
B
.
.
.
.
0.68
−0.06
.
*
F
0.65
0.60


Leu
359
.
.
B
.
.
.
.
0.12
−0.56
.
*
F
1.10
1.19


Lys
360
.
.
B
.
.
.
.
0.50
−0.34
.
*
F
0.65
0.55


Gly
361
.
.
B
.
.
.
.
0.63
−0.31
.
*
F
0.96
0.84


Arg
362
.
.
B
.
.
.
.
0.29
−0.39
.
*
F
1.42
1.58


Val
363
.
.
B
.
.
.
.
0.88
−0.64
.
*
F
1.88
0.78


Pro
364
.
.
B
.
.
T
.
1.39
−0.64
.
*
F
2.54
1.32


Pro
365
.
.
.
.
T
T
.
1.13
−0.69
.
*
F
3.10
0.90


Gly
366
.
.
.
.
T
T
.
1.27
−0.26
.
*
F
2.64
1.88


Asp
367
.
.
.
.
T
T
.
0.81
−0.47
.
.
F
2.33
1.88


Ser
368
.
.
.
.
.
.
C
1.67
−0.47
.
.
F
1.62
1.20


Pro
369
.
.
.
.
.
T
C
1.53
−0.50
.
.
F
1.81
1.95


Pro
370
.
.
.
.
T
T
.
1.44
−0.50
.
.
F
2.00
1.16


Gly
371
.
.
.
.
T
T
.
1.44
−0.11
.
.
F
2.00
1.16


Asn
372
.
.
.
.
T
T
.
1.23
−0.07
.
.
F
2.15
0.74


Gly
373
.
.
.
.
T
T
.
1.64
−0.07
.
.
F
2.45
0.74


Ser
374
.
.
.
.
.
T
C
1.82
−0.50
*
.
F
3.00
1.47


Gly
375
.
.
.
.
.
T
C
1.14
−0.43
*
.
F
2.40
1.24


Pro
376
.
.
B
.
.
T
.
1.49
−0.14
*
.
F
1.75
0.88


Arg
377
.
.
B
.
.
.
.
1.49
−0.17
*
.
F
1.40
1.05


His
378
.
.
B
.
.
.
.
1.53
−0.56
*
.
.
1.25
1.78


Ile
379
.
.
B
.
.
.
.
1.62
−0.60
*
.
.
1.20
1.54


Asn
380
.
.
B
.
.
.
.
1.27
−0.60
*
.
F
1.60
1.22


Asp
381
.
.
.
.
T
.
.
1.13
0.19
*
.
F
1.20
0.77


Ser
382
.
.
.
.
.
T
C
0.71
0.11
*
.
F
1.60
1.09


Pro
383
.
.
.
.
T
T
.
−0.07
−0.09
.
.
F
2.50
0.98


Phe
384
.
.
.
.
T
T
.
0.61
0.20
.
.
F
1.65
0.48


Gly
385
.
.
.
.
T
T
.
0.27
0.63
.
.
F
1.10
0.56


Thr
386
.
.
B
.
.
.
.
−0.03
0.67
.
.
F
0.49
0.36


Leu
387
.
.
.
.
.
T
C
−0.32
0.63
.
.
F
0.88
0.55


Pro
388
.
.
.
.
.
T
C
−0.11
0.34
.
*
F
1.17
0.57


Gly
389
.
.
.
.
T
T
.
0.38
−0.09
.
.
F
2.21
0.68


Ser
390
.
.
.
.
.
T
C
0.51
−0.14
.
*
F
2.40
1.27


Ala
391
.
.
.
.
.
.
C
0.23
−0.40
.
*
F
1.96
1.27


Glu
392
.
.
.
.
.
.
C
1.01
−0.33
.
*
F
1.72
1.30


Pro
393
.
.
B
.
.
.
.
0.56
−0.26
.
*
F
1.28
1.32


Pro
394
A
.
.
.
.
T
.
0.60
−0.07
.
*
F
1.09
0.70


Ala
395
A
.
.
.
.
T
.
0.31
−0.19
.
*
.
0.70
0.54


His
396
A
.
.
.
.
T
.
0.31
0.31
.
*
.
0.10
0.35


Cys
397
A
.
.
.
.
T
.
0.42
0.39
.
*
.
0.10
0.23


Ser
398
A
.
.
.
.
.
.
0.29
−0.04
*
.
.
0.50
0.45


Ala
399
A
.
.
.
.
.
.
−0.31
−0.11
*
.
.
0.50
0.33


Ala
400
A
.
.
.
.
.
.
0.39
0.07
*
.
.
−0.10
0.50


Arg
401
A
.
.
.
.
.
.
−0.17
−0.50
*
.
.
0.80
0.73


Gly
402
A
.
.
.
.
.
.
0.19
−0.39
*
.
.
0.50
0.73


Leu
403
.
.
B
B
.
.
.
0.60
−0.40
*
.
.
0.45
1.05


Arg
404
.
.
B
B
.
.
.
0.49
−0.90
*
.
.
0.75
1.05


Ala
405
.
.
B
B
.
.
.
0.87
−0.11
*
.
.
0.30
0.92


Thr
406
.
.
B
B
.
.
.
0.44
−0.11
*
.
F
0.60
1.72


Arg
407
.
.
B
B
.
.
.
0.49
−0.31
*
.
F
0.60
1.26


Phe
408
.
.
B
.
.
T
.
0.96
0.07
*
*
F
0.40
1.68


Pro
409
.
.
.
.
T
T
.
0.63
0.00
*
.
F
1.74
1.15


Thr
410
.
.
.
.
T
T
.
1.33
−0.06
*
*
F
1.93
0.91


Ser
411
.
.
.
.
.
T
C
1.76
−0.06
*
*
F
2.22
2.05


Gly
412
.
.
.
.
.
T
C
1.76
−0.84
*
*
F
2.86
2.60


Pro
413
.
.
.
.
T
T
.
2.24
−1.27
.
.
F
3.40
3.53


Arg
414
.
.
.
.
T
T
.
2.11
−1.33
.
.
F
3.06
4.08


Arg
415
.
.
.
.
T
T
.
1.76
−1.29
.
.
F
2.72
4.08


Arg
416
.
.
B
.
.
T
.
1.76
−1.14
.
.
F
1.98
1.41


Pro
417
.
.
.
.
T
T
.
2.21
−1.19
.
.
F
2.23
0.97


Gly
418
.
.
.
.
T
T
.
2.47
−1.19
.
.
F
2.23
0.97


Cys
419
.
.
.
.
T
T
.
2.36
−1.19
.
.
F
2.57
0.99


Ser
420
.
.
.
.
T
.
.
2.36
−0.79
.
.
F
2.86
1.03


Arg
421
.
.
.
.
T
T
.
1.93
−1.21
.
*
F
3.40
2.03


Lys
422
.
.
.
.
T
T
.
2.26
−1.16
.
*
F
3.06
5.47


Asn
423
.
.
.
.
T
T
.
2.30
−1.73
.
*
F
2.98
7.99


Arg
424
.
.
.
.
T
T
.
2.93
−1.73
.
*
F
2.90
5.47


Thr
425
.
.
.
.
T
.
.
2.57
−1.23
.
*
F
2.62
3.72


Arg
426
.
.
.
.
T
T
.
2.57
−0.66
.
*
F
2.74
1.24


Ser
427
.
.
B
.
.
T
.
1.71
−1.06
*
*
F
2.60
1.24


His
428
.
.
B
.
.
T
.
1.37
−0.37
*
*
.
1.74
0.71


Cys
429
.
.
B
.
.
T
.
1.26
−0.43
*
*
.
1.48
0.36


Arg
430
.
.
B
.
.
.
.
0.98
−0.03
*
*
.
1.02
0.46


Leu
431
.
.
B
.
.
.
.
0.52
0.09
*
*
.
0.16
0.34


Gly
432
.
.
B
.
.
.
.
0.52
0.01
.
*
.
−0.10
0.63


Gln
433
.
.
B
.
.
.
.
0.21
−0.17
*
*
F
0.65
0.43


Ala
434
.
.
B
.
.
.
.
0.53
0.26
*
.
F
0.05
0.52


Gly
435
.
.
.
.
.
T
C
0.08
0.00
*
.
F
1.05
0.52


Ser
436
.
.
.
.
.
T
C
0.54
0.00
.
.
F
1.05
0.30


Gly
437
.
.
.
.
.
T
C
0.58
0.03
.
.
F
0.45
0.29


Gly
438
.
.
.
.
.
T
C
0.23
0.01
.
.
F
0.71
0.43


Gly
439
.
.
.
.
.
T
C
0.82
0.01
.
.
F
0.97
0.31


Gly
440
.
.
.
.
.
T
C
0.87
−0.37
.
.
F
1.83
0.53


Thr
441
.
.
.
.
.
T
C
1.17
−0.41
.
.
F
2.09
0.72


Gly
442
.
.
B
.
.
T
.
1.17
−0.84
.
.
F
2.60
1.26


Asp
443
.
.
B
.
.
T
.
1.21
−0.84
.
*
F
2.34
1.26


Ser
444
.
.
B
.
.
T
.
1.21
−0.89
.
.
F
2.29
1.17


Glu
445
.
.
B
.
.
T
.
0.97
−0.94
.
*
F
2.24
1.17


Gly
446
.
.
.
.
T
T
.
0.47
−0.87
.
*
F
2.44
0.71


Ser
447
.
.
.
.
T
.
.
0.60
−0.19
.
*
F
1.89
0.43


Gly
448
.
.
.
.
T
.
.
0.30
−0.14
.
.
F
2.10
0.39


Ala
449
.
.
.
.
.
.
C
−0.21
0.24
.
.
F
1.09
0.52


Leu
450
.
.
B
.
.
T
.
−0.52
0.50
.
.
F
0.58
0.32


Pro
451
.
.
B
.
.
T
.
−0.84
0.60
.
.
F
0.37
0.47


Ser
452
.
.
B
.
.
T
.
−0.84
0.74
.
.
F
0.16
0.25


Leu
453
.
.
B
.
.
T
.
−1.31
0.63
.
.
.
−0.20
0.41


Thr
454
.
.
B
.
.
.
.
−1.03
0.63
.
.
.
−0.40
0.22


Cys
455
.
.
B
.
.
.
.
−0.43
0.69
*
.
.
−0.40
0.23


Ser
456
.
.
B
.
.
.
.
−1.03
0.73
.
.
.
−0.40
0.44


Leu
457
.
.
B
.
.
.
.
−1.08
0.73
.
.
.
−0.40
0.25


Thr
458
.
.
B
.
.
T
.
−1.08
0.67
.
.
F
−0.05
0.46


Pro
459
.
.
B
.
.
T
.
−1.36
0.79
.
.
F
−0.05
0.28


Leu
460
.
.
B
.
.
T
.
−1.50
0.90
.
.
.
−0.20
0.35


Gly
461
.
.
B
.
.
T
.
−2.06
0.90
.
.
.
−0.20
0.20


Leu
462
.
.
B
B
.
.
.
−2.06
1.06
.
.
.
−0.60
0.10


Ala
463
.
.
B
B
.
.
.
−2.03
1.31
.
.
.
−0.60
0.10


Leu
464
.
.
B
B
.
.
.
−2.13
1.54
.
.
.
−0.60
0.10


Val
465
.
.
B
B
.
.
.
−2.18
1.60
.
.
.
−0.60
0.18


Leu
466
.
.
B
B
.
.
.
−2.64
1.56
.
.
.
−0.60
0.13


Trp
467
.
.
B
B
.
.
.
−2.18
1.74
.
.
.
−0.60
0.13


Thr
468
.
.
B
B
.
.
.
−1.80
1.49
.
.
.
−0.60
0.17


Val
469
.
.
B
B
.
.
.
−1.66
1.27
.
.
.
−0.60
0.33


Leu
470
.
.
B
B
.
.
.
−1.19
1.16
.
.
.
−0.60
0.17


Gly
471
.
.
.
B
.
.
C
−0.77
0.67
.
.
.
−0.40
0.15


Pro
472
.
.
.
.
T
.
.
−0.87
0.61
.
.
.
0.00
0.25


Cys
473
.
.
.
.
T
.
.
−0.94
0.40
.
.
.
0.30
0.39































TABLE 10





Res
Position
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV






























Met
1
.
.
B
.
.
.
.
0.59
−0.19
.
*
.
0.86
1.62


Arg
2
.
.
B
.
.
.
.
0.77
−0.19
.
*
.
1.07
1.25


Thr
3
.
.
.
.
.
T
C
0.34
−0.19
.
*
.
1.68
1.52


Pro
4
.
.
.
.
.
T
C
0.52
0.07
.
*
.
1.29
1.26


Gly
5
.
.
.
.
.
T
C
0.06
−0.11
.
*
F
2.10
1.00


Pro
6
.
.
.
.
.
T
C
−0.16
0.53
.
*
F
0.99
0.51


Leu
7
.
A
B
.
.
.
.
−1.08
0.73
.
*
F
0.18
0.27


Pro
8
.
A
B
.
.
.
.
−1.58
0.99
.
.
.
−0.18
0.23


Val
9
.
A
B
.
.
.
.
−2.18
1.24
.
.
.
−0.39
0.12


Leu
10
.
A
B
.
.
.
.
−2.64
1.50
.
.
.
−0.60
0.12


Leu
11
.
A
B
.
.
.
.
−3.02
1.50
.
.
.
−0.60
0.06


Leu
12
.
A
B
.
.
.
.
−2.56
1.57
.
.
.
−0.60
0.09


Leu
13
.
A
B
.
.
.
.
−2.93
1.36
.
.
.
−0.60
0.11


Leu
14
.
A
B
.
.
.
.
−2.29
1.17
.
.
.
−0.60
0.13


Ala
15
.
A
B
.
.
.
.
−2.07
0.91
.
.
.
−0.60
0.24


Gly
16
.
A
B
.
.
.
.
−1.84
0.73
.
.
.
−0.60
0.30


Ala
17
.
.
B
.
.
.
.
−0.92
0.54
.
.
.
−0.40
0.37


Pro
18
.
.
B
.
.
.
.
−0.32
−0.14
.
.
.
0.74
0.71


Ala
19
.
.
.
.
T
.
.
0.18
−0.21
.
.
.
1.53
1.11


Ala
20
.
.
B
.
.
.
.
0.56
−0.16
.
.
.
1.37
1.58


Arg
21
.
.
B
.
.
.
.
0.69
−0.23
.
.
F
1.76
1.58


Pro
22
.
.
.
.
T
.
.
0.97
−0.23
.
.
F
2.40
2.42


Thr
23
.
.
.
.
.
.
C
0.51
−0.24
.
.
F
1.96
3.46


Pro
24
.
.
.
.
.
T
C
0.86
−0.17
.
.
F
1.77
0.95


Pro
25
.
.
.
.
T
T
.
1.14
0.59
.
*
F
0.83
0.96


Thr
26
.
.
.
.
T
T
.
1.14
0.54
.
*
F
0.59
0.89


Cys
27
.
.
B
.
.
T
.
0.76
0.06
.
*
.
0.25
1.13


Tyr
28
.
.
B
.
.
.
.
1.18
0.24
.
*
.
−0.10
0.72


Ser
29
.
A
B
.
.
.
.
0.80
−0.19
.
*
.
0.30
0.98


Arg
30
.
A
B
.
.
.
.
0.20
−0.17
.
*
.
0.45
1.85


Met
31
.
A
B
.
.
.
.
0.21
−0.06
.
*
.
0.30
0.97


Arg
32
.
A
B
.
.
.
.
0.88
−0.43
.
*
.
0.30
0.97


Ala
33
.
A
B
.
.
.
.
1.12
−0.41
*
*
.
0.30
0.86


Leu
34
.
A
.
.
.
.
C
0.53
−0.41
*
*
.
0.65
1.50


Ser
35
.
A
B
.
.
.
.
0.11
−0.34
*
*
F
0.45
0.54


Gln
36
.
A
B
.
.
.
.
0.82
0.14
*
.
F
−0.15
0.77


Glu
37
.
A
B
.
.
.
.
0.71
−0.36
*
.
F
0.60
1.83


Ile
38
.
A
B
.
.
.
.
0.60
−1.04
*
.
F
0.90
2.28


Thr
39
.
A
B
.
.
.
.
1.41
−0.64
*
*
F
0.90
1.14


Arg
40
.
A
B
.
.
.
.
0.90
−0.64
*
.
F
0.90
1.06


Asp
41
.
A
.
.
T
.
.
0.09
0.04
*
.
F
0.40
1.24


Phe
42
.
.
B
B
.
.
.
0.09
0.04
*
.
.
−0.30
0.71


Asn
43
.
.
B
B
.
.
.
0.12
−0.04
*
.
.
0.30
0.63


Leu
44
.
.
.
B
.
.
C
0.13
0.60
.
.
.
−0.40
0.28


Leu
45
.
.
B
B
.
.
.
0.02
0.99
.
.
.
−0.60
0.43


Gln
46
.
.
B
B
.
.
.
−0.19
0.20
.
.
.
0.04
0.47


Val
47
.
.
.
B
.
.
C
0.21
0.23
.
.
.
0.58
0.87


Ser
48
.
.
.
B
.
.
C
0.21
−0.07
.
.
F
1.82
1.42


Glu
49
.
.
.
.
.
T
C
0.81
−0.76
.
.
F
2.86
1.42


Pro
50
.
.
.
.
T
T
.
0.96
−0.73
.
.
F
3.40
2.95


Ser
51
.
.
.
.
T
T
.
0.10
−0.80
*
*
F
3.06
1.18


Glu
52
.
.
.
.
.
T
C
1.07
−0.54
*
*
F
2.37
0.51


Pro
53
.
.
.
B
T
.
.
1.12
−0.54
*
.
F
1.83
0.64


Cys
54
.
.
B
B
.
.
.
0.31
−0.21
*
.
.
0.64
0.75


Val
55
.
.
B
B
.
.
.
0.31
0.09
*
*
.
−0.30
0.36


Arg
56
.
.
B
B
.
.
.
0.72
0.51
*
*
.
−0.60
0.36


Tyr
57
.
.
B
B
.
.
.
−0.09
0.09
*
*
.
−0.15
1.30


Leu
58
.
.
B
B
.
.
.
−0.12
0.20
*
*
.
−0.15
1.45


Pro
59
.
.
B
B
.
.
.
−0.27
0.31
*
*
.
−0.15
1.16


Arg
60
.
.
B
B
.
.
.
0.59
1.00
*
*
.
−0.60
0.61


Leu
61
.
.
B
B
.
.
.
−0.41
0.24
*
*
.
−0.15
1.24


Tyr
62
.
.
B
B
.
.
.
−0.20
0.24
*
*
.
−0.30
0.56


Leu
63
.
.
B
B
.
.
.
0.61
0.31
*
*
.
−0.30
0.39


Asp
64
.
.
B
B
.
.
.
0.58
0.71
*
*
.
−0.60
0.76


Ile
65
.
.
B
B
.
.
.
−0.20
0.79
*
*
.
−0.60
0.76


His
66
.
.
B
.
.
T
.
−0.24
0.60
.
*
.
−0.20
0.49


Asn
67
.
.
B
.
.
T
.
−0.81
0.56
.
*
.
−0.20
0.22


Tyr
68
.
.
B
.
.
T
.
0.00
1.24
.
*
.
−0.20
0.26


Cys
69
.
.
B
.
.
T
.
0.04
0.56
.
.
.
−0.20
0.32


Val
70
.
A
B
B
.
.
.
0.12
0.06
.
*
.
−0.30
0.39


Leu
71
.
A
B
B
.
.
.
0.27
0.34
*
*
.
−0.30
0.21


Asp
72
.
A
B
B
.
.
.
0.27
−0.41
*
.
F
0.45
0.76


Lys
73
.
A
B
.
.
.
.
−0.19
−0.99
*
*
F
0.90
1.70


Leu
74
.
A
B
B
.
.
.
−0.38
−0.84
*
.
F
0.90
1.79


Arg
75
.
A
B
B
.
.
.
−0.11
−0.89
*
.
F
0.75
0.79


Asp
76
.
A
B
B
.
.
.
0.40
−0.39
*
.
.
0.30
0.40


Phe
77
.
A
B
B
.
.
.
0.19
0.00
*
.
.
0.30
0.65


Val
78
.
A
B
B
.
.
.
−0.07
−0.26
*
*
.
0.30
0.52


Ala
79
.
A
B
B
.
.
.
0.08
0.17
*
.
.
−0.30
0.48


Ser
80
.
A
.
B
.
.
C
−0.32
0.74
.
.
.
−0.40
0.30


Pro
81
.
.
.
.
.
T
C
−0.28
0.87
.
*
F
0.15
0.42


Pro
82
.
.
.
.
T
T
.
−0.43
0.23
.
.
F
0.65
0.83


Cys
83
.
.
.
.
T
T
.
−0.17
0.37
.
.
.
0.50
0.46


Trp
84
.
.
.
.
T
T
.
0.42
0.49
.
.
.
0.20
0.30


Lys
85
.
A
B
.
.
.
.
−0.13
0.46
.
.
.
−0.60
0.34


Val
86
.
A
B
.
.
.
.
0.08
0.67
.
.
.
−0.60
0.46


Ala
87
.
A
B
.
.
.
.
−0.01
0.10
.
.
.
−0.30
0.74


Gln
88
.
A
B
.
.
.
.
−0.16
−0.43
.
.
.
0.30
0.49


Val
89
.
A
B
.
.
.
.
0.18
0.26
.
.
.
−0.30
0.55


Asp
90
.
A
B
.
.
.
.
0.13
−0.39
.
.
F
0.60
1.09


Ser
91
.
A
B
.
.
.
.
1.03
−0.89
.
.
F
0.90
1.05


Leu
92
A
A
.
.
.
.
.
1.03
−1.29
*
*
F
0.90
2.83


Lys
93
A
A
.
.
.
.
.
1.14
−1.43
*
*
F
0.90
1.71


Asp
94
.
A
.
.
T
.
.
2.04
−1.43
*
.
F
1.30
2.50


Lys
95
A
A
.
.
.
.
.
1.23
−1.81
*
*
F
0.90
6.06


Ala
96
A
A
.
.
.
.
.
1.29
−1.81
*
*
F
0.90
2.50


Arg
97
.
A
B
.
.
.
.
1.79
−1.06
*
*
F
0.90
2.34


Lys
98
.
A
B
.
.
.
.
0.86
−0.57
*
*
F
0.90
1.69


Leu
99
.
A
B
.
.
.
.
0.26
0.11
*
.
.
−0.15
1.17


Tyr
100
.
A
B
.
.
.
.
0.21
0.23
*
.
.
−0.30
0.59


Thr
101
.
.
B
B
.
.
.
0.50
0.63
*
.
.
−0.60
0.48


Ile
102
.
.
B
B
.
.
.
−0.31
1.01
*
.
.
−0.60
0.77


Met
103
.
.
B
B
.
.
.
−1.02
1.11
*
.
.
−0.60
0.43


Asn
104
.
.
B
.
.
T
.
−0.10
0.93
*
.
.
0.04
0.16


Ser
105
.
.
B
.
.
T
.
0.26
0.44
*
.
.
0.28
0.44


Phe
106
.
.
B
.
.
T
.
0.57
−0.24
*
.
.
1.42
0.88


Cys
107
.
.
B
.
.
T
.
0.64
−0.86
.
.
.
1.96
0.91


Arg
108
.
.
.
.
T
.
.
0.39
−0.57
.
.
.
2.40
0.56


Arg
109
.
.
B
B
.
.
.
−0.31
−0.31
*
.
F
1.41
0.48


Asp
110
.
.
B
B
.
.
.
−0.82
−0.31
.
.
F
1.17
0.78


Leu
111
.
.
B
B
.
.
.
−0.93
−0.20
*
.
.
0.78
0.33


Val
112
.
.
B
B
.
.
.
−0.27
0.49
*
.
.
−0.36
0.14


Phe
113
.
.
B
B
.
.
.
−0.38
0.49
*
.
.
−0.60
0.14


Leu
114
.
.
B
B
.
.
.
−1.16
0.49
*
.
.
−0.60
0.28


Leu
115
.
.
B
B
.
.
.
−1.16
0.37
.
.
.
−0.02
0.20


Asp
116
.
.
.
.
T
T
.
−0.93
0.13
.
.
F
1.21
0.37


Asp
117
.
.
.
.
T
T
.
−0.89
−0.16
.
.
F
2.09
0.46


Cys
118
.
.
.
.
T
T
.
−0.19
−0.16
.
.
.
2.22
0.46


Asn
119
.
.
.
.
T
T
.
0.38
−0.84
.
.
.
2.80
0.48


Ala
120
.
A
B
.
.
.
.
0.98
−0.09
.
.
.
1.42
0.45


Leu
121
.
A
B
.
.
.
.
0.09
0.34
.
.
.
0.69
1.29


Glu
122
.
A
B
.
.
.
.
−0.12
0.46
.
*
.
−0.04
0.56


Tyr
123
.
A
B
.
.
.
.
−0.31
0.49
.
*
.
−0.32
0.86


Pro
124
.
.
B
B
.
.
.
−0.62
0.63
.
*
.
−0.60
0.77


Ile
125
.
.
B
B
.
.
.
−0.34
0.43
.
*
.
−0.60
0.64


Pro
126
.
.
B
B
.
.
.
−0.39
0.91
.
*
.
−0.60
0.59


Val
127
.
.
B
B
.
.
.
−1.20
0.80
.
.
.
−0.60
0.29


Thr
128
.
.
B
B
.
.
.
−1.17
1.06
.
.
.
−0.60
0.34


Thr
129
.
.
B
B
.
.
.
−0.96
0.80
.
.
F
−0.11
0.34


Val
130
.
.
B
B
.
.
.
0.04
0.37
.
.
F
0.53
0.75


Leu
131
.
.
B
.
.
T
.
0.26
−0.27
.
*
F
2.02
1.02


Pro
132
.
.
B
.
.
T
.
1.22
−0.36
.
*
F
2.36
1.23


Asp
133
.
.
.
.
T
T
.
1.14
−0.84
.
*
F
3.40
3.24


Arg
134
.
.
B
.
.
T
.
1.07
−1.06
.
*
.
2.51
5.03


Gln
135
.
.
B
.
.
.
.
1.53
−1.31
.
*
.
1.97
4.16


Arg
136
.
.
B
.
.
.
.
1.96
−1.31
.
*
.
1.63
3.18































TABLE 11





Res
Position
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV






























Met
1
A
A
.
.
.
.
.
−0.68
0.09
.
.
.
−0.30
0.62


Lys
2
A
A
.
.
.
.
.
−1.10
0.23
.
.
.
−0.30
0.26


Ala
3
A
A
.
.
.
.
.
−1.52
0.49
*
.
.
−0.60
0.17


Leu
4
A
A
.
.
.
.
.
−1.94
0.74
.
.
.
−0.60
0.14


Cys
5
A
A
.
.
.
.
.
−2.37
0.81
.
.
.
−0.60
0.06


Leu
6
A
A
.
.
.
.
.
−1.98
1.50
*
.
.
−0.60
0.05


Leu
7
.
A
B
.
.
.
.
−2.88
1.43
*
.
.
−0.60
0.09


Leu
8
.
A
B
.
.
.
.
−3.10
1.39
.
.
.
−0.60
0.12


Leu
9
.
A
B
.
.
.
.
−2.63
1.50
.
.
.
−0.60
0.12


Pro
10
.
A
B
.
.
.
.
−2.78
1.24
.
.
.
−0.60
0.15


Val
11
.
A
B
.
.
.
.
−2.78
1.24
.
.
.
−0.60
0.15


Leu
12
.
A
B
.
.
.
.
−2.82
1.24
.
.
.
−0.60
0.15


Gly
13
.
A
B
.
.
.
.
−2.31
1.20
*
.
.
−0.60
0.07


Leu
14
.
A
B
.
.
.
.
−1.80
1.16
.
.
.
−0.60
0.13


Leu
15
.
A
B
.
.
.
.
−1.54
0.90
.
.
.
−0.60
0.21


Val
16
.
.
B
.
.
T
.
−1.00
0.21
.
.
.
0.10
0.42


Ser
17
.
.
B
.
.
T
.
−1.00
0.27
.
.
F
0.25
0.73


Ser
18
A
.
.
.
.
T
.
−1.32
0.27
.
.
F
0.25
0.73


Lys
19
A
.
.
.
.
T
.
−0.81
0.16
.
.
F
0.25
0.53


Thr
20
A
.
.
B
.
.
.
−0.60
−0.10
.
.
F
0.45
0.53


Leu
21
A
.
.
B
.
.
.
0.26
0.13
*
.
.
−0.30
0.39


Cys
22
A
.
.
B
.
.
.
0.56
−0.26
*
.
.
0.30
0.34


Ser
23
A
.
.
B
.
.
.
0.27
−0.26
*
.
.
0.30
0.40


Met
24
A
A
.
.
.
.
.
−0.67
−0.24
*
.
.
0.30
0.49


Glu
25
A
A
.
.
.
.
.
−0.36
−0.24
*
.
.
0.30
0.65


Glu
26
A
A
.
.
.
.
.
0.46
−0.41
*
*
.
0.30
0.77


Ala
27
A
A
.
.
.
.
.
1.23
−0.80
*
*
.
0.75
1.36


Ile
28
A
A
.
.
.
.
.
0.64
−1.41
*
*
.
0.75
1.53


Asn
29
A
A
.
.
.
.
.
1.24
−0.73
*
*
.
0.60
0.62


Glu
30
A
A
.
.
.
.
.
1.24
−0.33
*
*
F
0.60
1.06


Arg
31
A
A
.
.
.
.
.
0.39
−0.83
*
*
F
0.90
2.63


Ile
32
A
A
.
.
.
.
.
0.39
−0.87
*
*
F
0.90
1.21


Gln
33
A
A
.
.
.
.
.
0.93
−0.77
*
*
F
0.75
0.71


Glu
34
A
A
.
.
.
.
.
0.63
−0.34
*
*
.
0.30
0.36


Val
35
A
A
.
.
.
.
.
−0.18
0.04
*
.
.
−0.30
0.68


Ala
36
A
A
.
.
.
.
.
−1.18
0.04
*
*
.
−0.30
0.33


Gly
37
A
A
.
B
.
.
.
−0.99
0.33
*
*
.
−0.30
0.13


Ser
38
A
A
.
B
.
.
.
−0.88
1.11
*
*
.
−0.60
0.15


Leu
39
A
A
.
B
.
.
.
−1.47
0.47
*
*
.
−0.60
0.30


Ile
40
.
A
B
B
.
.
.
−1.50
0.47
*
*
.
−0.60
0.30


Phe
41
.
A
B
B
.
.
.
−1.21
0.73
*
.
.
−0.60
0.16


Arg
42
.
A
B
B
.
.
.
−1.17
0.73
*
*
.
−0.60
0.26


Ala
43
.
A
B
B
.
.
.
−1.76
0.43
*
*
.
−0.60
0.49


Ile
44
.
A
B
B
.
.
.
−1.29
0.43
*
*
.
−0.60
0.40


Ser
45
.
A
.
B
.
.
C
−1.21
0.07
*
*
.
−0.10
0.20


Ser
46
.
.
.
B
.
.
C
−0.51
0.76
*
*
.
−0.40
0.17


Ile
47
.
.
.
B
T
.
.
−1.29
0.26
*
*
.
0.10
0.41


Gly
48
.
A
B
.
.
.
.
−0.70
0.14
.
.
.
−0.30
0.16


Leu
49
.
A
.
.
.
.
C
−0.11
0.16
*
.
.
−0.10
0.21


Glu
50
.
A
B
.
.
.
.
−0.67
0.16
.
.
.
−0.30
0.40


Cys
51
.
A
B
B
.
.
.
−0.68
0.11
.
.
.
−0.30
0.30


Gln
52
.
.
B
B
.
.
.
−0.09
0.17
*
*
.
−0.30
0.53


Ser
53
.
.
B
B
.
.
.
0.37
−0.13
*
*
F
0.45
0.41


Val
54
.
.
B
B
.
.
.
0.83
−0.13
.
*
F
0.91
1.50


Thr
55
.
.
B
B
.
.
.
0.83
−0.27
.
*
F
1.07
0.85


Ser
56
.
.
.
.
T
T
.
0.69
−0.67
.
*
F
2.63
1.07


Arg
57
.
.
.
.
T
T
.
0.10
−0.37
.
*
F
2.64
1.18


Gly
58
.
.
.
.
T
T
.
0.09
−0.51
.
*
F
3.10
0.83


Asp
59
.
.
.
.
T
T
.
0.28
−0.51
.
*
F
2.79
0.89


Leu
60
.
.
B
.
.
.
.
0.38
−0.33
.
*
F
1.58
0.24


Ala
61
.
.
B
.
.
.
.
0.79
0.10
*
*
.
0.52
0.38


Thr
62
.
.
B
.
.
.
.
0.33
−0.33
*
*
.
0.81
0.45


Cys
63
.
.
B
.
.
T
.
−0.02
0.10
*
*
.
0.10
0.54


Pro
64
.
.
.
.
T
T
.
−0.61
0.20
*
.
F
0.65
0.46


Arg
65
.
.
.
.
T
T
.
−0.66
0.20
*
.
F
0.65
0.32


Gly
66
.
.
.
.
T
T
.
−0.38
0.36
.
.
.
0.50
0.45


Phe
67
.
.
B
B
.
.
.
−0.41
0.27
*
.
.
−0.30
0.42


Ala
68
.
.
B
B
.
.
.
−0.41
0.27
*
.
.
−0.30
0.21


Val
69
.
.
B
B
.
.
.
−0.51
0.84
*
.
.
−0.60
0.11


Thr
70
.
.
B
B
.
.
.
−1.29
0.90
*
.
.
−0.60
0.19


Gly
71
.
.
B
B
.
.
.
−1.29
0.69
.
.
.
−0.60
0.10


Cys
72
.
.
.
.
T
T
.
−0.89
0.61
.
.
.
0.20
0.13


Thr
73
.
.
.
.
T
T
.
−0.89
0.36
.
.
.
0.50
0.12


Cys
74
.
.
.
.
T
T
.
−0.70
0.37
.
.
.
0.50
0.13


Gly
75
.
.
.
.
T
T
.
−0.73
0.51
.
.
.
0.20
0.13


Ser
76
.
.
.
.
T
.
.
−0.69
0.37
.
.
.
0.30
0.09


Ala
77
.
.
.
.
T
.
.
−0.31
0.27
.
.
.
0.30
0.22


Cys
78
.
.
.
.
T
T
.
0.00
0.61
.
*
.
0.20
0.23


Gly
79
.
.
.
.
T
T
.
−0.19
0.19
.
*
.
0.50
0.29


Ser
80
.
.
.
.
T
T
.
0.27
0.44
.
*
.
0.20
0.21


Trp
81
.
.
B
.
.
T
.
−0.02
−0.06
.
*
.
0.70
0.78


Asp
82
.
A
B
B
.
.
.
0.57
−0.13
.
*
.
0.30
0.79


Val
83
A
A
.
B
.
.
.
0.92
−0.56
.
*
.
0.75
1.02


Arg
84
A
A
.
B
.
.
.
0.96
−0.46
.
*
.
0.45
1.41


Ala
85
A
A
.
.
.
.
.
0.59
−0.89
.
*
F
0.90
1.21


Glu
86
.
A
.
.
T
.
.
0.84
−0.31
.
*
F
0.85
0.88


Thr
87
.
A
.
.
T
.
.
0.18
−0.46
.
*
F
0.85
0.61


Thr
88
.
A
.
.
T
.
.
1.03
0.11
.
*
.
0.10
0.32


Cys
89
.
.
.
.
T
T
.
0.26
0.01
.
*
.
0.50
0.32


His
90
.
.
.
.
T
T
.
0.26
0.59
.
.
.
0.20
0.12


Cys
91
.
.
B
.
.
T
.
−0.09
0.60
.
.
.
−0.20
0.08


Gln
92
.
.
B
.
.
T
.
−0.38
0.54
.
.
.
−0.20
0.16


Cys
93
.
.
.
.
T
.
.
−0.07
0.59
.
.
.
0.00
0.11


Ala
94
.
.
.
.
T
.
.
0.31
0.09
.
.
.
0.30
0.35


Gly
95
.
.
.
.
T
T
.
0.03
0.43
.
.
.
0.20
0.21


Met
96
.
.
.
.
T
T
.
0.36
0.51
.
.
.
0.42
0.57


Asp
97
.
.
.
.
T
T
.
−0.23
0.37
.
.
.
0.94
0.56


Trp
98
.
.
.
.
T
T
.
0.54
0.37
.
.
.
1.16
0.57


Thr
99
.
.
.
.
T
.
.
0.47
−0.06
.
.
.
1.93
1.14


Gly
100
.
.
.
.
T
T
.
0.14
−0.10
*
.
.
2.20
0.37


Ala
101
.
.
.
.
T
T
.
0.86
0.47
*
.
.
1.08
0.19


Arg
102
.
.
.
.
T
T
.
0.00
−0.44
*
.
.
1.76
0.25


Cys
103
.
.
.
.
T
T
.
0.29
−0.29
*
.
.
1.54
0.19


Cys
104
.
.
.
.
T
.
.
0.39
−0.31
*
.
.
1.12
0.32


Arg
105
.
.
B
.
.
.
.
0.34
−0.39
*
.
.
0.50
0.26


Val
106
.
.
B
.
.
.
.
0.54
0.04
*
.
.
−0.10
0.61


Gln
107
.
.
B
.
.
.
.
0.04
−0.10
*
.
.
0.65
1.46


Pro
108
.
.
B
.
.
.
.
0.32
−0.24
.
*
.
0.50
0.95































TABLE 12





Res
Position
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV






























Met
1
.
.
.
.
.
.
C
0.09
0.10
.
.
.
0.46
0.66


Ser
2
.
.
.
.
.
T
C
0.48
0.16
.
.
.
0.84
0.74


Ser
3
.
.
.
.
.
T
C
0.06
−0.27
*
.
.
1.77
1.01


Gly
4
.
.
.
.
.
T
C
−0.37
−0.01
.
.
.
1.80
0.84


Thr
5
.
.
.
.
.
T
C
−0.27
0.06
.
.
F
1.17
0.52


Glu
6
A
.
.
.
.
.
.
0.12
0.59
*
.
F
0.29
0.41


Leu
7
A
.
.
.
.
.
.
0.08
0.63
.
.
.
−0.04
0.63


Leu
8
A
.
.
.
.
.
.
−0.21
0.63
.
.
.
−0.22
0.43


Trp
9
A
.
.
.
.
T
.
−0.46
0.64
.
.
.
−0.20
0.25


Pro
10
A
.
.
.
.
T
.
−0.96
1.14
.
.
.
−0.20
0.31


Gly
11
A
.
.
.
.
T
.
−1.77
1.14
.
.
.
−0.20
0.31


Ala
12
A
.
.
.
.
T
.
−1.81
1.14
.
.
.
−0.20
0.24


Ala
13
A
.
.
B
.
.
.
−1.81
0.87
.
.
.
−0.60
0.12


Leu
14
A
.
.
B
.
.
.
−2.33
1.13
.
.
.
−0.60
0.10


Leu
15
A
.
.
B
.
.
.
−2.47
1.39
.
.
.
−0.60
0.08


Val
16
A
.
.
B
.
.
.
−2.98
1.31
.
.
.
−0.60
0.08


Leu
17
A
.
.
B
.
.
.
−2.98
1.46
.
.
.
−0.60
0.07


Leu
18
A
.
.
B
.
.
.
−2.98
1.27
.
.
.
−0.60
0.09


Gly
19
A
.
.
B
.
.
.
−2.47
1.09
.
*
.
−0.60
0.12


Val
20
A
.
.
B
.
.
.
−2.47
0.83
.
.
.
−0.60
0.19


Ala
21
A
.
.
B
.
.
.
−2.28
0.83
.
*
.
−0.60
0.19


Ala
22
A
.
.
B
.
.
.
−2.32
0.71
*
*
.
−0.60
0.10


Ser
23
A
.
.
B
.
.
.
−1.40
0.93
*
*
.
−0.60
0.10


Leu
24
A
.
.
B
.
.
.
−1.72
0.29
.
*
.
−0.30
0.20


Cys
25
.
.
B
B
.
.
.
−1.17
0.36
*
*
.
−0.30
0.11


Val
26
.
.
B
B
.
.
.
−0.47
0.24
*
*
.
−0.30
0.11


Arg
27
.
.
.
B
T
.
.
−0.09
−0.14
*
*
.
1.04
0.25


Cys
28
.
.
.
B
T
.
.
−0.13
−0.40
*
*
.
1.38
0.73


Ser
29
.
.
.
B
T
.
.
0.09
−0.54
*
*
F
2.17
0.97


Arg
30
.
.
.
.
.
T
C
0.80
−0.69
*
.
F
2.71
0.50


Pro
31
.
.
.
.
T
T
.
1.77
−0.69
*
.
F
3.40
1.86


Gly
32
.
.
.
.
T
T
.
1.36
−1.26
*
.
F
3.06
2.72


Ala
33
.
.
.
.
.
T
C
2.02
−1.26
*
.
F
2.52
1.86


Lys
34
.
A
.
.
.
.
C
2.37
−1.26
*
*
F
1.78
2.08


Arg
35
A
A
.
.
.
.
.
1.37
−1.69
*
*
F
1.24
4.21


Ser
36
A
A
.
.
.
.
.
1.33
−1.43
*
.
F
0.90
2.92


Glu
37
A
A
.
.
.
.
.
1.68
−1.17
*
.
F
0.90
2.29


Lys
38
A
A
.
.
.
.
.
2.27
−0.77
*
.
F
0.90
2.02


Ile
39
A
A
.
.
.
.
.
2.33
−0.37
*
.
F
0.60
2.62


Tyr
40
A
.
.
.
.
.
.
1.92
−0.76
.
.
F
1.40
2.96


Gln
41
A
.
.
.
.
T
.
1.41
−0.37
.
*
F
1.60
1.98


Gln
42
A
.
.
.
.
T
.
1.52
0.31
.
.
F
1.30
2.33


Arg
43
.
.
.
.
.
T
C
1.48
−0.37
.
*
F
2.40
2.91


Ser
44
.
.
.
.
.
T
C
2.37
−1.13
.
.
F
3.00
2.91


Leu
45
.
A
.
.
.
.
C
2.61
−1.53
*
*
F
2.30
2.81


Arg
46
.
A
.
.
T
.
.
2.61
−1.53
*
.
F
2.20
2.49


Glu
47
.
A
.
.
T
.
.
2.31
−1.13
*
.
F
1.90
3.21


Asp
48
.
A
.
.
T
.
.
1.50
−1.13
*
.
F
1.60
5.22


Gln
49
.
A
.
.
T
.
.
1.49
−1.03
*
*
F
1.30
2.31


Gln
50
.
A
.
.
T
.
.
1.96
−0.54
*
*
F
1.58
1.92


Ser
51
.
A
.
.
.
.
C
1.54
−0.11
*
.
F
1.36
1.14


Phe
52
.
.
.
.
T
T
.
1.66
0.27
.
.
F
1.49
0.88


Thr
53
.
.
.
.
T
T
.
1.34
−0.13
.
.
F
2.37
1.00


Gly
54
.
.
.
.
T
T
.
1.10
−0.04
*
.
F
2.80
1.07


Ser
55
.
.
.
.
T
T
.
0.80
0.33
.
.
F
1.92
1.94


Arg
56
.
.
.
B
T
.
.
0.29
−0.07
.
.
F
1.84
1.80


Thr
57
.
.
.
B
T
.
.
0.13
0.13
.
.
F
0.96
1.50


Tyr
58
.
.
.
B
T
.
.
0.10
0.34
*
.
.
0.38
0.83


Ser
59
.
.
.
B
.
.
C
0.44
0.39
*
.
.
−0.10
0.42


Leu
60
.
.
B
B
.
.
.
0.16
0.79
*
.
.
−0.60
0.50


Val
61
.
.
B
B
.
.
.
−0.24
0.80
*
.
.
−0.60
0.33


Gly
62
.
.
.
.
T
.
.
−0.14
0.96
.
.
.
0.00
0.26


Gln
63
.
.
.
.
T
.
.
−0.24
1.00
.
.
.
0.00
0.48


Ala
64
.
.
.
.
.
.
C
−0.16
0.74
.
.
.
−0.20
0.64


Trp
65
.
.
.
.
.
T
C
−0.16
0.53
.
.
F
0.15
1.00


Pro
66
.
.
.
.
.
T
C
0.11
0.79
*
.
F
0.15
0.48


Gly
67
.
.
.
.
.
T
C
0.46
0.89
*
.
F
0.15
0.48


Pro
68
.
.
.
.
.
T
C
−0.14
0.39
*
.
F
0.45
0.75


Leu
69
.
A
.
.
.
.
C
−0.14
0.09
.
.
F
0.05
0.48


Ala
70
.
A
.
.
.
.
C
−0.07
0.16
.
.
.
−0.10
0.49


Asp
71
A
A
.
.
.
.
.
−0.17
0.16
.
.
.
−0.30
0.49


Met
72
A
A
.
.
.
.
.
0.29
0.21
.
.
.
−0.30
0.86


Ala
73
A
A
.
.
.
.
.
0.54
−0.47
.
.
.
0.45
1.67


Pro
74
A
.
.
.
.
T
.
1.36
−0.97
.
.
F
1.30
2.00


Thr
75
A
.
.
.
.
T
.
1.99
−0.97
.
.
F
1.30
3.38


Arg
76
A
.
.
.
.
T
.
1.18
−1.59
.
.
F
1.30
6.69


Lys
77
A
.
.
.
.
T
.
0.97
−1.40
.
.
F
1.30
3.57


Asp
78
A
A
.
.
.
.
.
1.56
−1.14
.
.
F
0.90
2.04


Lys
79
A
A
.
.
.
.
.
1.07
−1.23
.
.
F
0.90
1.80


Leu
80
A
A
.
.
.
.
.
1.13
−0.44
.
.
.
0.30
0.78


Leu
81
.
A
B
.
.
.
.
0.81
0.31
.
.
.
−0.30
0.73


Gln
82
.
A
B
.
.
.
.
0.47
0.74
*
*
.
−0.60
0.57


Phe
83
.
A
B
.
.
.
.
−0.34
1.13
*
*
.
−0.60
0.92


Tyr
84
.
.
.
.
.
T
C
−0.39
1.13
*
*
.
0.00
0.92


Pro
85
.
.
.
.
.
T
C
0.42
0.44
*
*
.
0.00
0.92


Ser
86
.
.
.
.
.
T
C
1.02
0.04
*
*
F
0.60
1.78


Leu
87
.
.
.
.
.
T
C
0.43
−0.31
*
*
F
1.50
1.75


Glu
88
.
.
.
.
.
.
C
0.83
−0.57
*
*
F
1.90
1.14


Asp
89
.
.
.
.
.
.
C
0.78
−0.61
.
*
F
2.20
1.14


Pro
90
.
.
.
.
.
.
C
1.10
−0.61
.
*
F
2.50
1.86


Ala
91
.
.
.
.
T
.
.
1.16
−1.30
.
*
F
3.00
2.10


Ser
92
A
.
.
.
.
T
.
1.97
−0.54
.
*
F
2.50
1.97


Ser
93
A
.
.
.
.
T
.
1.97
−0.14
.
*
F
1.90
2.21


Arg
94
A
.
.
.
.
T
.
1.27
−0.17
*
.
F
1.60
3.52


Tyr
95
.
.
.
.
T
T
.
1.18
0.11
*
*
F
1.10
2.27


Gln
96
.
.
.
.
T
.
.
1.81
0.11
*
*
F
0.94
2.27


Asn
97
.
.
.
.
T
.
.
1.77
−0.27
*
*
F
1.88
2.32


Phe
98
.
.
.
.
T
.
.
1.77
0.16
*
*
F
1.62
1.47


Ser
99
.
.
.
.
T
T
.
1.77
−0.21
*
*
F
2.76
1.13


Lys
100
.
.
.
.
T
T
.
1.98
−0.61
*
.
F
3.40
1.38


Gly
101
.
.
.
.
.
T
C
1.63
−0.51
*
.
F
2.86
2.17


Ser
102
.
.
.
.
.
T
C
1.33
−0.87
*
.
F
2.82
1.60


Arg
103
.
.
.
.
.
.
C
2.03
−0.87
*
.
F
2.58
1.07


His
104
.
.
.
.
.
T
C
2.33
−0.87
*
.
F
2.74
1.88


Gly
105
.
.
.
.
.
T
C
1.70
−1.30
*
.
F
2.70
2.43


Ser
106
.
.
.
.
.
T
C
1.80
−1.19
*
.
F
3.00
1.25


Glu
107
A
.
.
.
.
T
.
1.21
−0.43
.
*
F
2.20
1.44


Glu
108
A
.
.
.
.
.
.
1.10
−0.24
.
*
F
1.70
1.02


Ala
109
A
.
.
.
.
.
.
0.92
−0.67
.
*
.
1.55
1.27


Tyr
110
A
.
.
.
.
.
.
0.38
−0.63
.
*
.
1.25
1.14


Ile
111
A
.
.
.
.
.
.
0.09
0.06
.
.
.
−0.10
0.46


Asp
112
A
.
.
.
.
.
.
−0.51
0.56
.
.
.
−0.40
0.46


Pro
113
A
A
.
.
.
.
.
−0.51
0.67
.
*
.
−0.60
0.29


Ile
114
A
A
.
.
.
.
.
−0.17
−0.09
.
.
.
0.30
0.72


Ala
115
A
A
.
.
.
.
.
−0.17
−0.01
*
.
.
0.30
0.67


Met
116
A
A
.
.
.
.
.
0.72
0.74
*
.
.
−0.60
0.68


Glu
117
A
A
.
.
.
.
.
0.43
0.71
*
.
.
−0.45
1.57


Tyr
118
A
.
.
.
.
T
.
0.30
0.94
*
*
.
−0.05
1.63


Tyr
119
.
.
.
.
T
T
.
1.30
0.87
*
*
.
0.35
1.63


Asn
120
.
.
.
.
T
T
.
1.19
0.26
*
*
.
0.65
1.84


Trp
121
.
.
.
.
T
T
.
1.49
1.04
*
*
.
0.35
1.02


Gly
122
.
.
.
.
T
.
.
1.53
0.67
*
.
.
0.00
0.87


Arg
123
.
.
.
.
T
.
.
1.57
−0.09
*
.
.
1.05
1.08


Phe
124
.
.
.
.
T
.
.
1.60
−0.06
*
.
F
1.20
1.59


Ser
125
.
.
.
.
.
.
C
1.60
−0.54
*
.
F
1.64
2.49


Lys
126
.
.
.
.
.
.
C
1.89
−0.97
*
*
F
1.98
2.20


Pro
127
.
.
.
.
.
T
C
2.23
−0.97
*
*
F
2.52
4.25


Pro
128
.
.
.
.
.
T
C
2.12
−1.76
*
*
F
2.86
5.29


Glu
129
.
.
.
.
T
T
.
2.23
−2.14
.
.
F
3.40
4.42


Asp
130
A
.
.
.
.
T
.
2.53
−1.64
.
*
F
2.66
2.89


Asp
131
A
.
.
.
.
.
.
2.19
−1.67
.
*
F
2.12
3.00


Asp
132
A
.
.
.
.
T
.
2.16
−1.71
.
.
F
1.98
2.32


Ala
133
A
.
.
.
.
T
.
2.37
−0.96
.
.
F
1.64
2.18


Asn
134
A
.
.
.
.
T
.
2.37
−0.96
.
.
F
1.30
2.26


Ser
135
A
.
.
.
.
T
.
1.51
−0.56
*
.
F
1.30
2.18


Tyr
136
A
.
.
.
.
.
.
0.70
0.09
*
.
F
0.20
1.60


Glu
137
A
.
.
.
.
.
.
−0.19
0.27
.
*
.
−0.10
0.82


Asn
138
A
.
.
B
.
.
.
−0.27
0.56
.
.
.
−0.60
0.43


Val
139
A
.
.
B
.
.
.
−0.22
0.74
.
.
.
−0.60
0.15


Leu
140
A
.
.
B
.
.
.
0.08
−0.01
.
.
.
0.30
0.17


Ile
141
A
.
.
B
.
.
.
0.37
0.39
.
.
.
−0.30
0.18


Cys
142
A
.
.
B
.
.
.
0.06
−0.01
.
.
.
0.56
0.49


Lys
143
A
.
.
B
.
.
.
−0.26
−0.17
.
.
F
0.97
0.86


Gln
144
A
.
.
.
.
.
.
0.60
−0.37
.
.
F
1.58
1.77


Lys
145
.
.
.
.
.
.
C
1.10
−1.06
.
.
F
2.34
5.73


Thr
146
.
.
.
.
.
.
C
1.64
−1.14
.
.
F
2.60
4.13


Thr
147
.
.
.
.
.
.
C
1.72
−0.71
.
*
F
2.34
2.36


Glu
148
.
.
.
.
.
.
C
1.68
−0.61
.
.
F
2.08
1.19


Thr
149
.
.
.
.
.
.
C
1.68
−0.21
.
.
F
1.52
1.43


Gly
150
.
A
.
.
.
.
C
1.63
−0.30
.
.
F
1.06
1.72


Ala
151
A
A
.
.
.
.
.
1.60
−0.79
.
.
F
0.90
1.72


Gln
152
A
A
.
.
.
.
.
1.02
−0.36
.
.
F
0.60
1.18


Gln
153
A
A
.
.
.
.
.
0.68
−0.16
.
.
F
0.45
0.83


Glu
154
.
A
.
.
T
.
.
0.64
−0.16
.
.
F
0.85
0.82


Gly
155
.
.
.
.
T
T
.
0.18
−0.23
.
.
F
1.25
0.47


Ile
156
.
.
.
.
T
T
.
0.10
0.06
*
*
F
0.65
0.22


Gly
157
.
.
.
.
T
T
.
0.21
0.23
*
*
F
0.90
0.07


Gly
158
.
.
.
.
T
T
.
−0.13
0.23
*
*
F
1.15
0.14


Leu
159
.
.
.
.
.
.
C
−0.13
0.23
*
*
.
0.85
0.19


Cys
160
.
.
B
.
.
T
.
−0.60
−0.46
*
*
.
1.70
0.32


Arg
161
.
.
.
.
T
T
.
−0.01
−0.20
*
*
F
2.50
0.27


Gly
162
.
.
.
.
T
T
.
−0.48
−0.24
.
*
F
2.25
0.44


Asp
163
.
.
.
.
T
T
.
−0.43
−0.24
.
*
F
2.00
0.68


Leu
164
A
A
.
.
.
.
.
−0.43
−0.43
.
*
F
0.95
0.46


Ser
165
A
A
.
.
.
.
.
−0.36
0.26
.
*
.
−0.05
0.39


Leu
166
A
A
.
.
.
.
.
−1.28
0.33
*
*
.
−0.30
0.23


Ser
167
A
A
.
.
.
.
.
−0.89
1.01
.
*
.
−0.60
0.23


Leu
168
A
A
.
.
.
.
.
−1.20
0.33
.
*
.
−0.30
0.35


Ala
169
A
A
.
.
.
.
.
−0.73
0.43
.
*
.
−0.60
0.61


Leu
170
A
A
.
.
.
.
.
−0.64
0.17
*
*
.
−0.05
0.45


Lys
171
.
A
.
.
T
.
.
−0.14
0.21
*
*
F
0.75
0.84


Thr
172
.
A
.
.
T
.
.
−0.14
0.01
.
*
F
1.15
1.20


Gly
173
.
.
.
.
.
T
C
0.32
−0.10
.
*
F
2.20
1.96


Pro
174
.
.
.
.
T
T
.
0.10
−0.36
.
.
F
2.50
0.97


Thr
175
.
.
.
.
T
T
.
0.24
0.33
.
*
F
1.65
0.55


Ser
176
.
.
.
.
T
T
.
−0.01
0.41
.
.
F
1.10
0.30


Gly
177
.
.
.
.
T
.
.
0.00
0.41
.
.
F
0.65
0.30


Leu
178
.
.
.
.
.
.
C
−0.24
0.37
.
.
F
0.50
0.28


Cys
179
.
.
.
.
.
T
C
−0.33
0.39
.
.
F
0.75
0.21


Pro
180
.
.
.
.
.
T
C
−0.23
0.39
.
.
F
1.05
0.28


Ser
181
.
.
.
.
.
T
C
0.07
0.39
.
.
F
1.35
0.53


Ala
182
.
.
.
.
.
T
C
0.41
−0.30
.
*
F
2.40
1.72


Ser
183
.
.
.
.
.
T
C
1.22
−0.87
.
*
F
3.00
1.93


Pro
184
.
.
.
.
.
T
C
1.89
−1.30
.
.
F
2.70
2.40


Glu
185
A
.
.
.
.
T
.
1.76
−1.69
.
.
F
2.20
4.12


Glu
186
A
.
.
.
.
T
.
1.17
−1.76
.
.
F
1.90
3.04


Asp
187
A
.
.
.
.
T
.
1.37
−1.46
.
.
F
1.60
1.38


Glu
188
A
.
.
.
.
T
.
1.28
−1.46
.
.
.
1.15
1.02


Gly
189
A
.
.
.
.
T
.
1.10
−1.03
.
.
.
1.00
0.75


Ile
190
A
.
.
.
.
T
.
0.71
−0.60
.
.
.
1.00
0.58































TABLE 13





Res
Position
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV






























Met
1
.
.
B
.
.
.
.
0.43
0.01
*
.
.
−0.10
0.79


Asp
2
.
.
B
.
.
.
.
0.48
−0.41
*
.
.
0.65
1.21


Leu
3
.
.
B
.
.
T
.
0.06
−0.41
*
.
.
0.70
0.94


Pro
4
A
.
.
.
.
T
.
−0.41
−0.16
*
.
.
0.70
0.78


Arg
5
.
.
B
.
.
T
.
−0.88
−0.13
*
.
F
0.85
0.35


Gly
6
.
.
B
.
.
T
.
−0.87
0.51
*
.
F
−0.05
0.31


Leu
7
.
.
B
B
.
.
.
−1.16
0.33
*
.
.
−0.30
0.20


Val
8
.
.
B
B
.
.
.
−0.93
0.81
*
.
.
−0.60
0.11


Val
9
.
.
B
B
.
.
.
−1.53
1.31
.
.
.
−0.60
0.11


Ala
10
.
.
B
B
.
.
.
−1.94
1.57
.
*
.
−0.60
0.11


Trp
11
.
.
B
B
.
.
.
−2.41
1.27
.
.
.
−0.60
0.20


Ala
12
.
.
B
B
.
.
.
−1.89
1.31
.
.
.
−0.60
0.22


Leu
13
.
.
B
B
.
.
.
−1.24
1.59
.
.
.
−0.60
0.23


Ser
14
.
.
.
.
.
.
C
−0.73
1.51
.
.
.
−0.20
0.34


Leu
15
.
.
.
.
.
.
C
−0.84
1.03
.
.
.
−0.20
0.34


Trp
16
.
.
.
.
.
T
C
−0.87
1.31
*
.
.
0.00
0.35


Pro
17
.
.
.
.
.
T
C
−0.28
1.11
*
.
.
0.00
0.38


Gly
18
.
.
.
.
T
T
.
0.22
0.73
*
.
F
0.35
0.77


Phe
19
.
.
.
.
T
T
.
−0.18
0.53
*
.
F
0.50
1.06


Thr
20
.
.
.
.
.
.
C
0.63
0.40
*
.
F
0.25
0.59


Asp
21
.
.
.
.
.
.
C
0.32
0.37
.
*
F
0.25
0.96


Thr
22
.
.
.
.
.
.
C
0.53
0.56
.
*
.
0.29
1.10


Phe
23
.
.
B
.
.
.
.
0.57
−0.23
*
*
.
1.33
1.27


Asn
24
.
.
B
.
.
T
.
1.38
−0.23
*
*
.
1.87
1.10


Met
25
.
.
.
.
T
T
.
1.73
−0.23
*
*
.
2.61
1.49


Asp
26
.
.
.
.
T
T
.
1.52
−0.71
*
.
F
3.40
3.44


Thr
27
.
.
.
.
T
T
.
1.94
−1.07
*
*
F
3.06
3.31


Arg
28
.
.
.
.
T
.
.
1.79
−1.47
*
*
F
2.52
6.55


Lys
29
.
.
B
.
.
.
.
0.90
−1.44
*
.
F
1.78
2.91


Pro
30
.
.
B
B
.
.
.
1.29
−0.76
*
.
F
1.24
1.41


Arg
31
.
.
B
B
.
.
.
0.94
−0.81
.
.
F
0.90
1.12


Val
32
.
.
B
B
.
.
.
0.96
−0.39
.
*
.
0.30
0.55


Ile
33
.
.
B
.
.
T
.
0.96
0.00
.
*
F
0.22
0.48


Pro
34
.
.
B
.
.
T
.
0.60
−0.43
.
*
F
0.79
0.48


Gly
35
.
.
.
.
T
T
.
0.22
0.06
*
*
F
0.56
0.93


Ser
36
.
.
B
.
.
T
.
−0.59
−0.09
*
*
F
0.88
1.34


Arg
37
.
.
B
B
.
.
.
−0.43
0.01
.
.
F
−0.30
0.75


Thr
38
.
.
B
B
.
.
.
0.11
0.37
.
.
F
−0.27
0.66


Ala
39
.
.
B
B
.
.
.
0.08
0.37
.
.
.
−0.39
0.49


Phe
40
.
.
B
B
.
.
.
0.11
0.74
.
.
.
−0.66
0.39


Phe
41
.
.
B
B
.
.
.
−0.44
1.23
.
.
.
−0.63
0.39


Gly
42
.
.
B
B
.
.
.
−0.56
1.39
.
.
.
−0.60
0.29


Tyr
43
.
.
B
B
.
.
.
−0.24
1.29
.
.
.
−0.60
0.57


Thr
44
.
.
B
B
.
.
.
0.31
0.90
.
.
.
−0.45
1.14


Val
45
.
.
B
B
.
.
.
1.01
0.61
.
.
.
−0.45
1.57


Gln
46
.
.
B
B
.
.
.
0.82
0.19
.
.
.
−0.15
1.67


Gln
47
.
.
B
B
.
.
.
0.87
0.11
.
.
.
−0.30
0.81


His
48
.
.
B
B
.
.
.
0.77
0.01
.
.
.
0.10
1.47


Asp
49
.
.
B
B
.
.
.
1.08
−0.20
.
.
F
0.95
0.84


Ile
50
.
.
.
B
T
.
.
1.98
−0.20
.
.
F
1.60
0.78


Ser
51
.
.
.
.
T
T
.
1.69
−0.60
.
.
F
2.70
1.14


Gly
52
.
.
.
.
T
T
.
0.88
−0.19
.
.
F
2.50
0.72


Asn
53
.
.
.
.
T
T
.
0.06
0.50
.
.
F
1.35
0.85


Lys
54
.
.
.
.
T
T
.
−0.80
0.46
.
.
F
1.10
0.47


Trp
55
.
.
B
B
.
.
.
−0.26
0.71
.
.
.
−0.10
0.35


Leu
56
.
.
B
B
.
.
.
−0.54
0.71
.
.
.
−0.35
0.22


Val
57
.
.
B
B
.
.
.
−0.41
0.81
.
.
.
−0.60
0.11


Val
58
.
.
B
B
.
.
.
−1.22
1.24
.
.
.
−0.60
0.16


Gly
59
.
.
B
B
.
.
.
−1.27
1.01
.
.
.
−0.60
0.16


Ala
60
.
.
B
.
.
.
.
−1.29
0.33
.
.
.
−0.10
0.38


Pro
61
.
.
.
.
.
.
C
−0.48
0.17
.
*
.
0.10
0.73


Leu
62
.
.
.
.
.
.
C
0.03
−0.07
.
.
F
1.34
1.19


Glu
63
A
.
.
.
.
T
.
0.64
−0.07
.
*
F
1.68
1.16


Thr
64
.
.
B
.
.
T
.
0.99
0.19
.
*
F
1.42
1.18


Asn
65
.
.
.
.
T
T
.
1.62
0.16
.
*
F
2.16
2.47


Gly
66
.
.
.
.
T
T
.
1.52
−0.53
.
.
F
3.40
2.86


Tyr
67
.
.
.
.
T
.
.
1.99
−0.04
.
.
F
2.56
2.86


Gln
68
.
.
.
.
T
.
.
1.99
−0.10
.
.
F
2.48
1.76


Lys
69
.
.
B
.
T
T
.
1.44
−0.50
*
.
F
2.60
2.97


Thr
70
.
.
B
.
.
T
.
1.20
−0.29
*
.
F
2.12
1.41


Gly
71
.
.
B
.
.
T
.
1.59
−0.29
*
.
F
2.04
1.27


Asp
72
.
.
B
.
.
T
.
1.17
−0.69
*
.
F
2.60
1.27


Val
73
.
.
B
.
.
.
.
0.96
−0.11
*
.
F
1.69
0.47


Tyr
74
.
.
B
.
.
T
.
0.06
−0.17
.
.
.
1.48
0.74


Lys
75
.
.
B
.
.
T
.
−0.52
0.04
.
.
.
0.62
0.33


Cys
76
.
.
B
.
.
T
.
−0.21
0.73
.
.
.
0.06
0.31


Pro
77
.
.
B
.
.
T
.
−0.56
0.59
.
.
.
−0.20
0.27


Val
78
.
.
B
.
.
.
.
0.30
0.26
.
.
.
−0.10
0.13


Ile
79
.
.
B
.
.
.
.
−0.12
0.66
.
.
.
−0.40
0.40


His
80
.
.
B
.
.
T
.
−0.48
0.66
.
.
.
−0.20
0.14


Gly
81
.
.
B
.
.
T
.
0.23
0.71
.
.
.
−0.20
0.27


Asn
82
.
.
.
.
T
T
.
−0.37
0.07
.
.
.
0.50
0.77


Cys
83
.
.
B
.
.
T
.
0.49
0.07
.
*
F
0.25
0.47


Thr
84
.
.
B
.
.
.
.
0.57
−0.03
.
*
F
0.65
0.76


Lys
85
.
.
B
.
.
.
.
0.26
0.23
.
.
F
0.05
0.39


Leu
86
.
.
B
.
.
.
.
0.71
0.26
.
.
.
−0.10
0.72


Asn
87
.
.
B
.
.
.
.
−0.14
−0.31
.
.
.
0.50
0.97


Leu
88
.
.
B
B
.
.
.
0.21
−0.16
.
*
.
0.30
0.36


Gly
89
.
.
B
B
.
.
.
−0.29
0.33
.
*
.
−0.30
0.63


Arg
90
.
.
B
B
.
.
.
−0.63
0.33
.
.
.
−0.30
0.32


Val
91
.
.
B
B
.
.
.
0.18
0.31
*
.
.
−0.30
0.53


Thr
92
.
.
B
B
.
.
.
−0.68
0.03
*
.
.
−0.30
0.85


Leu
93
.
.
B
B
.
.
.
−0.17
0.24
*
*
.
−0.30
0.32


Ser
94
.
.
B
B
.
.
.
0.18
0.63
.
*
.
−0.60
0.58


Asn
95
.
.
B
B
.
.
.
0.18
−0.01
.
*
F
0.45
0.70


Val
96
.
.
B
B
.
.
.
1.08
−0.50
.
.
F
0.60
1.67


Ser
97
A
.
.
.
.
.
.
1.39
−1.19
*
.
F
1.10
2.49


Glu
98
A
.
.
.
.
.
.
2.20
−1.57
*
.
F
1.10
2.58


Arg
99
A
.
.
.
.
T
.
1.90
−1.57
.
*
F
1.30
5.60


Lys
100
A
.
.
.
.
T
.
2.01
−1.60
.
*
F
1.30
4.13


Asp
101
A
.
.
.
.
T
.
2.06
−1.99
.
*
F
1.30
4.67


Asn
102
A
.
.
.
.
T
.
2.01
−1.30
.
*
.
1.15
1.97


Met
103
A
.
.
.
.
.
.
1.20
−0.87
.
*
.
0.80
0.97


Arg
104
A
.
.
.
.
.
.
0.79
−0.19
.
*
.
0.50
0.48


Leu
105
.
.
B
.
.
.
.
−0.07
0.20
*
*
.
−0.10
0.40


Gly
106
.
.
B
.
.
.
.
−0.66
0.49
*
*
.
−0.40
0.33


Leu
107
.
.
B
.
.
.
.
−0.97
0.37
*
*
.
−0.10
0.17


Ser
108
.
.
B
.
.
.
.
−0.37
0.86
*
*
.
−0.40
0.30


Leu
109
.
.
B
.
.
.
.
−0.69
0.57
*
*
.
−0.06
0.49


Ala
110
.
.
B
.
.
.
.
0.17
0.57
.
*
.
0.28
0.92


Thr
111
.
.
B
.
.
.
.
0.51
−0.11
.
.
F
1.82
1.37


Asn
112
.
.
.
.
.
T
C
1.32
−0.50
.
*
F
2.56
2.78


Pro
113
.
.
.
.
T
T
.
1.32
−0.79
.
.
F
3.40
4.42


Lys
114
.
.
.
.
T
T
.
1.43
−0.90
.
.
F
3.06
4.10


Asp
115
.
.
.
.
T
T
.
1.21
−0.60
.
.
F
2.72
2.21


Asn
116
.
.
.
.
T
T
.
0.93
−0.31
.
.
F
2.08
1.18


Ser
117
.
.
B
.
.
T
.
0.27
−0.24
.
.
F
1.19
0.60


Phe
118
.
.
B
.
.
T
.
0.18
0.33
.
.
.
0.10
0.19


Leu
119
.
.
B
.
.
T
.
−0.08
0.71
.
.
.
−0.20
0.16


Ala
120
.
.
B
.
.
.
.
−0.89
0.74
.
.
.
−0.40
0.18


Cys
121
.
.
B
.
.
.
.
−1.18
1.04
.
.
.
−0.40
0.17


Ser
122
.
.
.
.
.
T
C
−1.18
1.17
.
.
.
0.00
0.22


Pro
123
.
.
.
.
T
T
.
−0.51
0.87
.
.
.
0.20
0.30


Leu
124
.
.
.
.
T
T
.
0.30
0.87
.
.
.
0.20
0.75


Trp
125
.
.
.
.
T
T
.
0.22
0.30
.
.
.
0.50
0.97


Ser
126
.
.
B
.
.
.
.
0.54
0.49
.
.
.
−0.27
0.34


His
127
.
.
B
.
.
T
.
0.54
0.49
.
.
.
0.06
0.40


Glu
128
.
.
.
.
T
T
.
0.46
0.19
.
.
.
0.89
0.51


Cys
129
.
.
.
.
T
T
.
1.02
−0.34
.
.
F
1.77
0.51


Gly
130
.
.
.
.
T
T
.
1.07
0.03
.
.
F
1.30
0.59


Ser
131
.
.
.
.
T
T
.
1.06
0.29
.
.
F
1.17
0.54


Ser
132
.
.
.
.
T
T
.
0.78
0.77
.
.
F
0.89
1.44


Tyr
133
.
.
.
.
T
T
.
0.43
0.69
.
.
F
0.76
2.11


Tyr
134
.
.
B
.
.
T
.
0.50
0.69
.
.
F
0.23
1.55


Thr
135
.
.
B
.
.
.
.
0.18
0.91
.
.
F
−0.10
1.15


Thr
136
.
.
B
.
.
.
.
0.18
1.10
.
.
.
−0.40
0.39


Gly
137
.
.
B
.
.
T
.
0.59
0.73
.
*
.
−0.20
0.34


Met
138
.
.
B
.
.
T
.
−0.02
−0.03
.
*
.
0.70
0.46


Cys
139
.
.
B
.
.
T
.
0.22
0.13
.
*
.
0.10
0.23


Ser
140
.
.
B
.
.
T
.
0.23
0.04
.
*
.
0.10
0.38


Arg
141
.
.
B
.
.
.
.
0.54
0.00
*
*
F
0.05
0.52


Val
142
.
.
B
.
.
.
.
0.19
−0.21
*
*
F
0.80
1.55


Asn
143
.
.
B
.
.
T
.
0.90
0.00
*
*
F
0.61
1.00


Ser
144
.
.
.
.
.
T
C
0.87
−0.39
*
*
F
1.62
1.00


Asn
145
.
.
.
.
T
T
.
0.87
0.40
*
*
F
1.13
1.17


Phe
146
.
.
.
.
T
T
.
0.80
0.14
*
*
.
1.34
0.97


Arg
147
.
.
.
.
T
.
.
1.34
−0.26
*
*
.
2.10
1.45


Phe
148
.
.
.
B
T
.
.
0.49
−0.16
*
*
.
1.69
1.30


Ser
149
.
.
B
B
.
.
.
0.20
0.09
*
*
F
0.63
1.12


Lys
150
.
.
.
B
.
.
C
−0.01
−0.20
*
*
F
1.07
0.58


Thr
151
.
.
.
B
T
.
.
0.10
0.23
*
*
F
0.61
1.03


Val
152
.
.
.
B
.
.
C
−0.82
−0.06
*
.
.
0.50
0.77


Ala
153
.
.
B
B
.
.
C
−0.12
0.24
*
.
.
−0.10
0.32


Pro
154
A
A
.
.
.
.
.
0.29
0.64
*
.
.
−0.60
0.38


Ala
155
A
A
.
.
.
.
.
−0.42
0.16
*
.
.
−0.15
1.01


Leu
156
A
A
.
B
.
.
.
−0.11
0.09
*
.
.
−0.30
0.54


Gln
157
A
A
.
B
.
.
.
0.43
−0.01
*
.
.
0.30
0.60


Arg
158
.
A
B
B
.
.
.
0.78
0.04
*
.
F
−0.15
0.86


Cys
159
.
A
B
B
.
.
.
0.39
0.30
*
*
F
0.00
1.63


Gln
160
.
.
B
B
.
.
.
0.98
0.23
*
*
.
−0.30
0.93


Thr
161
.
.
B
B
.
.
.
0.90
−0.17
*
*
.
0.30
0.80


Tyr
162
.
.
B
B
.
.
.
0.04
0.51
*
*
.
−0.45
1.04


Met
163
.
.
B
B
.
.
.
−0.96
0.59
.
*
.
−0.60
0.45


Asp
164
.
.
B
B
.
.
.
−1.14
0.87
.
*
.
−0.60
0.22


Ile
165
.
.
B
B
.
.
.
−1.96
1.03
.
*
.
−0.60
0.10


Val
166
.
.
B
B
.
.
.
−1.64
0.96
.
*
.
−0.60
0.09


Ile
167
.
.
B
B
.
.
.
−1.74
0.34
.
*
.
−0.30
0.09


Val
168
.
.
B
B
.
.
.
−1.44
0.77
.
*
.
−0.35
0.12


Leu
169
.
.
B
B
.
.
.
−1.44
0.47
.
*
.
−0.10
0.22


Asp
170
.
.
.
B
T
.
.
−0.86
0.23
*
*
F
1.00
0.50


Gly
171
.
.
.
.
T
T
.
−0.89
−0.07
.
.
F
2.25
0.90


Ser
172
.
.
.
.
T
T
.
−0.24
−0.03
*
*
F
2.50
0.77


Asn
173
.
.
.
.
.
T
C
0.40
0.04
*
.
F
1.45
0.72


Ser
174
.
.
.
.
.
T
C
0.92
0.47
*
.
F
1.05
1.12


Ile
175
.
.
.
B
.
.
C
0.07
0.96
*
.
.
0.10
0.88


Tyr
176
.
.
.
B
.
.
C
0.41
1.21
*
.
.
−0.15
0.41


Pro
177
.
.
B
B
.
.
.
−0.14
0.81
.
*
.
−0.60
0.53


Trp
178
.
.
B
B
.
.
.
−0.14
1.07
.
*
.
−0.60
0.56


Val
179
.
.
B
B
.
.
.
0.12
0.79
.
.
.
−0.60
0.62


Glu
180
.
.
B
B
.
.
.
0.31
0.53
.
*
.
−0.60
0.54


Val
181
A
.
.
B
.
.
.
−0.26
0.89
.
.
.
−0.60
0.45


Gln
182
A
.
.
B
.
.
.
−0.93
0.66
.
.
.
−0.60
0.50


His
183
A
.
.
B
.
.
.
−0.64
0.70
*
*
.
−0.60
0.20


Phe
184
A
.
.
B
.
.
.
−0.68
1.10
*
.
.
−0.60
0.43


Leu
185
A
.
.
B
.
.
.
−1.49
1.14
*
.
.
−0.60
0.18


Ile
186
A
.
.
B
.
.
.
−0.59
1.43
*
.
.
−0.60
0.11


Asn
187
A
.
.
B
.
.
.
−0.54
0.93
*
.
.
−0.60
0.25


Ile
188
A
.
.
B
.
.
.
−1.21
0.14
*
*
.
−0.30
0.60


Leu
189
A
.
.
B
.
.
.
−0.76
0.24
*
*
.
−0.30
0.74


Lys
190
.
.
B
B
.
.
.
−0.83
0.31
*
.
.
−0.30
0.72


Lys
191
.
.
B
B
.
.
.
−0.29
0.60
*
*
.
−0.60
0.72


Phe
192
.
.
B
B
.
.
.
−0.50
0.34
*
.
.
−0.30
0.86


Tyr
193
.
.
B
B
.
.
.
0.04
0.09
*
.
.
−0.30
0.67


Ile
194
.
.
B
B
.
.
.
0.86
0.51
*
.
.
−0.60
0.33


Gly
195
.
.
.
.
.
T
C
−0.08
0.91
*
.
.
0.00
0.66


Pro
196
.
.
.
.
T
T
.
−0.12
0.81
.
*
F
0.35
0.30


Gly
197
.
.
.
.
T
T
.
−0.28
0.46
.
*
F
0.35
0.73


Gln
198
.
.
B
.
.
T
.
−0.38
0.41
.
*
F
−0.05
0.55


Ile
199
.
.
B
B
.
.
.
−0.34
0.41
.
*
.
−0.60
0.35


Gln
200
.
.
B
B
.
.
.
−0.86
0.63
.
*
.
−0.60
0.26


Val
201
.
.
B
B
.
.
.
−0.64
0.84
.
*
.
−0.60
0.11


Gly
202
.
.
B
B
.
.
.
−0.54
0.84
.
*
.
−0.60
0.28


Val
203
.
.
B
B
.
.
.
−0.89
0.91
.
*
.
−0.60
0.25


Val
204
.
.
B
B
.
.
.
0.00
0.94
.
.
.
−0.60
0.34


Gln
205
.
.
B
B
.
.
.
0.00
0.30
.
.
.
−0.30
0.59


Tyr
206
.
.
B
B
.
.
.
0.00
−0.13
*
.
.
0.45
1.32


Gly
207
.
A
B
B
.
.
.
−0.51
−0.13
*
.
F
0.60
1.32


Glu
208
A
A
.
B
.
.
.
0.31
−0.13
*
.
F
0.45
0.57


Asp
209
A
A
.
B
.
.
.
1.17
−0.03
*
.
F
0.45
0.49


Val
210
A
A
.
B
.
.
.
0.47
−0.79
*
.
.
0.60
0.86


Val
211
A
A
.
B
.
.
.
0.68
−0.43
*
.
.
0.30
0.43


His
212
A
A
.
B
.
.
.
0.21
0.07
*
*
.
−0.30
0.35


Glu
213
A
A
.
.
.
.
.
0.21
0.76
.
.
.
−0.60
0.39


Phe
214
A
A
.
.
.
.
.
0.21
0.51
.
*
.
−0.60
0.84


His
215
A
A
.
.
.
.
.
0.82
−0.13
*
.
.
0.45
1.04


Leu
216
A
A
.
.
.
.
.
1.79
0.13
*
.
.
0.04
0.94


Asn
217
A
.
.
.
.
T
.
1.52
0.13
*
.
.
0.93
2.12


Asp
218
A
.
.
.
.
T
.
0.67
−0.27
*
.
F
2.02
2.09


Tyr
219
.
.
.
.
T
T
.
1.41
−0.13
*
.
F
2.76
1.88


Arg
220
.
.
.
.
T
T
.
1.44
−0.81
*
.
F
3.40
2.34


Ser
221
A
.
.
.
.
.
.
1.40
−1.21
*
.
F
2.46
2.34


Val
222
.
A
B
.
.
.
.
0.54
−0.57
*
.
F
1.92
1.11


Lys
223
.
A
B
.
.
.
.
0.54
−0.69
*
.
F
1.43
0.42


Asp
224
.
A
B
.
.
.
.
0.20
−0.69
*
.
F
1.09
0.54


Val
225
A
A
.
.
.
.
.
−0.50
−0.57
*
.
.
0.60
0.74


Val
226
A
A
.
.
.
.
.
−0.50
−0.71
*
.
.
0.60
0.37


Glu
227
A
A
.
.
.
.
.
0.32
−0.33
*
.
.
0.30
0.30


Ala
228
A
A
.
.
.
.
.
−0.61
0.17
*
.
.
−0.30
0.55


Ala
229
A
A
.
.
.
.
.
−0.61
0.21
*
.
.
−0.30
0.52


Ser
230
A
A
.
.
.
.
.
0.24
−0.43
*
*
.
0.30
0.52


His
231
A
A
.
.
.
.
.
1.21
−0.03
*
*
.
0.64
0.89


Ile
232
A
A
.
.
.
.
.
0.87
−0.53
*
*
.
1.43
1.72


Glu
233
A
A
.
.
.
.
.
1.11
−0.60
*
*
F
1.92
1.27


Gln
234
.
.
.
.
T
T
.
1.39
−0.56
*
*
F
2.91
0.92


Arg
235
.
.
.
.
T
T
.
1.69
−0.57
.
*
F
3.40
1.90


Gly
236
.
.
.
.
T
T
.
1.41
−1.26
*
*
F
3.06
1.90


Gly
237
.
.
.
.
.
T
C
2.41
−0.77
*
*
F
2.68
1.59


Thr
238
.
.
.
.
.
.
C
2.10
−1.17
*
*
F
2.30
1.59


Glu
239
.
.
.
.
.
.
C
1.51
−0.69
*
*
F
2.12
2.31


Thr
240
.
.
B
.
.
.
.
0.70
−0.61
*
*
F
1.74
2.36


Arg
241
.
.
B
.
.
.
.
0.70
−0.26
.
*
F
1.60
1.42


Thr
242
.
.
B
.
.
.
.
0.16
−0.31
.
*
F
1.29
0.81


Ala
243
A
A
.
.
.
.
.
0.47
0.37
.
*
.
0.18
0.39


Phe
244
A
A
.
.
.
.
.
−0.23
−0.11
.
*
.
0.62
0.35


Gly
245
A
A
.
.
.
.
.
−0.51
0.67
.
*
.
−0.44
0.21


Ile
246
A
A
.
.
.
.
.
−0.51
0.69
.
*
.
−0.60
0.21


Glu
247
A
A
.
.
.
.
.
−0.50
0.19
.
.
.
−0.30
0.47


Phe
248
A
A
.
.
.
.
.
0.09
−0.21
.
.
.
0.30
0.64


Ala
249
A
A
.
.
.
.
.
0.20
−0.64
.
.
.
0.75
1.58


Arg
250
A
A
.
.
.
.
.
−0.16
−0.83
.
.
F
0.75
0.92


Ser
251
A
A
.
.
.
.
.
0.73
−0.04
.
.
F
0.45
0.92


Glu
252
A
A
.
.
.
.
.
0.78
−0.43
.
.
F
0.60
1.58


Ala
253
A
A
.
.
.
.
.
1.13
−0.93
.
.
F
0.90
1.61


Phe
254
A
A
.
.
.
.
.
1.38
−0.50
*
*
F
0.60
1.19


Gln
255
A
.
.
.
.
T
.
1.38
−0.46
*
.
F
0.85
0.68


Lys
256
A
.
.
.
.
T
.
1.72
−0.46
*
.
F
1.00
1.32


Gly
257
A
.
.
.
.
T
.
1.38
−0.96
*
.
F
1.30
3.05


Gly
258
A
.
.
.
.
T
.
1.38
−1.31
*
.
F
1.30
1.74


Arg
259
A
A
.
.
.
.
.
2.12
−1.21
*
.
F
0.75
0.88


Lys
260
A
A
.
.
.
.
.
2.17
−1.21
*
.
F
0.90
1.78


Gly
261
A
A
.
.
.
.
.
1.27
−1.64
*
.
F
0.90
3.59


Ala
262
A
A
.
.
.
.
.
1.01
−1.43
*
.
F
0.90
1.36


Lys
263
A
.
.
B
.
.
.
0.47
−0.81
*
.
F
0.75
0.67


Lys
264
.
.
B
B
.
.
.
−0.50
−0.13
*
.
.
0.30
0.48


Val
265
.
.
B
B
.
.
.
−1.43
0.09
*
.
.
−0.30
0.35


Met
266
.
.
B
B
.
.
.
−1.40
0.27
*
.
.
−0.30
0.12


Ile
267
.
.
B
B
.
.
.
−0.81
0.76
*
.
.
−0.60
0.09


Val
268
.
.
B
B
.
.
.
−1.20
0.76
*
.
.
−0.60
0.20


Ile
269
.
.
B
.
.
T
.
−1.24
0.54
.
.
.
−0.20
0.20


Thr
270
.
.
B
.
.
T
.
−0.69
−0.07
.
.
F
0.85
0.49


Asp
271
.
.
B
.
.
T
.
−0.12
−0.37
.
.
F
0.85
0.89


Gly
272
.
.
.
.
.
T
C
0.77
−0.51
.
.
F
1.50
1.73


Glu
273
.
.
.
.
.
.
C
1.32
−1.20
.
.
F
1.60
2.00


Ser
274
.
.
.
.
.
.
C
2.00
−1.30
.
.
F
1.90
1.60


His
275
.
.
.
.
.
.
C
2.31
−0.87
.
.
F
2.20
2.51


Asp
276
.
.
.
.
.
.
C
1.50
−1.30
.
*
F
2.50
2.42


Ser
277
.
.
.
.
.
T
C
1.84
−0.61
.
.
F
3.00
1.49


Pro
278
A
.
.
.
.
T
.
1.89
−1.00
.
.
F
2.50
1.89


Asp
279
A
.
.
.
.
T
.
1.33
−1.50
*
.
F
2.20
2.27


Leu
280
A
.
.
.
.
T
.
0.48
−0.86
*
.
F
1.90
1.25


Glu
281
A
A
.
.
.
.
.
0.48
−0.56
*
.
F
1.05
0.57


Lys
282
A
A
.
.
.
.
.
0.78
−0.59
*
.
F
0.75
0.59


Val
283
A
A
.
.
.
.
.
0.69
−0.19
*
.
.
0.45
1.24


Ile
284
A
A
.
.
.
.
.
0.69
−0.49
*
.
F
0.45
0.96


Gln
285
.
A
B
.
.
.
.
1.61
−0.49
*
.
F
0.79
0.83


Gln
286
A
A
.
.
.
.
.
1.61
−0.49
*
.
F
1.28
2.19


Ser
287
A
A
.
.
.
.
.
1.57
−1.13
*
*
F
1.92
5.22


Glu
288
.
.
.
.
.
T
C
1.57
−1.41
*
.
F
2.86
4.85


Arg
289
.
.
.
.
T
T
.
2.14
−1.17
*
.
F
3.40
2.08


Asp
290
.
.
.
.
T
T
.
2.26
−1.09
.
.
F
3.06
2.24


Asn
291
.
.
.
.
T
T
.
2.01
−1.47
*
.
F
2.72
2.53


Val
292
.
.
B
B
.
.
.
1.72
−0.71
.
.
F
1.58
2.02


Thr
293
.
.
B
B
.
.
.
0.87
−0.21
.
.
F
0.94
1.22


Arg
294
.
.
B
B
.
.
.
0.17
0.43
*
.
.
−0.60
0.57


Tyr
295
.
.
B
B
.
.
.
−0.69
0.53
*
.
.
−0.60
0.77


Ala
296
.
.
B
B
.
.
.
−1.50
0.53
*
.
.
−0.60
0.40


Val
297
.
.
B
B
.
.
.
−0.99
0.73
*
.
.
−0.60
0.17


Ala
298
.
.
B
B
.
.
.
−0.92
1.16
*
.
.
−0.60
0.11


Val
299
.
.
B
B
.
.
.
−1.28
1.16
*
.
.
−0.60
0.16


Leu
300
.
.
B
B
.
.
.
−1.03
1.41
*
.
.
−0.60
0.34


Gly
301
.
.
B
B
.
.
.
−0.33
1.17
*
.
.
−0.60
0.55


Tyr
302
.
.
B
B
.
.
.
0.63
0.67
*
.
.
−0.45
1.45


Tyr
303
.
.
B
.
.
.
.
0.88
0.03
*
.
.
0.39
3.44


Asn
304
.
.
B
.
.
T
.
0.84
−0.23
*
.
.
1.53
3.44


Arg
305
.
.
.
.
T
T
.
1.66
0.03
*
.
F
1.82
1.54


Arg
306
.
.
.
.
T
T
.
1.79
−0.33
*
.
F
2.76
1.58


Gly
307
.
.
.
.
T
T
.
2.03
−0.66
*
.
F
3.40
1.52


Ile
308
.
.
.
.
.
.
C
1.97
−1.06
.
.
F
2.66
1.34


Asn
309
.
.
.
.
.
T
C
1.27
−0.57
*
.
F
2.37
0.99


Pro
310
.
.
.
.
.
T
C
0.34
0.21
*
.
F
1.13
0.86


Glu
311
.
.
B
.
.
T
.
0.23
0.47
.
.
F
0.44
1.02


Thr
312
.
.
B
.
.
T
.
0.58
0.19
*
.
F
0.40
1.02


Phe
313
A
A
.
.
.
.
.
0.58
−0.21
.
*
.
0.45
1.14


Leu
314
A
A
.
.
.
.
.
0.62
0.04
*
*
.
−0.30
0.46


Asn
315
A
A
.
.
.
.
.
0.59
0.04
*
.
.
−0.30
0.64


Glu
316
A
A
.
.
.
.
.
−0.30
0.31
*
.
F
0.00
1.16


Ile
317
A
A
.
.
.
.
.
−0.58
0.21
*
.
.
−0.30
0.98


Lys
318
A
A
.
.
.
.
.
−0.18
0.03
*
.
.
−0.30
0.62


Tyr
319
.
A
B
.
.
.
.
0.63
0.01
*
.
.
−0.30
0.48


Ile
320
.
A
B
.
.
.
.
0.42
0.01
*
.
.
−0.15
1.14


Ala
321
.
A
B
.
.
.
.
0.42
−0.24
*
.
.
0.64
0.88


Ser
322
.
A
B
.
.
.
.
1.31
−0.24
*
.
.
0.98
0.94


Asp
323
.
.
.
.
.
T
C
1.31
−1.00
.
.
F
2.52
2.23


Pro
324
A
.
.
.
.
T
.
1.52
−1.69
.
.
F
2.66
4.42


Asp
325
.
.
.
.
T
T
.
1.71
−1.69
.
.
F
3.40
4.49


Asp
326
A
.
.
.
.
T
.
1.60
−1.29
.
.
F
2.66
2.33


Lys
327
A
A
.
.
.
.
.
1.90
−0.50
.
.
F
1.62
1.30


His
328
A
A
.
.
.
.
.
1.04
−0.53
.
*
.
1.43
1.26


Phe
329
.
A
B
.
.
.
.
0.94
0.11
*
.
.
0.04
0.56


Phe
330
.
A
B
.
.
.
.
0.94
0.60
*
.
.
−0.60
0.40


Asn
331
A
A
.
.
.
.
.
0.94
0.60
*
.
.
−0.60
0.49


Val
332
A
A
.
.
.
.
.
0.31
0.10
*
.
.
−0.30
0.99


Thr
333
A
A
.
.
.
.
.
−0.24
−0.19
*
.
F
0.60
1.15


Asp
334
A
A
.
.
.
.
.
−0.36
−0.47
*
*
F
0.45
0.72


Glu
335
A
A
.
.
.
.
.
0.39
−0.19
.
*
.
0.30
0.81


Ala
336
A
A
.
.
.
.
.
0.39
−0.83
.
.
.
0.75
1.12


Ala
337
A
A
.
.
.
.
.
0.36
−1.31
.
.
.
0.75
1.12


Leu
338
A
A
.
.
.
.
.
−0.19
−0.63
*
.
.
0.60
0.45


Lys
339
A
A
.
.
.
.
.
−0.19
0.01
*
.
.
−0.30
0.33


Asp
340
A
A
.
.
.
.
.
−0.78
−0.49
*
.
.
0.30
0.55


Ile
341
A
A
.
.
.
.
.
−1.00
−0.49
*
.
.
0.30
0.67


Val
342
A
A
.
.
.
.
.
−0.76
−0.49
*
.
.
0.30
0.28


Asp
343
A
A
.
.
.
.
.
0.06
−0.06
*
.
.
0.30
0.16


Ala
344
A
A
.
.
.
.
.
0.12
−0.06
*
.
.
0.30
0.39


Leu
345
A
.
.
.
.
T
.
−0.77
−0.74
*
.
.
1.15
1.03


Gly
346
A
.
.
.
.
T
.
−0.58
−0.70
*
.
.
1.00
0.43


Asp
347
A
.
.
.
.
T
.
−0.02
0.09
*
.
.
0.10
0.37


Arg
348
.
.
B
.
.
T
.
−0.83
−0.03
*
.
.
0.70
0.60


Ile
349
.
.
B
.
.
.
.
−0.24
−0.03
*
.
.
0.50
0.50


Phe
350
.
.
B
.
.
.
.
0.22
−0.46
*
.
.
0.50
0.52


Ser
351
.
.
B
.
.
.
.
0.26
−0.03
*
.
.
0.50
0.26


Leu
352
.
.
B
.
.
.
.
0.26
0.46
.
.
.
−0.10
0.54


Glu
353
.
.
.
.
.
.
C
0.19
0.17
.
.
F
1.00
1.01


Gly
354
.
.
.
.
.
.
C
1.08
−0.61
.
.
F
2.20
1.50


Thr
355
.
.
.
.
.
.
C
1.78
−0.60
.
*
F
2.50
2.93


Asn
356
.
.
.
.
.
T
C
1.77
−1.29
.
.
F
3.00
2.93


Lys
357
.
.
.
.
.
T
C
2.28
−0.80
.
*
F
2.70
4.28


Asn
358
.
.
.
.
.
T
C
1.58
−0.84
.
.
F
2.40
3.97


Glu
359
A
.
.
.
.
T
.
1.58
−0.54
.
.
F
1.90
2.14


Thr
360
A
.
.
.
.
T
.
1.08
−0.51
.
*
F
1.60
1.06


Ser
361
A
.
.
.
.
T
.
1.08
0.17
.
*
F
0.25
0.54


Phe
362
A
.
.
.
.
T
.
0.43
−0.23
.
*
.
0.70
0.54


Gly
363
A
.
.
.
.
T
.
0.13
0.39
.
.
.
0.10
0.37


Leu
364
A
.
.
.
.
.
.
0.13
0.29
.
*
.
−0.10
0.37


Glu
365
A
.
.
.
.
.
.
0.13
0.30
.
.
.
−0.10
0.74


Met
366
A
.
.
.
.
.
.
0.09
0.00
.
.
.
0.05
1.09


Ser
367
A
.
.
.
.
T
.
0.09
0.00
.
.
F
0.40
1.30


Gln
368
A
.
.
.
.
T
.
0.13
0.10
.
.
F
0.25
0.65


Thr
369
.
.
.
.
.
T
C
0.64
0.49
.
.
F
0.15
0.88


Gly
370
.
.
.
.
.
T
C
0.61
0.26
.
.
F
0.45
0.88


Phe
371
.
.
.
.
.
.
C
0.36
0.37
.
.
F
0.25
0.69


Ser
372
.
.
.
B
.
.
C
−0.20
0.61
.
.
F
−0.25
0.36


Ser
373
.
.
.
B
.
.
C
−0.20
0.77
.
.
.
−0.40
0.27


His
374
.
.
B
B
.
.
.
0.11
0.34
.
.
.
−0.30
0.53


Val
375
.
.
B
B
.
.
.
0.11
−0.44
.
.
.
0.30
0.67


Val
376
.
.
B
.
.
T
.
−0.04
−0.40
.
.
.
0.70
0.49


Glu
377
.
.
B
.
.
T
.
−0.56
−0.14
.
.
.
0.70
0.27


Asp
378
A
.
.
.
.
T
.
−1.07
0.04
.
.
.
0.10
0.30


Gly
379
A
.
.
.
.
T
.
−1.38
0.09
.
.
.
0.10
0.33


Val
380
A
A
.
.
.
.
.
−1.11
−0.13
.
.
.
0.30
0.19


Leu
381
A
A
.
.
.
.
.
−1.11
0.37
.
.
.
−0.30
0.11


Leu
382
.
A
B
.
.
.
.
−1.46
1.01
.
.
.
−0.60
0.09


Gly
383
.
A
B
.
.
.
.
−2.04
1.01
.
.
.
−0.60
0.11


Ala
384
.
A
B
.
.
.
.
−1.94
0.87
.
.
.
−0.60
0.14


Val
385
.
A
B
.
.
.
.
−1.09
0.94
.
.
.
−0.60
0.27


Gly
386
.
.
B
.
.
.
.
−0.57
0.26
.
.
.
−0.10
0.45


Ala
387
.
.
B
.
.
.
.
0.24
0.74
.
.
.
−0.40
0.47


Tyr
388
.
.
B
.
.
.
.
0.24
0.64
.
.
.
−0.25
1.01


Asp
389
.
.
.
.
T
.
.
0.24
0.43
.
.
.
0.15
1.01


Trp
390
A
.
.
.
.
.
.
0.24
0.50
.
.
.
−0.25
1.01


Asn
391
A
.
.
.
.
.
.
−0.22
0.64
*
.
.
−0.40
0.48


Gly
392
A
A
.
.
.
.
.
0.41
0.57
*
.
.
−0.60
0.24


Ala
393
A
A
.
.
.
.
.
0.66
0.57
.
.
.
−0.60
0.45


Val
394
A
A
.
.
.
.
.
0.34
−0.34
.
.
.
0.30
0.49


Leu
395
A
A
.
.
.
.
.
0.33
−0.26
.
.
F
0.45
0.71


Lys
396
A
A
.
.
.
.
.
−0.26
−0.30
.
.
F
0.45
0.94


Glu
397
A
A
.
.
.
.
.
−0.26
−0.30
.
.
F
0.60
1.28


Thr
398
A
A
.
.
.
.
.
0.38
−0.51
.
.
F
0.90
1.54


Ser
399
A
.
.
.
.
T
.
0.38
−1.20
.
.
F
1.30
1.54


Ala
400
A
.
.
.
.
T
.
0.30
−0.56
.
.
F
1.15
0.66


Gly
401
A
.
.
.
.
T
.
0.04
0.13
.
.
F
0.25
0.32


Lys
402
.
.
B
.
.
T
.
−0.77
0.07
.
.
F
0.25
0.37


Val
403
.
.
B
.
.
.
.
−0.34
0.37
.
.
.
−0.10
0.30


Ile
404
.
.
B
.
.
.
.
−0.04
−0.13
.
.
.
0.50
0.60


Pro
405
.
.
B
.
.
.
.
0.24
−0.56
.
.
.
0.80
0.52


Leu
406
.
.
B
.
.
.
.
0.34
−0.17
.
.
.
0.50
0.93


Arg
407
A
.
.
.
.
.
.
−0.51
−0.06
.
.
F
0.80
2.08


Glu
408
A
.
.
.
.
.
.
0.39
−0.06
.
.
F
0.80
1.11


Ser
409
A
.
.
.
.
.
.
1.28
−0.49
.
*
F
0.80
2.69


Tyr
410
A
A
.
.
.
.
.
0.79
−1.17
.
.
F
0.90
2.38


Leu
411
A
A
.
.
.
.
.
1.39
−0.39
.
.
F
0.60
1.19


Lys
412
A
A
.
.
.
.
.
1.28
0.04
.
.
F
0.00
1.37


Glu
413
A
A
.
.
.
.
.
1.28
−0.34
*
.
F
0.60
1.52


Phe
414
A
A
.
.
.
.
.
0.77
−1.10
*
.
F
0.90
3.19


Pro
415
A
A
.
.
.
.
.
1.06
−1.10
*
.
F
0.90
1.31


Glu
416
A
A
.
.
.
.
.
1.87
−1.10
*
.
F
0.90
1.52


Glu
417
A
A
.
.
.
.
.
1.79
−0.70
*
*
F
0.90
2.82


Leu
418
A
A
.
.
.
.
.
1.44
−0.99
*
*
F
0.90
2.48


Lys
419
A
A
.
.
.
.
.
1.56
−0.99
*
*
F
0.90
1.42


Asn
420
A
.
.
.
.
T
.
1.52
−0.49
*
.
.
0.70
0.83


His
421
A
.
.
.
.
T
.
0.71
0.27
*
.
.
0.25
1.57


Gly
422
A
.
.
.
.
T
.
0.37
0.27
.
.
.
0.10
0.65


Ala
423
.
.
B
.
.
T
.
0.93
0.70
.
.
.
−0.20
0.40


Tyr
424
.
.
B
B
.
.
.
0.58
1.06
.
.
.
−0.60
0.46


Leu
425
.
.
B
B
.
.
.
−0.28
1.04
.
.
.
−0.60
0.67


Gly
426
.
.
B
B
.
.
.
−0.56
1.26
.
.
.
−0.60
0.49


Tyr
427
.
.
B
B
.
.
.
−0.51
1.24
.
.
.
−0.60
0.45


Thr
428
.
.
B
B
.
.
.
−0.78
0.87
.
.
.
−0.60
0.74


Val
429
.
.
B
B
.
.
.
−1.39
0.83
*
.
.
−0.60
0.55


Thr
430
.
.
B
B
.
.
.
−0.88
1.04
*
.
.
−0.60
0.26


Ser
431
.
.
B
B
.
.
.
−0.83
0.67
.
.
.
−0.60
0.24


Val
432
.
.
B
B
.
.
.
−0.48
0.57
.
.
.
−0.60
0.44


Val
433
.
.
B
B
.
.
.
−0.17
−0.07
.
.
F
0.79
0.60


Ser
434
.
.
B
.
.
T
.
0.34
−0.16
.
.
F
1.53
0.77


Ser
435
.
.
B
.
.
T
.
0.77
−0.11
.
.
F
2.02
1.03


Arg
436
.
.
B
.
.
T
.
0.21
−0.76
.
.
F
2.66
2.71


Gln
437
.
.
.
.
T
T
.
0.82
−0.76
.
*
F
3.40
1.50


Gly
438
.
.
B
B
.
.
.
0.82
−0.39
*
*
F
1.96
1.75


Arg
439
.
.
B
B
.
.
.
0.53
−0.13
*
*
F
1.47
0.66


Val
440
.
.
B
B
.
.
.
0.49
0.37
*
*
.
0.38
0.39


Tyr
441
.
.
B
B
.
.
.
−0.21
0.40
*
*
.
−0.26
0.39


Val
442
.
.
B
B
.
.
.
−0.42
0.47
*
*
.
−0.60
0.20


Ala
443
.
.
B
B
.
.
.
0.03
0.90
*
*
.
−0.60
0.42


Gly
444
.
.
B
B
.
.
.
−0.78
0.26
*
*
.
−0.30
0.52


Ala
445
.
.
B
.
.
.
.
0.08
0.29
.
*
.
−0.10
0.61


Pro
446
.
.
.
.
.
.
C
0.29
0.04
*
*
F
0.25
0.97


Arg
447
.
.
B
.
.
.
.
0.83
0.04
*
*
F
0.20
1.33


Phe
448
.
.
B
.
.
.
.
1.08
0.10
*
*
.
0.18
1.90


Asn
449
.
.
.
.
T
.
.
1.47
0.03
*
*
.
0.71
1.22


His
450
.
.
.
.
T
T
.
1.20
−0.40
*
*
F
1.79
1.24


Thr
451
.
.
.
.
.
T
C
0.52
0.24
*
*
F
1.12
1.06


Gly
452
.
.
.
.
T
T
.
−0.40
0.14
*
*
F
1.30
0.46


Lys
453
.
.
B
.
.
T
.
−0.40
0.43
*
*
F
0.47
0.28


Val
454
.
.
B
B
.
.
.
−0.71
0.71
.
.
.
−0.21
0.17


Ile
455
.
.
B
B
.
.
.
−1.28
0.71
.
.
.
−0.34
0.25


Leu
456
.
.
B
B
.
.
.
−1.00
0.90
.
.
.
−0.47
0.12


Phe
457
.
.
B
B
.
.
.
−0.66
1.40
.
.
.
−0.60
0.22


Thr
458
.
.
B
B
.
.
.
−0.70
1.16
.
.
.
−0.60
0.51


Met
459
.
.
B
B
.
.
.
0.27
0.87
.
.
.
−0.32
1.00


His
460
.
.
.
.
.
T
C
0.86
0.19
.
.
.
1.01
2.26


Asn
461
.
.
.
.
.
T
C
0.86
−0.21
.
.
F
2.04
2.10


Asn
462
.
.
.
.
.
T
C
1.24
−0.01
.
.
F
2.32
1.75


Arg
463
.
.
.
.
T
T
.
0.67
−0.14
.
.
F
2.80
1.85


Ser
464
.
.
.
.
.
.
C
1.23
0.04
.
.
F
1.37
0.81


Leu
465
A
A
.
.
.
.
.
1.27
0.14
.
.
.
0.54
0.68


Thr
466
.
A
B
.
.
.
.
0.68
0.14
.
.
.
0.26
0.60


Ile
467
.
A
B
.
.
.
.
0.08
0.64
.
*
.
−0.32
0.46


His
468
.
A
B
.
.
.
.
0.08
0.87
.
*
.
−0.60
0.55


Gln
469
.
A
B
.
.
.
.
0.03
0.19
*
*
.
−0.30
0.74


Ala
470
.
A
B
.
.
.
.
0.84
0.13
*
*
.
0.02
1.05


Met
471
.
.
B
.
.
T
.
1.16
−0.16
*
.
.
1.19
1.33


Arg
472
.
.
B
.
.
T
.
1.16
−0.26
*
*
F
1.51
1.33


Gly
473
.
.
B
.
.
T
.
0.84
0.03
*
.
F
0.93
0.92


Gln
474
.
.
B
.
.
T
.
0.54
−0.04
.
.
F
1.70
0.92


Gln
475
.
.
B
.
.
.
.
0.89
−0.27
*
.
F
1.33
0.63


Ile
476
.
.
B
.
.
.
.
0.79
0.49
*
.
F
0.41
1.00


Gly
477
.
.
B
.
.
T
.
0.33
0.84
*
.
F
0.29
0.50


Ser
478
.
.
B
.
.
T
.
0.38
0.87
*
.
F
0.12
0.29


Tyr
479
.
.
.
.
.
T
C
0.38
0.86
*
.
.
0.00
0.55


Phe
480
.
.
B
.
.
T
.
−0.51
0.17
*
.
F
0.25
0.96


Gly
481
.
.
.
B
.
.
C
0.07
0.43
*
.
F
−0.25
0.50


Ser
482
.
.
.
B
.
.
C
0.11
0.53
*
.
F
−0.25
0.46


Glu
483
.
.
B
B
.
.
.
−0.44
0.16
*
.
F
−0.15
0.71


Ile
484
.
.
B
B
.
.
.
−0.20
0.01
.
.
F
−0.15
0.54


Thr
485
.
.
B
B
.
.
.
−0.39
−0.41
.
*
F
0.45
0.67


Ser
486
.
.
B
B
.
.
.
−0.04
−0.11
.
*
F
0.45
0.27


Val
487
.
.
B
B
.
.
.
−0.09
−0.11
.
*
F
0.76
0.64


Asp
488
.
.
B
B
.
.
.
−0.09
−0.37
.
*
F
1.07
0.44


Ile
489
.
.
B
.
.
.
.
0.46
−0.86
.
*
F
1.88
0.55


Asp
490
.
.
.
.
T
T
.
−0.09
−0.81
.
*
F
2.79
0.73


Gly
491
.
.
.
.
T
T
.
−0.10
−0.81
.
*
F
3.10
0.33


Asp
492
.
.
.
.
T
T
.
0.76
−0.33
*
*
F
2.49
0.67


Gly
493
.
.
B
.
.
T
.
−0.10
−1.01
*
*
F
2.08
0.67


Val
494
.
.
B
B
.
.
.
−0.02
−0.37
.
*
F
1.07
0.50


Thr
495
.
.
B
B
.
.
.
−0.83
−0.11
.
.
F
0.76
0.25


Asp
496
.
.
B
B
.
.
.
−1.34
0.57
*
.
F
−0.45
0.21


Val
497
.
.
B
B
.
.
.
−1.69
0.79
*
.
.
−0.60
0.21


Leu
498
.
.
B
B
.
.
.
−1.93
0.57
.
.
.
−0.60
0.14


Leu
499
.
.
B
B
.
.
.
−1.29
0.59
.
.
.
−0.60
0.09


Val
500
.
.
B
B
.
.
.
−1.58
1.01
.
.
.
−0.60
0.18


Gly
501
.
.
B
B
.
.
.
−1.82
0.99
.
.
.
−0.60
0.21


Ala
502
.
.
B
.
.
.
.
−1.67
1.06
.
.
.
−0.40
0.41


Pro
503
.
.
B
.
.
.
.
−0.86
1.16
.
.
.
−0.40
0.48


Met
504
.
.
B
.
.
.
.
−0.04
0.91
.
.
.
−0.40
0.77


Tyr
505
.
.
B
.
.
.
.
0.47
0.49
.
.
.
−0.25
1.33


Phe
506
.
.
B
.
.
.
.
0.92
0.41
*
.
.
−0.40
0.85


Asn
507
A
.
.
.
.
T
.
1.51
−0.01
.
*
.
0.85
1.68


Glu
508
A
.
.
.
.
T
.
1.83
−0.63
.
*
F
1.30
1.86


Gly
509
A
.
.
.
.
T
.
2.09
−1.39
.
*
F
1.30
4.20


Arg
510
A
.
.
.
.
T
.
2.38
−1.74
.
*
F
1.30
2.58


Glu
511
A
.
.
.
.
.
.
2.22
−2.14
.
*
F
1.10
2.98


Arg
512
.
.
.
B
T
.
.
1.98
−1.50
.
*
F
1.30
2.24


Gly
513
.
.
.
B
T
.
.
1.12
−1.17
.
*
F
1.30
1.79


Lys
514
.
.
B
B
.
.
.
1.22
−0.53
.
*
F
0.75
0.77


Val
515
.
.
B
B
.
.
.
1.11
0.23
.
*
.
−0.30
0.61


Tyr
516
.
.
B
B
.
.
.
0.30
0.23
.
*
.
−0.15
1.07


Val
517
.
.
B
B
.
.
.
0.30
0.49
.
*
.
−0.60
0.44


Tyr
518
.
.
B
.
.
.
.
0.64
0.49
*
*
.
0.01
1.17


Glu
519
.
.
B
.
.
.
.
0.60
0.24
*
.
.
0.57
1.29


Leu
520
.
.
B
.
.
.
.
1.57
−0.11
.
.
.
1.43
2.80


Arg
521
A
.
.
.
.
T
.
1.11
−0.76
.
.
F
2.34
3.50


Gln
522
.
.
B
.
.
T
.
1.11
−0.73
.
.
F
2.60
1.75


Asn
523
.
.
B
.
.
T
.
1.11
−0.09
*
.
F
2.04
1.57


Arg
524
.
.
B
.
.
T
.
1.11
−0.01
*
*
F
1.78
1.26


Phe
525
.
.
B
.
.
.
.
1.58
0.39
*
.
.
0.57
1.17


Val
526
.
.
B
.
.
T
.
1.16
0.41
*
*
.
0.06
0.72


Tyr
527
.
.
B
.
.
T
.
0.34
0.50
.
*
.
−0.20
0.53


Asn
528
.
.
B
.
.
T
.
0.39
1.19
.
*
F
0.29
0.50


Gly
529
.
.
.
.
T
T
.
0.28
0.40
*
*
F
1.18
1.36


Thr
530
.
.
.
.
.
.
C
0.68
−0.24
.
.
F
2.02
1.45


Leu
531
.
.
.
.
.
T
C
1.50
−0.61
.
.
F
2.86
1.21


Lys
532
.
.
.
.
T
T
.
1.44
−0.51
.
*
F
3.40
1.66


Asp
533
.
.
B
.
.
T
.
1.20
−0.56
.
*
F
2.66
1.54


Ser
534
.
.
B
.
.
T
.
1.54
−0.29
.
.
F
2.02
2.93


His
535
.
.
B
.
.
T
.
1.86
−0.57
.
.
.
1.83
2.54


Ser
536
.
.
B
.
.
T
.
2.08
−0.17
.
*
.
1.19
2.44


Tyr
537
.
.
B
.
.
T
.
2.14
0.33
.
*
.
0.25
1.84


Gln
538
.
.
B
.
.
T
.
1.44
−0.06
.
*
.
0.85
2.65


Asn
539
.
.
B
.
.
.
.
1.40
0.23
.
*
.
0.05
1.71


Ala
540
.
.
B
.
.
.
.
1.13
0.27
.
*
.
0.05
1.08


Arg
541
.
.
B
.
.
.
.
1.13
−0.10
*
*
.
0.50
0.84


Phe
542
.
.
B
.
.
.
.
0.49
−0.11
*
*
F
0.65
0.70


Gly
543
.
.
.
.
T
T
.
−0.10
0.17
*
*
F
0.65
0.48


Ser
544
.
.
.
.
.
T
C
−0.40
0.17
*
*
F
0.45
0.25


Ser
545
.
.
B
.
.
T
.
−0.67
0.56
.
*
F
−0.05
0.39


Ile
546
.
.
B
.
.
T
.
−0.67
0.41
*
*
.
−0.20
0.29


Ala
547
.
.
B
B
.
.
.
0.03
−0.01
*
.
.
0.30
0.42


Ser
548
.
.
B
B
.
.
.
−0.43
−0.40
.
.
.
0.30
0.53


Val
549
.
.
B
B
.
.
.
−0.13
−0.10
*
.
.
0.64
0.62


Arg
550
.
.
B
B
.
.
.
0.17
−0.39
*
.
F
1.13
0.99


Asp
551
.
.
B
.
.
.
.
1.06
−0.49
*
.
F
1.82
1.28


Leu
552
.
.
B
.
.
.
.
1.34
−0.87
*
.
F
2.46
2.88


Asn
553
.
.
.
.
T
T
.
1.40
−1.13
*
.
F
3.40
1.97


Gln
554
.
.
.
.
T
T
.
2.26
−0.37
*
.
F
2.76
1.85


Asp
555
.
.
.
.
T
T
.
2.14
0.03
*
.
F
1.82
3.60


Ser
556
.
.
.
.
T
T
.
1.29
−0.66
*
.
F
2.38
3.74


Tyr
557
.
.
.
B
T
.
.
1.24
−0.41
.
.
F
1.34
1.60


Asn
558
.
.
B
B
.
.
.
0.39
−0.17
.
.
F
0.45
0.71


Asp
559
.
.
B
B
.
.
.
0.04
0.47
.
.
.
−0.60
0.39


Val
560
.
.
B
B
.
.
.
−0.54
0.51
*
.
.
−0.60
0.25


Val
561
.
.
B
B
.
.
.
−0.46
0.26
.
.
.
−0.30
0.16


Val
562
.
.
B
B
.
.
.
−1.02
0.29
.
.
.
−0.30
0.14


Gly
563
.
.
B
B
.
.
.
−1.02
0.97
.
.
.
−0.60
0.16


Ala
564
.
A
B
.
.
.
.
−1.02
0.33
.
.
.
−0.30
0.38


Pro
565
A
A
.
.
.
.
.
−0.17
−0.31
.
.
.
0.30
0.85


Leu
566
A
A
.
.
.
.
.
0.66
−0.56
.
.
.
0.75
1.37


Glu
567
A
A
.
.
.
.
.
0.92
−0.49
.
.
F
0.60
1.85


Asp
568
A
A
.
.
.
.
.
0.92
−0.49
.
.
.
0.45
1.21


Asn
569
A
A
.
.
.
.
.
0.92
−0.49
.
.
.
0.45
1.45


His
570
A
A
.
.
.
.
.
0.24
−0.67
.
.
.
0.60
0.85


Ala
571
A
.
.
B
.
.
.
0.81
0.01
.
.
.
−0.30
0.36


Gly
572
A
.
.
B
.
.
.
−0.08
0.77
.
.
.
−0.60
0.35


Ala
573
A
.
.
B
.
.
.
−0.78
1.06
.
.
.
−0.60
0.18


Ile
574
.
.
B
B
.
.
.
−0.81
1.34
.
.
.
−0.60
0.15


Tyr
575
.
.
B
B
.
.
.
−1.12
1.34
.
.
.
−0.60
0.21


Ile
576
.
.
B
B
.
.
.
−1.23
1.34
*
*
.
−0.60
0.21


Phe
577
.
.
B
B
.
.
.
−0.78
1.63
*
*
.
−0.60
0.25


His
578
.
.
B
B
.
.
.
−0.53
0.94
*
*
.
−0.60
0.32


Gly
579
.
.
.
B
T
.
.
0.06
0.61
*
*
.
−0.20
0.45


Phe
580
.
.
.
.
T
T
.
−0.59
0.31
*
*
.
0.50
0.69


Arg
581
.
.
.
.
T
T
.
−0.51
0.21
*
*
F
0.65
0.36


Gly
582
.
.
.
.
T
T
.
0.23
0.40
.
.
F
0.35
0.30


Ser
583
.
.
.
.
T
T
.
−0.04
−0.03
.
.
F
1.25
0.69


Ile
584
.
.
.
.
.
.
C
0.09
−0.33
*
*
F
1.15
0.51


Leu
585
.
.
.
.
.
.
C
0.83
0.10
*
*
F
0.85
0.79


Lys
586
.
.
.
.
.
.
C
0.72
−0.33
.
*
F
1.90
1.18


Thr
587
.
.
.
.
.
T
C
1.18
−0.31
*
*
F
2.40
2.92


Pro
588
.
.
.
.
.
T
C
0.59
−1.00
*
*
F
3.00
6.94


Lys
589
.
.
B
.
.
T
.
1.17
−1.00
*
*
F
2.50
2.43


Gln
590
.
.
B
.
.
T
.
1.39
−0.51
*
*
F
2.20
2.43


Arg
591
.
.
B
B
.
.
.
1.04
−0.50
.
*
F
1.20
1.59


Ile
592
.
.
B
B
.
.
.
1.36
−0.54
.
*
F
1.20
1.06


Thr
593
.
A
B
B
.
.
.
0.76
−0.54
.
*
F
0.90
1.06


Ala
594
.
A
B
B
.
.
.
0.12
−0.26
.
*
F
0.45
0.45


Ser
595
.
A
B
.
.
.
.
−0.19
0.24
.
.
F
−0.15
0.65


Glu
596
.
A
B
.
.
.
.
−0.64
0.04
.
*
F
−0.15
0.65


Leu
597
A
A
.
B
.
.
.
−0.57
−0.01
.
.
F
0.45
0.63


Ala
598
A
A
.
B
.
.
.
−0.26
0.17
*
.
.
−0.30
0.39


Thr
599
A
A
.
B
.
.
.
0.09
0.19
*
.
.
−0.30
0.39


Gly
600
A
A
.
B
.
.
.
−0.31
0.94
*
.
.
−0.60
0.74


Leu
601
.
.
B
B
.
.
.
−0.66
1.04
*
.
.
−0.60
0.63


Gln
602
.
.
B
B
.
.
.
−0.51
0.97
*
.
.
−0.60
0.44


Tyr
603
.
.
.
.
T
T
.
−0.22
1.06
*
.
.
0.20
0.24


Phe
604
.
.
B
.
.
T
.
−0.80
1.01
*
.
.
−0.20
0.38


Gly
605
.
.
B
.
.
T
.
−0.49
1.01
*
.
.
−0.20
0.15


Cys
606
.
.
B
.
.
T
.
−0.02
1.11
.
*
.
−0.20
0.13


Ser
607
.
.
B
B
.
.
.
−0.02
0.79
.
*
.
−0.60
0.15


Ile
608
.
.
B
B
.
.
.
−0.59
0.40
.
*
.
−0.60
0.27


His
609
.
.
B
B
.
.
.
0.11
0.66
.
*
.
−0.60
0.41


Gly
610
.
.
B
B
.
.
.
−0.36
0.09
.
*
.
−0.30
0.52


Gln
611
.
.
B
.
.
.
.
0.31
0.39
.
*
.
−0.10
0.61


Leu
612
.
.
B
.
.
.
.
0.61
0.10
.
*
.
−0.10
0.72


Asp
613
.
.
B
.
.
.
.
1.50
−0.40
.
*
.
0.65
1.26


Leu
614
.
.
B
.
.
.
.
1.19
−0.83
.
*
.
0.95
1.21


Asn
615
A
.
.
.
.
T
.
0.72
−0.80
.
*
F
1.30
1.45


Glu
616
A
.
.
.
.
T
.
−0.17
−0.80
.
*
F
1.15
0.72


Asp
617
A
.
.
.
.
T
.
0.64
−0.11
.
*
F
0.85
0.61


Gly
618
A
.
.
.
.
T
.
−0.17
−0.80
.
*
F
1.15
0.63


Leu
619
A
.
.
B
.
.
.
0.06
−0.51
.
*
.
0.60
0.30


Ile
620
A
.
.
B
.
.
.
−0.80
−0.01
.
*
.
0.30
0.18


Asp
621
.
.
B
B
.
.
.
−1.14
0.63
.
*
.
−0.60
0.14


Leu
622
.
.
B
B
.
.
.
−1.73
0.63
*
*
.
−0.60
0.16


Ala
623
.
.
B
B
.
.
.
−2.20
0.44
.
.
.
−0.60
0.24


Val
624
A
.
.
B
.
.
.
−1.73
0.44
*
.
.
−0.60
0.12


Gly
625
A
A
.
.
.
.
.
−0.84
0.87
*
*
.
−0.60
0.14


Ala
626
A
A
.
.
.
.
.
−1.43
0.59
.
.
.
−0.60
0.22


Leu
627
A
A
.
.
.
.
.
−1.48
0.59
.
.
.
−0.60
0.30


Gly
628
A
A
.
.
.
.
.
−1.78
0.59
.
.
.
−0.60
0.23


Asn
629
.
A
B
B
.
.
.
−1.73
0.84
.
.
.
−0.60
0.16


Ala
630
.
A
B
B
.
.
.
−1.68
1.03
.
.
.
−0.60
0.16


Val
631
.
A
B
B
.
.
.
−1.39
1.26
*
.
.
−0.60
0.17


Ile
632
.
A
B
B
.
.
.
−0.47
1.21
*
.
.
−0.60
0.14


Leu
633
.
A
B
B
.
.
.
−0.33
0.81
*
.
.
−0.60
0.27


Trp
634
.
A
B
B
.
.
.
−1.19
0.74
*
.
.
−0.60
0.57


Ser
635
.
A
B
B
.
.
.
−1.46
0.74
*
.
.
−0.60
0.60


Arg
636
.
.
B
B
.
.
.
−0.60
0.70
*
*
F
−0.45
0.54


Pro
637
.
.
B
B
.
.
.
−0.60
0.41
*
*
.
−0.60
0.89


Val
638
.
.
B
B
.
.
.
0.21
0.19
*
*
.
−0.30
0.46


Val
639
.
.
B
B
.
.
.
−0.09
0.20
*
*
.
−0.30
0.38


Gln
640
.
.
B
B
.
.
.
−0.09
0.70
*
*
.
−0.60
0.25


Ile
641
.
.
B
B
.
.
.
−1.01
0.66
*
*
.
−0.60
0.45


Asn
642
.
.
B
.
.
T
.
−0.83
0.70
.
*
.
−0.20
0.50


Ala
643
.
.
B
.
.
T
.
−0.68
0.56
.
*
.
−0.20
0.39


Ser
644
.
.
B
.
.
T
.
0.18
0.94
.
*
.
−0.20
0.48


Leu
645
A
.
.
.
.
T
.
−0.03
0.26
.
*
.
0.10
0.52


His
646
A
.
.
.
.
.
.
0.56
0.29
.
*
.
0.18
0.80


Phe
647
A
.
.
.
.
.
.
0.60
0.17
.
*
.
0.46
0.80


Glu
648
A
.
.
.
.
T
.
0.30
−0.21
.
*
F
1.84
1.94


Pro
649
A
.
.
.
.
T
.
0.60
−0.21
.
*
F
1.97
1.00


Ser
650
.
.
.
.
T
T
.
0.52
−0.31
.
*
F
2.80
1.85


Lys
651
A
.
.
.
.
T
.
−0.14
−0.41
.
.
F
1.97
0.75


Ile
652
A
.
.
B
.
.
.
0.52
0.37
*
*
F
0.69
0.42


Asn
653
A
.
.
B
.
.
.
0.63
0.44
*
*
.
−0.04
0.43


Ile
654
A
.
.
B
.
.
.
0.84
0.06
*
*
.
−0.02
0.42


Phe
655
.
.
B
B
.
.
.
0.48
0.06
*
*
.
0.04
1.00


His
656
.
.
B
.
.
T
.
0.48
−0.06
*
*
.
1.38
0.33


Arg
657
.
.
B
.
.
T
.
1.48
−0.46
*
*
.
1.72
0.95


Asp
658
.
.
.
.
T
T
.
1.18
−1.14
*
.
F
3.06
2.14


Cys
659
.
.
.
.
T
T
.
1.72
−1.54
*
.
F
3.40
2.11


Lys
660
.
.
.
.
T
.
.
2.53
−1.61
*
.
F
2.86
1.07


Arg
661
.
.
.
.
T
T
.
2.57
−1.61
*
.
F
3.03
1.25


Ser
662
.
.
.
.
T
T
.
1.87
−1.61
*
.
F
3.00
3.90


Gly
663
.
.
.
.
T
T
.
1.56
−1.69
*
.
F
2.97
1.97


Arg
664
.
.
.
.
T
T
.
1.56
−1.20
*
.
F
2.94
1.45


Asp
665
.
.
.
.
T
T
.
0.70
−0.63
*
.
F
3.10
0.58


Ala
666
.
.
B
.
.
T
.
0.00
−0.33
*
.
F
2.09
0.48


Thr
667
.
.
B
.
.
T
.
−0.29
−0.26
.
.
.
1.63
0.25


Cys
668
.
.
B
.
.
T
.
−0.64
0.24
*
.
.
0.72
0.15


Leu
669
A
A
.
.
.
.
.
−1.57
1.03
*
.
.
−0.29
0.13


Ala
670
A
A
.
.
.
.
.
−2.23
1.21
.
.
.
−0.60
0.07


Ala
671
A
A
.
.
.
.
.
−2.34
1.30
.
.
.
−0.60
0.07


Phe
672
A
A
.
.
.
.
.
−2.34
1.51
.
.
.
−0.60
0.08


Leu
673
A
A
.
.
.
.
.
−1.89
1.31
.
.
.
−0.60
0.11


Cys
674
.
A
B
.
.
.
.
−1.97
1.24
.
.
.
−0.60
0.17


Phe
675
.
.
B
B
.
.
.
−2.08
1.43
.
.
.
−0.60
0.14


Thr
676
.
.
B
B
.
.
.
−2.30
1.43
.
.
.
−0.60
0.14


Pro
677
.
.
B
B
.
.
.
−2.19
1.43
.
.
.
−0.60
0.22


Ile
678
.
A
.
B
T
.
.
−1.59
1.36
.
.
.
−0.20
0.26


Phe
679
.
A
B
B
.
.
.
−0.96
1.00
.
.
.
−0.60
0.28


Leu
680
.
A
B
B
.
.
.
−0.96
1.01
.
.
.
−0.60
0.24


Ala
681
.
A
.
B
.
.
C
−0.64
1.37
.
.
.
−0.40
0.30


Pro
682
.
A
.
B
.
.
C
−0.74
1.09
.
.
.
−0.40
0.60


His
683
.
A
.
B
T
.
.
−0.17
0.79
.
.
.
−0.05
1.05


Phe
684
.
A
.
B
T
.
.
0.22
0.59
.
.
.
−0.05
1.51


Gln
685
.
A
B
B
.
.
.
0.18
0.57
.
.
F
−0.30
1.41


Thr
686
.
A
B
B
.
.
.
0.42
0.79
.
.
F
−0.45
0.77


Thr
687
.
.
B
B
.
.
.
−0.26
0.71
*
*
F
−0.45
0.88


Thr
688
.
.
B
B
.
.
.
−0.11
0.61
*
*
F
−0.45
0.35


Val
689
.
.
B
B
.
.
.
0.34
0.21
.
*
.
−0.30
0.48


Gly
690
.
.
B
B
.
.
.
0.34
0.49
.
*
.
−0.60
0.52


Ile
691
.
.
B
B
.
.
.
0.07
0.40
.
*
.
−0.60
0.58


Arg
692
.
.
B
B
.
.
.
0.07
0.41
.
*
.
−0.60
0.79


Tyr
693
.
.
B
B
.
.
.
−0.22
0.26
.
*
.
−0.15
1.16


Asn
694
.
A
B
B
.
.
.
0.63
0.44
.
*
.
−0.45
1.63


Ala
695
.
A
B
B
.
.
.
0.98
−0.24
*
*
.
0.45
1.39


Thr
696
.
A
B
B
.
.
.
1.98
−0.24
.
*
.
0.45
1.54


Met
697
.
A
B
B
.
.
.
1.98
−1.00
.
*
F
0.90
1.87


Asp
698
.
A
B
.
.
.
.
1.98
−1.40
.
.
F
0.90
3.63


Glu
699
.
A
B
.
.
.
.
1.67
−1.14
.
.
F
1.20
3.94


Arg
700
.
A
B
.
.
.
.
2.04
−1.14
*
*
F
1.50
5.75


Arg
701
.
A
.
.
T
.
.
2.47
−1.33
*
*
F
2.20
5.33


Tyr
702
.
A
.
.
T
.
.
2.48
−1.33
*
*
F
2.50
6.02


Thr
703
.
.
.
.
.
T
C
2.44
−0.83
.
*
F
3.00
3.11


Pro
704
.
.
.
.
.
T
C
1.63
−0.33
.
*
F
2.40
2.16


Arg
705
.
.
B
.
.
T
.
1.52
0.36
.
*
.
1.15
1.14


Ala
706
.
.
B
.
.
T
.
1.41
−0.40
*
*
.
1.45
1.31


His
707
.
.
B
.
.
.
.
1.31
−0.89
.
*
.
1.59
1.47


Leu
708
.
.
B
.
.
.
.
1.28
−0.89
*
*
F
1.63
0.74


Asp
709
.
.
B
.
.
T
.
1.49
−0.46
.
*
F
1.87
0.73


Glu
710
.
.
.
.
T
T
.
1.49
−0.96
*
*
F
2.91
0.89


Gly
711
.
.
.
.
T
T
.
1.38
−1.46
*
*
F
3.40
2.12


Gly
712
.
.
.
.
T
T
.
1.10
−1.36
*
.
F
3.06
1.10


Asp
713
.
.
.
.
T
.
.
1.91
−0.87
*
*
F
2.37
0.92


Arg
714
A
.
.
.
.
.
.
2.02
−0.47
*
*
F
1.48
1.49


Phe
715
A
.
.
.
.
.
.
1.43
−0.90
*
*
F
1.44
2.95


Thr
716
.
.
B
.
.
.
.
0.92
−0.83
*
*
F
1.10
1.79


Asn
717
.
A
B
.
.
.
.
0.46
−0.19
*
*
F
0.45
0.68


Arg
718
.
A
B
.
.
.
.
−0.36
0.50
*
*
F
−0.45
0.64


Ala
719
.
A
B
.
.
.
.
−0.77
0.40
*
*
.
−0.60
0.37


Val
720
.
A
B
.
.
.
.
−0.37
0.30
.
.
.
−0.06
0.31


Leu
721
.
A
B
.
.
.
.
−0.40
0.29
.
.
.
0.18
0.21


Leu
722
.
A
B
.
.
.
.
−0.40
0.71
.
.
F
0.27
0.21


Ser
723
.
.
.
.
.
T
C
−0.51
0.61
.
.
F
1.11
0.48


Ser
724
.
.
.
.
.
T
C
−0.73
−0.03
.
.
F
2.40
1.01


Gly
725
.
.
.
.
.
T
C
−0.54
−0.03
*
.
F
2.16
1.01


Gln
726
A
.
.
.
.
T
.
0.27
−0.14
*
.
F
1.57
0.40


Glu
727
A
A
.
.
.
.
.
1.19
−0.53
*
*
F
1.23
0.52


Leu
728
A
A
.
.
.
.
.
0.60
−0.91
*
*
F
1.14
1.03


Cys
729
A
A
.
.
.
.
.
0.90
−0.66
*
*
.
0.60
0.42


Glu
730
A
A
.
.
.
.
.
0.54
−0.66
*
*
.
0.60
0.39


Arg
731
A
A
.
.
.
.
.
0.51
0.13
*
*
.
−0.30
0.41


Ile
732
A
A
.
.
.
.
.
−0.34
−0.06
*
*
.
0.45
1.03


Asn
733
A
A
.
.
.
.
.
−0.34
0.01
*
*
.
−0.30
0.44


Phe
734
A
A
.
.
.
.
.
0.32
0.70
*
*
.
−0.60
0.19


His
735
.
A
B
.
.
.
.
0.01
0.70
*
*
.
−0.60
0.44


Val
736
.
A
B
.
.
.
.
−0.69
0.50
*
*
.
−0.60
0.40


Leu
737
.
A
B
.
.
.
.
0.20
0.60
.
*
.
−0.60
0.46


Asp
738
A
A
.
.
.
.
.
−0.04
−0.19
.
.
F
0.62
0.57


Thr
739
A
.
.
.
.
T
.
−0.20
0.07
.
.
F
0.74
1.20


Ala
740
A
.
.
.
.
T
.
−0.12
0.07
*
.
F
0.91
1.08


Asp
741
A
.
.
.
.
T
.
0.52
−0.61
*
.
.
1.83
1.30


Tyr
742
.
.
B
.
.
T
.
0.48
−0.19
*
.
.
1.70
1.39


Val
743
.
.
B
B
.
.
.
0.17
−0.03
.
.
.
1.13
1.02


Lys
744
.
.
B
B
.
.
.
−0.22
−0.04
.
.
F
0.96
0.88


Pro
745
.
.
B
B
.
.
.
0.07
0.74
*
.
F
−0.11
0.49


Val
746
.
.
B
B
.
.
.
−0.79
0.37
*
.
.
−0.13
0.88


Thr
747
.
.
B
B
.
.
.
−0.54
0.37
*
*
.
−0.30
0.33


Phe
748
.
.
B
B
.
.
.
0.07
0.37
.
*
.
−0.30
0.37


Ser
749
.
.
B
B
.
.
.
−0.28
0.70
.
*
.
−0.60
0.77


Val
750
.
.
B
B
.
.
.
−0.88
0.44
.
*
.
−0.60
0.72


Glu
751
.
.
B
B
.
.
.
−0.02
0.64
.
*
.
−0.60
0.68


Tyr
752
.
.
B
.
.
.
.
0.29
−0.14
.
*
.
0.50
0.88


Ser
753
.
.
.
.
.
.
C
0.78
−0.53
.
*
.
1.49
1.99


Leu
754
.
.
.
.
T
.
.
1.08
−0.74
*
.
.
2.03
1.78


Glu
755
A
.
.
.
.
.
.
1.90
−0.74
*
.
F
2.12
1.89


Asp
756
A
.
.
.
.
T
.
1.56
−1.00
.
.
F
2.66
1.92


Pro
757
.
.
.
.
T
T
.
1.59
−0.96
.
.
F
3.40
2.31


Asp
758
.
.
.
.
T
T
.
1.29
−1.21
.
.
F
3.06
2.06


His
759
.
.
.
.
.
T
C
1.29
−0.60
.
.
F
2.52
1.22


Gly
760
.
.
.
.
.
.
C
1.29
0.09
.
.
F
0.93
0.65


Pro
761
.
.
B
.
.
.
.
1.29
−0.34
.
.
F
0.99
0.65


Met
762
.
.
B
.
.
.
.
1.16
−0.34
*
.
.
0.50
0.80


Leu
763
.
.
B
.
.
.
.
0.87
−0.41
*
.
F
0.89
0.80


Asp
764
.
.
.
.
T
T
.
0.69
0.07
.
.
F
1.13
0.54


Asp
765
.
.
.
.
T
T
.
0.72
0.07
.
.
F
1.37
0.85


Gly
766
.
.
.
.
T
T
.
0.62
−0.06
.
.
F
2.36
1.48


Trp
767
.
.
.
.
.
T
C
0.41
−0.26
*
*
F
2.40
1.28


Pro
768
.
.
.
B
.
.
C
1.33
0.43
*
*
F
0.71
0.63


Thr
769
.
.
B
B
.
.
.
0.48
0.43
*
*
F
0.42
1.25


Thr
770
.
.
B
B
.
.
.
0.18
0.64
*
*
F
0.03
0.89


Leu
771
.
.
B
B
.
.
.
−0.33
0.11
.
*
.
−0.06
0.77


Arg
772
.
.
B
B
.
.
.
−0.26
0.33
*
*
.
−0.30
0.39


Val
773
.
.
B
B
.
.
.
−0.74
0.27
*
*
.
−0.30
0.42


Ser
774
.
.
B
B
.
.
.
−0.72
0.57
*
*
.
−0.60
0.44


Val
775
.
.
B
B
.
.
.
−0.41
0.80
*
*
.
−0.60
0.24


Pro
776
.
.
B
B
.
.
.
0.06
1.20
*
*
.
−0.60
0.52


Phe
777
.
.
.
B
T
.
.
−0.72
0.99
*
*
.
−0.20
0.38


Trp
778
.
.
.
.
T
T
.
0.13
1.17
.
.
.
0.20
0.28


Asn
779
.
.
.
.
.
T
C
0.43
0.93
.
.
.
0.00
0.29


Gly
780
.
.
.
.
T
T
.
1.29
0.50
.
.
.
0.20
0.57


Cys
781
.
.
.
.
T
T
.
1.50
−0.29
.
.
F
1.25
0.91


Asn
782
.
A
.
.
T
.
.
2.17
−1.20
.
.
F
1.15
0.98


Glu
783
.
A
.
.
T
.
.
1.79
−1.10
.
.
F
1.30
1.35


Asp
784
.
A
.
.
T
.
.
0.93
−0.96
.
.
F
1.30
1.35


Glu
785
.
A
.
.
T
.
.
1.07
−0.89
.
.
F
1.15
0.62


His
786
.
A
.
.
T
.
.
1.73
−0.86
*
.
.
1.00
0.56


Cys
787
A
A
.
.
.
.
.
0.92
−0.86
*
.
.
0.60
0.56


Val
788
A
.
.
.
.
.
.
0.07
−0.17
.
.
.
0.50
0.26


Pro
789
A
.
.
.
.
.
.
−0.74
0.47
.
.
F
−0.25
0.14


Asp
790
A
A
.
.
.
.
.
−0.74
0.66
*
.
F
−0.45
0.22


Leu
791
A
A
.
.
.
.
.
−1.30
0.09
*
*
.
−0.30
0.50


Val
792
A
A
.
.
.
.
.
−0.52
−0.06
*
*
.
0.30
0.33


Leu
793
A
A
.
.
.
.
.
0.03
−0.49
*
*
.
0.30
0.38


Asp
794
.
A
B
.
.
.
.
0.24
−0.10
.
*
.
0.30
0.62


Ala
795
A
A
.
.
.
.
.
−0.57
−0.79
.
*
F
0.90
1.40


Arg
796
A
.
.
.
.
T
.
0.03
−0.74
.
*
F
1.30
1.40


Ser
797
A
.
.
.
.
T
.
0.58
−1.00
.
*
F
1.30
1.29


Asp
798
A
.
.
.
.
T
C
0.80
−0.51
.
*
F
1.50
1.85


Leu
799
.
.
.
.
.
T
C
0.20
−0.51
*
*
F
1.35
0.95


Pro
800
.
A
.
.
.
.
C
0.79
0.10
*
*
F
0.05
0.70


Thr
801
A
A
.
.
.
.
.
0.43
−0.29
.
*
.
0.30
0.73


Ala
802
A
A
.
.
.
.
.
0.07
0.47
.
.
.
−0.45
1.39


Met
803
A
A
.
.
.
.
.
0.07
0.36
*
.
.
−0.30
0.48


Glu
804
A
A
.
.
.
.
.
0.99
0.33
*
*
.
−0.30
0.58


Tyr
805
A
.
.
B
.
.
.
0.34
−0.16
*
.
.
0.45
1.12


Cys
806
A
.
.
B
.
.
.
−0.16
−0.01
*
.
.
0.30
0.84


Gln
807
A
.
.
B
.
.
.
0.54
0.06
*
.
.
−0.30
0.40


Arg
808
A
.
.
B
.
.
.
1.19
0.06
*
.
.
−0.30
0.50


Val
809
A
.
.
B
.
.
.
0.98
−0.70
*
.
.
0.75
1.86


Leu
810
.
.
B
B
.
.
.
0.63
−0.84
*
.
F
1.20
1.66


Arg
811
.
.
B
B
.
.
.
1.30
−0.74
*
.
F
1.35
0.86


Lys
812
.
.
B
B
.
.
.
1.30
−0.34
*
.
F
1.50
2.00


Pro
813
.
.
.
.
T
.
.
0.52
−0.99
*
.
F
2.70
4.06


Ala
814
.
.
.
.
T
.
.
1.08
−1.10
*
.
F
3.00
1.11


Gln
815
.
.
B
.
.
T
.
1.30
−0.71
*
.
F
2.35
0.74


Asp
816
.
.
B
.
.
T
.
0.94
−0.21
*
.
F
1.75
0.49


Cys
817
.
.
B
.
.
T
.
0.59
0.11
.
.
.
0.70
0.75


Ser
818
.
.
B
.
.
T
.
−0.01
0.10
.
.
.
0.40
0.63


Ala
819
.
.
B
B
.
.
.
0.28
0.39
.
.
.
−0.30
0.31


Tyr
820
.
.
B
B
.
.
.
−0.42
0.77
.
.
.
−0.60
0.78


Thr
821
.
.
B
B
.
.
.
−0.42
0.99
.
.
.
−0.60
0.50


Leu
822
.
.
B
B
.
.
.
−0.07
0.60
.
.
.
−0.60
0.83


Ser
823
.
.
B
B
.
.
.
−0.08
0.59
.
*
.
−0.60
0.76


Phe
824
.
.
B
B
.
.
.
−0.34
0.31
.
*
.
−0.30
0.76


Asp
825
.
.
B
B
.
.
.
−0.80
0.47
.
*
F
−0.45
0.69


Thr
826
.
.
B
B
.
.
.
−1.38
0.57
.
*
F
−0.45
0.44


Thr
827
.
.
B
B
.
.
.
−1.46
0.87
.
*
F
−0.45
0.36


Val
828
.
.
B
B
.
.
.
−1.16
0.77
.
.
.
−0.60
0.15


Phe
829
.
.
B
B
.
.
.
−0.76
0.77
.
.
.
−0.60
0.18


Ile
830
.
.
B
B
.
.
.
−1.07
0.67
.
.
.
−0.60
0.17


Ile
831
.
.
B
B
.
.
.
−0.64
0.67
.
.
.
−0.60
0.33


Glu
832
A
.
.
B
.
.
.
−0.33
0.03
.
*
F
−0.15
0.74


Ser
833
A
.
.
.
.
T
.
0.63
−0.36
.
*
F
1.00
1.83


Thr
834
A
.
.
.
.
T
.
0.48
−1.04
.
*
F
1.30
5.10


Arg
835
A
.
.
.
.
T
.
0.78
−1.09
.
*
F
1.30
2.19


Gln
836
A
.
.
.
.
T
.
0.81
−0.59
.
*
F
1.30
1.65


Arg
837
A
A
.
.
.
.
.
0.81
−0.33
.
*
F
0.45
0.85


Val
838
.
A
B
.
.
.
.
0.52
−0.81
.
*
.
0.60
0.75


Ala
839
.
A
B
.
.
.
.
0.52
−0.31
.
*
.
0.30
0.44


Val
840
.
A
B
.
.
.
.
−0.40
−0.23
.
*
.
0.30
0.32


Glu
841
A
A
.
.
.
.
.
−0.40
0.46
.
*
.
−0.60
0.36


Ala
842
A
A
.
.
.
.
.
−0.51
−0.19
.
*
.
0.30
0.61


Thr
843
A
A
.
.
.
.
.
0.46
−0.29
.
*
.
0.45
1.33


Leu
844
A
A
.
.
.
.
.
0.70
−0.93
.
*
F
0.90
1.50


Glu
845
A
A
.
.
.
.
.
1.56
−0.50
.
*
F
0.60
1.47


Asn
846
A
.
.
.
.
T
.
1.56
−1.00
.
*
F
1.60
1.77


Arg
847
A
.
.
.
.
T
.
1.56
−1.09
*
*
F
1.90
3.45


Gly
848
A
.
.
.
.
T
.
1.62
−1.27
*
*
F
2.20
2.01


Glu
849
A
.
.
.
.
T
.
2.13
−0.51
*
*
F
2.50
1.96


Asn
850
.
.
.
.
.
T
C
1.82
−0.53
*
.
F
3.00
1.34


Ala
851
A
.
.
.
.
T
.
0.97
−0.04
*
.
F
2.20
1.95


Tyr
852
.
.
B
.
.
T
.
0.04
0.17
*
.
.
1.00
0.84


Ser
853
.
.
B
.
.
T
.
0.39
0.86
*
.
.
0.40
0.43


Thr
854
.
.
B
B
.
.
.
−0.50
0.86
*
.
.
−0.30
0.68


Val
855
.
.
B
B
.
.
.
−0.80
1.04
*
.
.
−0.60
0.31


Leu
856
.
.
B
B
.
.
.
−0.21
0.67
*
.
.
−0.60
0.31


Asn
857
.
.
B
B
.
.
.
−0.27
0.69
*
.
.
−0.60
0.37


Ile
858
.
.
B
B
.
.
.
−0.56
0.59
*
.
F
−0.45
0.66


Ser
859
.
.
B
.
.
.
.
−0.24
0.44
.
*
F
−0.25
0.81


Gln
860
.
.
B
.
.
.
C
−0.20
0.16
.
*
F
0.25
0.81


Ser
861
.
.
.
.
.
T
C
0.61
0.44
.
*
F
0.15
0.96


Ala
862
.
.
B
.
.
T
.
−0.09
0.16
.
*
F
0.40
1.23


Asn
863
.
.
B
.
.
T
.
0.21
0.56
.
*
.
−0.20
0.62


Leu
864
A
.
.
.
.
T
.
0.21
0.66
.
.
.
−0.20
0.47


Gln
865
A
A
.
.
.
.
.
−0.60
0.66
.
*
.
−0.60
0.62


Phe
866
A
A
.
.
.
.
.
−1.19
0.84
.
*
.
−0.60
0.32


Ala
867
A
A
.
.
.
.
.
−0.60
1.13
*
*
.
−0.60
0.27


Ser
868
A
A
.
.
.
.
.
−0.56
0.84
*
*
.
−0.60
0.27


Leu
869
A
A
.
.
.
.
.
0.26
0.44
*
.
.
−0.60
0.62


Ile
870
A
A
.
.
.
.
.
0.26
−0.34
*
.
.
0.45
1.07


Gln
871
A
A
.
.
.
.
.
0.66
−0.84
*
.
F
1.24
1.33


Lys
872
A
A
.
.
.
.
.
1.24
−0.84
*
.
F
1.58
2.16


Glu
873
A
A
.
.
.
.
.
1.20
−1.53
*
.
F
1.92
5.14


Asp
874
.
.
.
.
T
T
.
1.71
−1.79
.
*
F
3.06
2.94


Ser
875
.
.
.
.
T
T
.
1.71
−1.80
.
*
F
3.40
1.97


Asp
876
.
.
.
.
T
T
.
1.71
−1.11
.
*
F
2.91
0.80


Gly
877
.
.
.
.
T
T
.
1.00
−1.11
.
*
F
2.57
0.83


Ser
878
A
A
.
.
.
.
.
0.14
−0.54
.
.
F
1.43
0.33


Ile
879
A
A
.
.
.
.
.
0.14
−0.29
.
*
.
0.64
0.15


Glu
880
A
A
.
.
.
.
.
0.44
0.11
.
.
.
−0.30
0.24


Cys
881
.
A
B
.
.
.
.
0.44
−0.31
.
*
.
0.30
0.31


Val
882
A
A
.
.
.
.
.
0.90
−0.70
*
.
.
0.60
0.76


Asn
883
A
A
.
.
.
.
.
1.31
−1.39
*
.
F
0.75
0.86


Glu
884
A
A
.
.
.
.
.
1.39
−1.39
*
*
F
0.90
3.15


Glu
885
A
A
.
.
.
.
.
1.39
−1.27
*
.
F
0.90
3.50


Arg
886
A
A
.
.
.
.
.
2.10
−1.51
*
.
F
0.90
3.77


Arg
887
A
A
.
.
.
.
.
2.96
−1.91
*
*
F
0.90
4.35


Leu
888
A
A
.
.
.
.
.
2.10
−1.51
*
.
F
0.90
4.35


Gln
889
A
A
.
.
.
.
.
1.43
−0.87
*
.
F
0.90
1.65


Lys
890
A
A
.
.
.
.
.
1.43
−0.30
*
.
F
0.45
0.45


Gln
891
.
A
B
.
.
.
.
0.47
0.10
*
.
.
−0.30
0.88


Val
892
.
A
B
.
.
.
.
0.06
0.06
*
*
.
−0.30
0.38


Cys
893
.
A
B
.
.
.
.
0.62
0.04
*
.
.
−0.30
0.25


Asn
894
.
.
B
.
.
T
.
0.41
0.80
*
.
.
−0.20
0.23


Val
895
.
.
B
.
.
T
.
−0.33
0.83
*
.
.
−0.20
0.48


Ser
896
.
.
B
.
.
T
.
−1.03
0.97
*
.
.
−0.20
0.77


Tyr
897
.
.
B
.
.
T
.
−0.07
1.19
*
.
.
−0.20
0.41


Pro
898
.
.
B
B
.
.
.
0.01
0.79
*
*
.
−0.45
1.09


Phe
899
A
A
.
B
.
.
.
0.06
0.64
*
*
.
−0.60
0.82


Phe
900
A
A
.
B
.
.
.
0.32
0.26
*
*
.
−0.15
1.05


Arg
901
A
A
.
B
.
.
.
0.67
0.00
*
*
.
−0.30
0.69


Ala
902
A
A
.
.
.
.
.
0.06
−0.43
*
*
.
0.45
1.59


Lys
903
A
A
.
.
.
.
.
−0.32
−0.57
*
*
F
0.90
1.36


Ala
904
A
A
.
.
.
.
.
−0.32
−0.86
*
*
F
0.75
0.70


Lys
905
A
A
.
B
.
.
.
0.49
−0.07
*
*
.
0.30
0.60


Val
906
A
A
.
B
.
.
.
−0.43
−0.57
*
*
.
0.60
0.59


Ala
907
A
A
.
B
.
.
.
0.16
0.11
.
*
.
−0.30
0.48


Phe
908
A
A
.
B
.
.
.
−0.59
−0.39
.
*
.
0.30
0.40


Arg
909
A
A
.
B
.
.
.
0.00
0.40
*
*
.
−0.60
0.47


Leu
910
A
A
.
.
.
.
.
−0.74
−0.24
*
*
.
0.30
0.80


Asp
911
A
A
.
.
.
.
.
−0.19
0.04
*
*
.
−0.30
0.80


Phe
912
A
A
.
.
.
.
.
0.44
−0.36
*
*
.
0.30
0.55


Glu
913
A
A
.
.
.
.
.
0.84
−0.36
*
*
.
0.45
1.33


Phe
914
A
A
.
.
.
.
.
−0.16
−0.66
*
*
.
0.75
1.07


Ser
915
A
.
.
.
.
T
.
−0.04
0.03
.
*
F
0.25
0.87


Lys
916
A
.
.
.
.
T
.
−0.86
0.03
.
*
F
0.25
0.43


Ser
917
A
.
.
.
.
T
.
−0.19
0.71
.
.
.
−0.20
0.41


Ile
918
A
.
.
.
.
T
.
−0.22
0.43
.
.
.
−0.20
0.42


Phe
919
A
A
.
.
.
.
.
−0.33
0.54
.
.
.
−0.60
0.28


Leu
920
A
A
.
.
.
.
.
−0.03
1.23
.
.
.
−0.60
0.18


His
921
A
A
.
.
.
.
.
−0.97
0.84
.
*
.
−0.60
0.43


His
922
A
A
.
.
.
.
.
−0.67
0.84
.
*
.
−0.60
0.35


Leu
923
A
A
.
.
.
.
.
−0.59
0.06
.
*
.
−0.30
0.74


Glu
924
A
A
.
.
.
.
.
−0.48
0.06
.
*
.
−0.30
0.45


Ile
925
A
A
.
.
.
.
.
−0.26
0.06
.
*
.
−0.30
0.33


Glu
926
A
A
.
.
.
.
.
−0.57
0.06
.
*
.
−0.30
0.41


Leu
927
A
A
.
.
.
.
.
−0.83
−0.20
.
*
.
0.30
0.23


Ala
928
A
A
.
.
.
.
.
−0.02
0.19
.
*
.
−0.30
0.44


Ala
929
A
A
.
.
.
.
.
−0.32
−0.50
.
*
.
0.30
0.43


Gly
930
A
.
.
.
.
T
.
0.57
−0.11
.
*
F
0.85
0.69


Ser
931
.
.
.
.
.
T
C
0.57
−0.40
*
*
F
1.20
1.11


Asp
932
.
.
.
.
.
T
C
1.49
−0.90
.
.
F
1.50
1.89


Ser
933
.
.
.
.
.
T
C
2.08
−1.40
.
.
F
1.84
3.75


Asn
934
.
.
.
.
.
.
C
2.37
−1.83
.
.
F
1.98
4.67


Glu
935
A
.
.
.
.
.
.
2.40
−1.83
*
*
F
2.12
3.75


Arg
936
A
.
.
.
.
.
.
2.74
−1.34
.
.
F
2.46
4.04


Asp
937
.
.
.
.
T
T
.
2.74
−1.73
.
*
F
3.40
5.02


Ser
938
.
.
.
.
.
T
C
3.04
−2.13
.
.
F
2.86
5.02


Thr
939
A
.
.
.
.
T
.
3.04
−2.13
.
.
F
2.32
4.28


Lys
940
A
.
.
.
.
T
.
2.19
−1.73
*
.
F
1.98
4.12


Glu
941
A
A
.
.
.
.
.
1.49
−1.09
*
.
F
1.24
2.28


Asp
942
A
A
.
.
.
.
.
1.28
−0.97
.
.
F
0.90
1.60


Asn
943
A
A
.
.
.
.
.
0.77
−1.03
.
*
F
0.90
1.24


Val
944
A
A
.
.
.
.
.
1.19
−0.34
.
*
.
0.30
0.59


Ala
945
A
A
.
.
.
.
.
0.44
−0.34
.
*
.
0.30
0.69


Pro
946
A
A
.
.
.
.
.
0.41
0.44
.
*
.
−0.60
0.37


Leu
947
A
A
.
.
.
.
.
−0.40
0.54
.
*
.
−0.60
0.68


Arg
948
A
A
.
.
.
.
.
−0.36
0.59
.
*
.
−0.60
0.56


Phe
949
A
A
.
.
.
.
.
0.26
0.09
.
*
.
−0.30
0.72


His
950
A
A
.
.
.
.
.
0.84
0.41
.
*
.
−0.45
1.37


Leu
951
A
A
.
.
.
.
.
0.47
−0.27
*
*
.
0.45
1.21


Lys
952
A
A
.
.
.
.
.
1.28
0.23
*
*
.
−0.15
1.41


Tyr
953
A
A
.
.
.
.
.
0.31
−0.56
*
*
.
0.75
1.73


Glu
954
A
A
.
.
.
.
.
0.20
−0.41
.
*
.
0.45
1.56


Ala
955
A
.
.
B
.
.
.
−0.47
−0.41
.
*
.
0.30
0.64


Asp
956
A
.
.
B
.
.
.
0.03
0.37
.
*
.
−0.30
0.35


Val
957
A
.
.
B
.
.
.
0.10
0.10
.
*
.
−0.30
0.30


Leu
958
A
.
.
B
.
.
.
0.04
0.10
.
.
.
−0.30
0.57


Phe
959
A
.
.
B
.
.
.
−0.26
−0.01
.
.
.
0.30
0.46


Thr
960
A
.
.
B
.
.
.
0.03
0.37
.
.
F
0.06
0.83


Arg
961
A
.
.
B
.
.
.
−0.78
0.11
.
.
F
0.42
1.35


Ser
962
.
.
.
.
T
T
.
−0.22
0.11
.
.
F
1.43
1.29


Ser
963
.
.
.
.
.
T
C
0.56
−0.29
.
.
F
2.04
1.19


Ser
964
.
.
.
.
.
T
C
1.01
−0.27
.
.
F
2.10
0.83


Leu
965
.
.
.
.
.
T
C
1.32
0.49
.
.
F
0.99
0.97


Ser
966
.
.
.
.
.
.
C
0.36
0.10
.
*
.
0.88
1.25


His
967
.
.
B
.
.
.
.
0.70
0.36
.
*
.
0.32
0.69


Tyr
968
.
.
B
.
.
.
.
0.19
−0.03
.
*
.
0.86
1.68


Glu
969
.
.
B
.
.
.
.
0.49
−0.03
.
*
.
0.65
1.04


Val
970
A
.
.
.
.
.
.
1.00
−0.01
.
*
.
0.65
1.22


Lys
971
A
.
.
.
.
.
.
1.00
−0.13
.
*
F
0.80
1.05


Leu
972
A
.
.
.
.
.
.
0.22
−0.50
.
*
F
0.65
0.81


Asn
973
.
.
.
.
.
T
C
0.47
0.19
*
*
F
0.45
0.90


Ser
974
A
.
.
.
.
T
.
0.58
−0.46
*
*
F
0.85
0.78


Ser
975
A
.
.
.
.
T
.
1.19
−0.46
*
*
F
1.00
1.85


Leu
976
.
.
B
.
.
T
.
1.14
−0.39
*
*
F
1.31
1.80


Glu
977
.
.
B
.
.
.
.
1.61
−0.79
*
.
F
1.72
2.25


Arg
978
.
.
B
.
.
T
.
0.72
−0.74
*
.
F
2.23
1.66


Tyr
979
.
.
B
.
.
T
.
0.68
−0.44
.
.
F
2.24
1.41


Asp
980
.
.
.
.
T
T
.
0.77
−0.70
.
.
F
3.10
0.81


Gly
981
.
.
.
.
T
T
.
1.37
−0.27
*
.
F
2.49
0.64


Ile
982
.
.
.
.
T
.
.
0.67
0.16
*
.
F
1.38
0.63


Gly
983
.
.
.
.
.
.
C
0.26
0.19
.
*
F
0.87
0.33


Pro
984
.
.
.
.
.
T
C
−0.17
0.57
*
.
F
0.46
0.44


Pro
985
.
.
.
.
T
T
.
−1.06
0.71
*
.
F
0.35
0.34


Phe
986
.
.
.
.
T
T
.
−1.41
0.71
*
*
.
0.20
0.24


Ser
987
.
.
B
.
.
T
.
−0.41
1.07
*
*
.
−0.20
0.13


Cys
988
.
.
B
B
.
.
.
−0.96
0.64
*
*
.
−0.60
0.17


Ile
989
.
.
B
B
.
.
.
−0.74
0.90
*
*
.
−0.60
0.14


Phe
990
.
.
B
B
.
.
.
−0.53
0.51
*
*
.
−0.60
0.18


Arg
991
.
.
B
B
.
.
.
−0.64
0.53
*
*
.
−0.60
0.53


Ile
992
.
.
B
B
.
.
.
−0.69
0.64
*
*
.
−0.60
0.63


Gln
993
.
.
B
B
.
.
.
−0.83
0.39
*
*
.
−0.30
0.72


Asn
994
.
.
.
B
T
.
.
−0.64
0.29
*
*
.
0.10
0.30


Leu
995
.
.
.
B
T
.
.
−0.16
1.07
.
*
.
−0.20
0.37


Gly
996
.
.
.
B
T
.
.
−1.16
0.81
*
*
.
−0.20
0.33


Leu
997
.
.
.
.
.
.
C
−0.30
1.10
.
.
.
−0.20
0.14


Phe
998
.
.
B
.
.
.
.
−0.64
1.20
.
.
.
−0.40
0.24


Pro
999
.
.
B
.
.
.
.
−1.53
0.94
.
.
.
−0.40
0.24


Ile
1000
A
.
.
B
.
.
.
−1.32
1.20
.
.
.
−0.60
0.20


His
1001
A
.
.
B
.
.
.
−1.58
1.13
.
.
.
−0.60
0.23


Gly
1002
A
.
.
B
.
.
.
−0.72
0.96
*
.
.
−0.60
0.15


Ile
1003
A
.
.
B
.
.
.
−0.91
0.53
.
*
.
−0.60
0.42


Met
1004
A
.
.
B
.
.
.
−1.01
0.53
.
*
.
−0.60
0.22


Met
1005
.
.
B
B
.
.
.
−1.01
0.51
.
*
.
−0.60
0.32


Lys
1006
.
.
B
B
.
.
.
−1.19
0.77
.
*
.
−0.60
0.32


Ile
1007
.
.
B
B
.
.
.
−1.73
0.51
.
*
.
−0.60
0.50


Thr
1008
.
.
B
B
.
.
.
−1.43
0.59
.
*
.
−0.60
0.35


Ile
1009
.
.
B
B
.
.
.
−1.14
0.47
*
*
.
−0.60
0.18


Pro
1010
.
.
B
B
.
.
.
−0.43
0.96
*
*
.
−0.60
0.37


Ile
1011
.
.
B
B
.
.
.
−0.78
0.27
*
*
.
0.04
0.50


Ala
1012
.
.
B
B
.
.
.
−0.23
0.17
*
*
.
0.38
0.95


Thr
1013
.
.
B
.
.
T
.
0.08
−0.09
*
*
F
1.87
0.61


Arg
1014
.
.
.
.
T
T
.
1.08
−0.11
*
*
F
2.76
1.40


Ser
1015
.
.
.
.
T
T
.
0.48
−0.80
*
*
F
3.40
2.71


Gly
1016
.
.
.
.
T
T
.
0.56
−0.61
*
*
F
3.06
1.55


Asn
1017
.
A
.
.
T
.
.
1.19
−0.41
*
.
F
1.87
0.65


Arg
1018
.
A
B
.
.
.
.
0.69
−0.41
*
*
F
1.13
0.97


Leu
1019
.
A
B
.
.
.
.
0.69
−0.11
*
*
F
0.79
0.81


Leu
1020
.
A
B
.
.
.
.
0.99
−0.54
.
.
F
0.75
0.99


Lys
1021
.
A
B
.
.
.
.
0.63
−0.94
*
.
F
0.75
0.84


Leu
1022
.
A
B
.
.
.
.
−0.18
−0.16
*
.
F
0.45
0.88


Arg
1023
.
A
B
.
.
.
.
−0.60
−0.16
*
.
F
0.45
0.88


Asp
1024
.
A
B
.
.
.
.
0.21
−0.36
*
*
F
0.45
0.64


Phe
1025
.
A
B
.
.
.
.
1.02
−0.36
*
*
.
0.45
1.29


Leu
1026
A
A
.
.
.
.
.
0.12
−1.04
*
*
F
0.90
1.14


Thr
1027
A
A
.
.
.
.
.
0.34
−0.40
*
.
F
0.45
0.51


Asp
1028
A
A
.
.
.
.
.
0.23
0.10
.
.
F
−0.15
0.59


Glu
1029
A
A
.
.
.
.
.
−0.08
−0.29
*
.
.
0.45
1.15


Val
1030
A
A
.
.
.
.
.
0.32
−0.49
.
.
.
0.45
1.15


Ala
1031
A
A
.
.
.
.
.
0.47
−0.59
*
.
.
0.60
0.93


Asn
1032
.
.
.
.
T
T
.
0.78
−0.01
*
.
F
1.25
0.29


Thr
1033
.
.
.
.
T
T
.
−0.11
0.39
*
.
F
0.65
0.62


Ser
1034
.
.
B
.
.
T
.
−0.40
0.43
*
.
F
−0.05
0.43


Cys
1035
.
.
B
.
.
T
.
0.11
0.84
*
*
.
−0.20
0.28


Asn
1036
.
.
.
.
T
.
.
0.70
0.87
.
*
.
0.16
0.19


Ile
1037
.
.
.
.
T
.
.
0.40
0.79
.
*
.
0.32
0.23


Trp
1038
.
.
.
.
T
T
.
0.40
0.79
.
.
.
0.68
0.58


Gly
1039
.
.
.
.
.
T
C
0.70
0.70
.
.
F
0.79
0.52


Asn
1040
.
.
.
.
T
T
.
1.12
0.30
.
*
F
1.60
1.28


Ser
1041
.
.
.
.
.
T
C
1.23
0.37
.
*
F
1.24
1.91


Thr
1042
.
.
.
.
.
.
C
1.91
−0.54
.
*
F
1.78
3.79


Glu
1043
.
.
.
.
T
.
.
1.89
−0.54
.
*
F
1.82
3.64


Tyr
1044
.
.
.
.
T
.
.
2.02
−0.46
.
.
F
1.66
3.92


Arg
1045
.
.
.
.
.
T
C
1.17
−0.41
.
.
F
1.80
4.20


Pro
1046
.
.
.
.
.
T
C
1.47
−0.26
.
.
F
2.10
1.80


Thr
1047
.
.
.
.
.
T
C
1.78
−0.26
.
.
F
2.40
1.99


Pro
1048
.
.
.
.
.
T
C
1.78
−1.01
.
*
F
3.00
1.76


Val
1049
A
A
.
.
.
.
.
1.21
−1.01
.
*
F
2.10
1.90


Glu
1050
A
A
.
.
.
.
.
1.21
−0.76
*
.
F
1.80
1.09


Glu
1051
A
A
.
.
.
.
.
1.53
−1.24
*
.
F
1.50
1.38


Asp
1052
A
A
.
.
.
.
.
1.26
−1.67
*
.
F
1.20
3.63


Leu
1053
A
A
.
.
.
.
.
1.26
−1.81
*
.
F
0.90
2.12


Arg
1054
A
A
.
.
.
.
.
2.11
−1.39
*
*
F
0.90
1.89


Arg
1055
A
A
.
.
.
.
.
1.30
−0.99
*
*
F
0.90
1.96


Ala
1056
A
A
.
.
.
.
.
1.30
−0.30
*
.
F
0.60
1.96


Pro
1057
A
A
.
.
.
.
.
1.27
−0.59
*
*
F
0.90
1.61


Gln
1058
A
A
.
.
.
.
.
1.78
−0.09
*
.
F
0.60
1.12


Leu
1059
.
A
B
.
.
.
.
1.67
0.30
*
.
.
0.13
1.48


Asn
1060
.
A
.
.
.
.
C
1.26
0.20
.
.
.
0.61
1.54


His
1061
.
.
.
.
.
T
C
1.84
0.16
.
.
F
1.44
1.19


Ser
1062
.
.
.
.
.
T
C
1.20
−0.24
.
.
F
2.32
2.42


Asn
1063
.
.
.
.
T
T
.
0.34
−0.29
*
.
F
2.80
1.12


Ser
1064
.
.
.
.
T
T
.
0.86
−0.04
*
.
F
2.37
0.61


Asp
1065
.
.
B
B
.
.
.
−0.03
−0.16
.
.
F
1.29
0.61


Val
1066
.
.
B
B
.
.
.
0.00
0.14
.
.
F
0.41
0.27


Val
1067
.
.
B
B
.
.
.
−0.37
0.14
.
.
.
−0.02
0.32


Ser
1068
.
.
B
.
.
T
.
−0.37
0.33
.
*
.
0.10
0.10


Ile
1069
.
.
B
.
.
T
.
−0.96
0.73
*
*
.
−0.20
0.22


Asn
1070
.
.
B
.
.
T
.
−0.84
0.77
.
*
.
−0.20
0.21


Cys
1071
.
.
B
.
.
T
.
−0.80
0.13
*
*
.
0.10
0.31


Asn
1072
.
.
B
B
.
.
.
−0.80
0.43
*
*
.
−0.60
0.36


Ile
1073
.
.
B
B
.
.
.
−0.71
0.39
*
*
.
−0.30
0.17


Arg
1074
.
.
B
B
.
.
.
0.18
0.41
*
*
.
−0.60
0.48


Leu
1075
.
.
B
B
.
.
.
0.18
0.24
*
*
.
−0.30
0.48


Val
1076
.
.
B
.
.
T
.
0.84
0.24
.
*
F
0.40
1.18


Pro
1077
.
.
.
.
.
T
C
−0.04
−0.44
*
*
F
1.20
1.05


Asn
1078
.
.
.
.
T
T
.
0.84
0.24
*
*
F
0.65
0.89


Gln
1079
A
.
.
.
.
T
.
0.03
−0.04
*
*
F
1.00
1.93


Glu
1080
.
A
B
.
.
.
.
0.81
0.10
.
*
F
0.00
1.08


Ile
1081
.
A
B
.
.
.
.
0.86
0.17
.
*
F
−0.15
0.91


Asn
1082
.
A
B
.
.
.
.
0.26
0.46
.
*
.
−0.60
0.43


Phe
1083
.
A
B
.
.
.
.
−0.09
0.74
.
*
.
−0.60
0.21


His
1084
.
A
B
.
.
.
.
−0.09
1.17
.
*
.
−0.60
0.29


Leu
1085
.
A
B
.
.
.
.
−0.90
0.89
.
*
.
−0.60
0.29


Leu
1086
.
A
.
.
.
.
C
−0.30
1.17
.
*
.
−0.40
0.28


Gly
1087
.
A
.
.
T
.
.
−1.11
1.30
*
*
.
−0.20
0.22


Asn
1088
A
A
.
.
.
.
.
−0.30
1.49
*
*
.
−0.60
0.22


Leu
1089
A
A
.
.
.
.
.
−0.57
0.80
*
*
.
−0.60
0.51


Trp
1090
A
A
.
.
.
.
.
−0.57
0.50
*
*
.
−0.60
0.69


Leu
1091
A
A
.
.
.
.
.
0.29
0.76
*
.
.
−0.60
0.35


Arg
1092
A
A
.
.
.
.
.
0.04
0.36
*
*
.
−0.30
0.86


Ser
1093
A
A
.
.
.
.
.
−0.77
0.17
*
.
.
−0.30
0.83


Leu
1094
A
A
.
.
.
.
.
0.09
−0.06
*
.
F
0.45
0.83


Lys
1095
A
A
.
.
.
.
.
0.13
−0.74
*
.
F
0.75
0.84


Ala
1096
A
A
.
.
.
.
.
0.99
0.01
*
*
.
−0.30
0.99


Leu
1097
A
A
.
.
.
.
.
0.58
−0.37
*
.
.
0.45
2.39


Lys
1098
A
A
.
.
.
.
.
0.28
−0.67
.
.
F
0.90
1.60


Tyr
1099
A
A
.
.
.
.
.
1.13
−0.06
*
.
F
0.60
1.57


Lys
1100
A
A
.
.
.
.
.
0.20
−0.56
*
.
F
0.90
3.81


Ser
1101
A
A
.
.
.
.
.
0.19
−0.56
.
*
F
0.90
1.34


Met
1102
A
A
.
.
.
.
.
0.14
0.06
.
*
.
−0.30
0.84


Lys
1103
A
A
.
.
.
.
.
0.10
−0.06
.
*
.
0.30
0.31


Ile
1104
.
A
B
.
.
.
.
−0.24
0.34
*
*
.
−0.30
0.38


Met
1105
A
A
.
.
.
.
.
−0.88
0.46
*
*
.
−0.60
0.38


Val
1106
A
A
.
.
.
.
.
−1.39
0.34
*
*
.
−0.30
0.19


Asn
1107
A
A
.
.
.
.
.
−0.79
1.03
.
*
.
−0.60
0.23


Ala
1108
A
A
.
.
.
.
.
−0.72
0.74
.
*
.
−0.60
0.40


Ala
1109
A
A
.
.
.
.
.
0.17
0.13
.
*
.
−0.15
1.05


Leu
1110
A
A
.
.
.
.
.
0.07
−0.11
*
.
.
0.45
1.13


Gln
1111
A
A
.
.
.
.
.
0.89
0.27
*
.
.
−0.30
0.97


Arg
1112
A
A
.
.
.
.
.
0.59
0.27
*
.
.
−0.15
1.31


Gln
1113
.
A
B
.
.
.
.
0.97
0.16
*
.
.
−0.15
2.13


Phe
1114
.
A
.
.
T
.
.
0.86
−0.10
*
.
.
0.85
1.90


His
1115
.
A
.
.
.
.
C
0.78
0.29
*
.
.
−0.10
0.84


Ser
1116
.
A
.
.
.
.
C
0.08
0.97
.
*
.
−0.40
0.34


Pro
1117
.
.
.
B
.
.
C
0.08
1.36
.
*
.
−0.40
0.34


Phe
1118
.
A
.
B
.
.
C
0.08
0.57
.
*
.
−0.40
0.49


Ile
1119
.
A
B
B
.
.
.
0.78
0.07
.
*
.
−0.30
0.63


Phe
1120
.
A
B
B
.
.
.
0.81
−0.31
.
.
.
0.30
0.71


Arg
1121
A
A
.
B
.
.
.
0.90
−0.74
.
.
F
1.24
1.37


Glu
1122
.
A
.
.
T
.
.
0.81
−1.10
*
*
F
1.98
3.01


Glu
1123
.
A
.
.
.
.
C
1.62
−1.40
*
*
F
2.12
4.66


Asp
1124
.
.
.
.
.
T
C
2.51
−2.19
*
.
F
2.86
4.66


Pro
1125
.
.
.
.
T
T
.
2.32
−1.79
*
*
F
3.40
4.66


Ser
1126
.
.
.
.
T
T
.
1.36
−1.10
*
.
F
3.06
1.89


Arg
1127
A
.
.
.
.
T
.
0.66
−0.46
.
.
F
1.87
0.84


Gln
1128
A
.
.
B
.
.
.
0.66
0.33
*
.
F
0.53
0.47


Ile
1129
A
.
.
B
.
.
.
−0.23
−0.10
*
.
.
0.64
0.61


Val
1130
A
.
.
B
.
.
.
−0.32
0.20
*
.
.
−0.30
0.22


Phe
1131
.
.
B
B
.
.
.
0.02
0.59
*
.
.
−0.60
0.17


Glu
1132
.
.
B
B
.
.
.
−0.09
0.19
*
.
.
−0.30
0.48


Ile
1133
.
.
B
B
.
.
.
−0.09
−0.10
*
*
F
0.60
1.12


Ser
1134
.
A
.
.
.
.
C
0.80
−0.74
.
*
F
1.10
2.24


Lys
1135
.
A
.
.
T
.
.
1.37
−1.53
.
.
F
1.30
2.16


Gln
1136
.
A
.
.
T
.
.
2.07
−0.61
.
*
F
1.30
3.24


Glu
1137
A
A
.
.
.
.
.
1.21
−0.90
.
*
F
0.90
4.18


Asp
1138
.
A
.
B
T
.
.
1.89
−0.64
.
*
F
1.30
1.55


Trp
1139
.
A
.
B
T
.
.
1.30
−0.21
.
*
.
0.85
1.39


Gln
1140
.
A
B
B
.
.
.
0.97
0.07
.
*
.
−0.30
0.56


Val
1141
.
A
B
B
.
.
.
0.08
0.99
.
*
.
−0.60
0.35


Pro
1142
.
A
B
B
.
.
.
−0.81
1.67
.
*
.
−0.60
0.24


Ile
1143
.
.
B
B
.
.
.
−1.67
1.44
.
*
.
−0.60
0.10


Trp
1144
.
.
B
B
.
.
.
−1.72
1.69
.
*
.
−0.60
0.10


Ile
1145
.
.
B
B
.
.
.
−2.02
1.47
.
.
.
−0.60
0.06


Ile
1146
.
.
B
B
.
.
.
−1.48
1.43
.
.
.
−0.60
0.12


Val
1147
.
.
B
B
.
.
.
−2.08
1.23
.
.
.
−0.60
0.16


Gly
1148
.
.
B
B
.
.
.
−1.53
1.00
.
.
F
−0.45
0.19


Ser
1149
.
.
.
B
.
.
C
−1.59
0.74
.
.
F
−0.25
0.27


Thr
1150
.
.
.
B
.
.
C
−1.51
0.49
.
.
F
−0.25
0.35


Leu
1151
.
.
.
B
.
.
C
−1.43
0.53
.
.
F
−0.25
0.30


Gly
1152
.
.
.
B
T
.
.
−1.39
0.79
.
.
F
−0.05
0.18


Gly
1153
.
A
B
B
.
.
.
−1.86
1.09
.
.
.
−0.60
0.10


Leu
1154
.
A
B
B
.
.
.
−2.14
1.29
.
.
.
−0.60
0.10


Leu
1155
.
A
B
B
.
.
.
−2.64
1.10
.
.
.
−0.60
0.11


Leu
1156
A
A
.
B
.
.
.
−2.64
1.36
.
.
.
−0.60
0.09


Leu
1157
A
A
.
B
.
.
.
−3.16
1.61
.
.
.
−0.60
0.09


Ala
1158
A
A
.
B
.
.
.
−3.62
1.57
.
.
.
−0.60
0.08


Leu
1159
A
A
.
B
.
.
.
−3.40
1.57
.
.
.
−0.60
0.08


Leu
1160
A
A
.
B
.
.
.
−3.40
1.39
.
.
.
−0.60
0.10


Val
1161
A
A
.
B
.
.
.
−2.88
1.39
.
.
.
−0.60
0.08


Leu
1162
A
A
.
B
.
.
.
−2.02
1.80
*
.
.
−0.60
0.10


Ala
1163
A
A
.
B
.
.
.
−2.24
1.11
.
.
.
−0.60
0.25


Leu
1164
A
A
.
B
.
.
.
−1.78
1.11
.
*
.
−0.60
0.27


Trp
1165
A
A
.
B
.
.
.
−1.67
0.90
.
.
.
−0.60
0.33


Lys
1166
A
A
.
B
.
.
.
−1.51
1.00
.
.
.
−0.60
0.28


Leu
1167
A
A
.
B
.
.
.
−0.59
1.29
.
.
.
−0.60
0.29


Gly
1168
A
.
.
B
.
.
.
−0.30
0.60
.
.
.
−0.60
0.55


Phe
1169
.
.
B
B
.
.
.
−0.08
0.07
*
.
.
−0.30
0.37


Phe
1170
.
.
B
B
.
.
.
0.32
0.57
*
.
.
−0.60
0.45


Arg
1171
.
.
B
B
.
.
.
0.39
−0.11
*
.
.
0.30
0.89


Ser
1172
.
.
.
B
.
.
C
1.31
−0.54
*
.
F
1.10
2.02


Ala
1173
.
.
.
.
.
.
C
1.77
−1.33
*
.
F
1.30
4.57


Arg
1174
.
.
.
.
.
.
C
2.47
−2.11
*
.
F
1.30
4.57


Arg
1175
.
.
.
.
T
.
.
2.96
−2.11
*
.
F
1.84
5.90


Arg
1176
.
.
.
.
T
.
.
2.50
−2.07
*
.
F
2.18
9.03


Arg
1177
.
.
.
.
T
.
.
1.99
−2.14
.
.
F
2.52
4.56


Glu
1178
.
.
.
.
.
T
C
2.58
−1.46
.
.
F
2.86
1.92


Pro
1179
.
.
.
.
T
T
.
2.26
−1.46
.
.
F
3.40
1.64


Gly
1180
.
.
.
.
T
T
.
1.83
−1.03
.
.
F
3.06
1.29


Leu
1181
.
.
.
.
.
T
C
1.51
−0.54
.
*
F
2.69
1.08


Asp
1182
.
.
.
.
.
T
C
1.44
−0.11
.
*
F
2.22
1.08


Pro
1183
.
.
.
.
.
T
C
0.59
−0.54
*
*
F
2.35
2.18


Thr
1184
.
.
.
.
.
T
C
−0.01
−0.33
*
.
F
1.88
1.96


Pro
1185
.
.
B
.
.
T
.
0.33
−0.33
*
.
F
1.70
0.97


Lys
1186
.
A
B
.
.
.
.
0.76
−0.33
*
*
F
1.28
1.08


Val
1187
.
A
B
.
.
.
.
0.37
−0.33
*
.
F
0.96
0.96


Leu
1188
A
A
.
.
.
.
.
0.19
−0.39
*
.
.
0.64
0.79


Glu
1189
A
A
.
.
.
.
.
0.11
−0.39
*
.
.
0.47
0.51









INCORPORATION BY REFERENCE
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. In addition, the sequence listing submitted herewith is incorporated herein by reference in its entirety. The specification and sequence listing of each of the following U.S. and PCT applications are herein incorporated by reference in their entirety: U.S. Provisional Appln. No. 60/243,792 filed on Oct. 30, 2000, U.S. Provisional Appln. No. 60/198,407 filed on Apr. 19, 2000, U.S. Provisional Appln. No. 60/105,971 filed on Oct. 28, 1998, U.S. application Ser. No. 09/836,353 filed on Apr. 18, 2001, PCT Appln. No. PCT/US99/25031 filed on Oct. 27, 1999, U.S. Provisional Appln. No. 60/304,417 filed on Jul. 12, 2001, U.S. Provisional Appln. No. 60/270,625 filed on Feb. 23, 2001, U.S. Provisional Appln. No. 60/277,340 filed on 21 Mar. 2001, U.S. Provisional Appln. No. 60/306,171 filed on 19 Jul. 2001, U.S. Provisional Appln. No. 60/278,650 filed on 27 Mar. 2001, U.S. Provisional Appln. No. 60/331,287 filed on 13 Nov. 2001, U.S. application Ser. No. 09/950,082 filed on 12 Sep. 2001, U.S. application Ser. No. 09/950,083 filed on 12 Sep. 2001, U.S. Provisional Appln. No. 60/040,162 filed on 7 Mar. 1997, U.S. Provisional Appln. No. 60/043,576 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,601 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,845 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,580 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,599 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,664 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,314 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,632 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,892 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,568 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,595 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,632 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,578 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/040,333 filed on 7 Mar. 1997, U.S. Provisional Appln. No. 60/043,670 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,596 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,864 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,674 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,612 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,631 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,569 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,588 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,876 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,671 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/043,311 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/038,621 filed on 7 Mar. 1997, U.S. Provisional Appln. No. 60/043,672 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,613 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,636 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,669 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,582 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,910 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,315 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,598 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,874 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,312 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,585 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,881 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,313 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,586 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,909 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/040,161 filed on 7 Mar. 1997, U.S. Provisional Appln. No. 60/047,587 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,879 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/047,500 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,880 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/047,584 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,894 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/047,492 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,911 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/040,626 filed on 7 Mar. 1997, U.S. Provisional Appln. No. 60/047,503 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,903 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/047,501 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,637 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/047,590 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,875 filed on 22 Aug. 1997, U.S. Provisional Appln. 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## Claims

1. An isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 128;
(b) the amino acid sequence comprising amino acid residues 2-212 of SEQ ID NO: 128;
(c) the amino acid sequence comprising amino acid residues 31-212 of SEQ ID NO: 128;
(d) the amino acid sequence of SEQ ID NO: 130;
(e) the amino acid sequence comprising amino acid residues 2-101 of SEQ ID NO: 130;
(f) the amino acid sequence comprising amino acid residues 31-101 of SEQ ID NO: 130;
(g) the amino acid sequence of the polypeptide encoded by the HWBAO62 cDNA contained in ATCC Deposit No. 209603; and
(h) the amino acid sequence of the polypeptide encoded by the HWBAO62 cDNA contained in in ATCC Deposit No. 209603, excepting the N-terminal methionine.

2. The polypeptide of claim 1, wherein said polypeptide further comprises a heterologous polypeptide.

3. The polypeptide of claim 2, wherein said heterologous amino acid sequence is the amino acid sequence of an immunoglobulin Fc domain.

4. The polypeptide of claim 2, wherein said heterologous amino acid sequence is the amino acid sequence of albumin.

5. The polypeptide of claim 1, wherein said polypeptide is glycosylated.

6. The polypeptide of claim 1, wherein said polypeptide is labeled.

7. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

8. An isolated polypeptide produced by the method comprising:
(a) expressing the polypeptide of claim 1 in a cell; and
(b) recovering said polypeptide.

9. An isolated polypeptide comprising an amino acid sequence that consists of at least contiguous amino acid residues of SEQ ID NO: 128.

10. The isolated polypeptide of claim 9, wherein the polypeptide consists of at least 50 contiguous amino acid residues of SEQ ID NO: 128.