# Patent

## Title
Secreted protein HHTLF25

## Abstract

The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. 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
This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/239,899, filed Oct. 13, 2000, which is hereby incorporated by reference; this application is also a continuation-in-part of, and claims benefit under 35 U.S.C. § 120 of U.S. patent application Ser. No. 09/227,357, filed Jan. 8, 1999 (now U.S. Pat. No. 6,342,581 issued on Jan. 29, 2002), which is hereby incorporated by reference, and which is a continuation-in-part, and claims benefit under 35 U.S.C. § 120 of copending International patent application Ser. No.: PCT/US98/13684, filed Jul. 7, 1998 and published in English, which is hereby incorporated by reference, which claims benefit under 35 U.S.C. § 119(e) based on U.S. Provisional Applications, all of which are hereby incorporated by reference:













Filing Date
Appln No.

















1.
08 Jul. 1997
60/051,926


2.
08 Jul. 1997
60/052,793


3.
08 Jul. 1997
60/051,925


4.
08 Jul. 1997
60/051,929


5.
08 Jul. 1997
60/052,803


6.
08 Jul. 1997
60/052,732


7.
08 Jul. 1997
60/051,931


8.
08 Jul. 1997
60/051,932


9.
08 Jul. 1997
60/051,916


10.
08 Jul. 1997
60/051,930


11.
08 Jul. 1997
60/051,918


12.
08 Jul. 1997
60/051,920


13.
08 Jul. 1997
60/052,733


14.
08 Jul. 1997
60/052,795


15.
08 Jul. 1997
60/051,919


16.
08 Jul. 1997
60/051,928


17.
18 Aug. 1997
60/055,722


18.
18 Aug. 1997
60/055,723


19.
18 Aug. 1997
60/055,948


20.
18 Aug. 1997
60/055,949


21.
18 Aug. 1997
60/055,953


22.
18 Aug. 1997
60/055,950


23.
18 Aug. 1997
60/055,947


24.
18 Aug. 1997
60/055,964


25.
18 Aug. 1997
60/056,360


26.
18 Aug. 1997
60/055,684


27.
18 Aug. 1997
60/055,984


28.
18 Aug. 1997
60/055,954


29.
12 Sep. 1997
60/058,785


30.
12 Sep. 1997
60/058,664


31.
12 Sep. 1997
60/058,660


32.
12 Sep. 1997
60/058,661











FIELD OF THE INVENTION
The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel 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 and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. 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.
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, and numerous other compounds. Additionally, knowledge of gene sequences can provide the key to treatment or cure of genetic diseases (such as muscular dystrophy and cystic fibrosis).
SUMMARY OF THE INVENTION
The present invention relates to novel secreted proteins. 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 and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. 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.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
FIGS. 1(A)–(C) show the nucleotide sequence (SEQ ID NO: 32) and deduced amino acid sequence (SEQ ID NO: 172) corresponding to Gene No: 22.



DETAILED DESCRIPTION
Polynucleotides and Polypeptides
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, “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 ATCC Deposit Number 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 ATCC, 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), pBluescript (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 Stratagene 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 Stratagene.
Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, 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 Technologies. 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 Technologies. 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 (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 ATCC. 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 ATCC 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 ATCC 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 ATCC 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 ATCC Deposit No.Z.
Description of Table 1B
Table 1B summarizes 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 identifier (SEQ ID NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. 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 and/or 1B. The third column provides a unique contig identifier, “Contig ID:” for each of the contig sequences disclosed in Table 1B. The fourth column provides the sequence identifier, “SEQ ID NO:X”, for each of the contig sequences disclosed in Table 1A and/or 1B. The fifth column, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineate the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1B as SEQ ID NO:Y (column 6). Column 7 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 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. 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, “Tissue Distribution” shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleoti des of the invention. The first number in column 8 (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. For those identifier codes in which the first two letters are not “AR”, the second number in column 8 (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 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. Column 9 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 Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIMTM. 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 URL: http://www.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 column 10 labeled “OMIM Disease Reference(s)”. A key to the OMIM reference identification numbers is provided in Table 5.

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).

Description of Table 1D

Table 1D: In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “FEATURES OF PROTEIN” sections (below) and also as listed in the “Preferred Indications” column of Table 1D (below); 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 seccond columns of Table 1D show the “Gene No.” and “Clone ID”, 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, a typical 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, hypematremia, 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., hypemephroma, 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.
Description of Table 1E
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 (“Clone ID”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B, 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, 1B, and 2). 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, diagnosing, preventing, and/or treating.
Table 1E describes the use of FMAT technology, inter alia, 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 flurophore 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 highthroughput screening using flourometric 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)).
Table 1E also describes 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 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 Table 1A or 1B. The second column provides the unique contig identifier, “Contig ID:” corresponding to contigs in Table 1B and allowing for correlation with the information in Table 1B. 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, “Clone ID”, for a cDNA clone related to contig sequences disclosed in Table 1B. The second column provides the sequence identifier, “SEQ ID NO:X”, for contig sequences disclosed in Table 1A and/or 1B. The third column provides the unique contig identifier, “Contig ID:”, for contigs disclosed in Table 1B. The fourth column provides a 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 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. For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be substituted into the general formula of a−b, and used to describe polynucleotides which may be preferably excluded from the invention. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, 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, 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).

Description of Table 4

Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B, column 8. Column 1 provides the tissue/cell source identifier code disclosed in Table 1B, Column 8. Columns 2–5 provide a description of the tissue or cell source. 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, column 10. OMM 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 URL: http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B, column 9, as determined using the Morbid Map database.

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 contained in SEQ ID NO:X (as described in column 5 of Table 1A), or cDNA clone (as described in column 2 of Table 1A and contained within a pool of plasmids deposited with the ATCC in ATCC Deposit No:Z). 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, a representative plasmid containing the sequence of SEQ ID NO:X was deposited with the American Type Culture Collection (“ATCC”) and/or described in Table 1A. As shown in Table 1A, each cDNA is identified by a cDNA clone identifier and the ATCC Deposit Number (ATCC Deposit No:Z). Plasmids that were pooled and deposited as a single deposit have the same ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
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) and/or sequences of the cDNA contained in the deposited clone (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein). “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 included within “polynucleotides” of the present invention 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 polynucleotides of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, 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).
“SEQ ID NO:X” refers to a polynucleotide sequence described in column 5 of Table 1A, while “SEQ ID NO:Y” refers to a polypeptide sequence described in column 10 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 6 of Table 1A. The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. The polynucleotide sequences are shown in the sequence listing immediately followed by all of the polypeptide sequences. Thus, a polypeptide sequence corresponding to polynucleotide sequence SEQ ID NO:2 is the first polypeptide sequence shown in the sequence listing. The second polypeptide sequence corresponds to the polynucleotide sequence shown as SEQ ID NO:3, and so on.
The polypeptides 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 NY Acad Sci 663:48–62 (1992)).
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.
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) protein of the invention. Such functional activities include, but are not limited to, biological activity, 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.
“A polypeptide having functional activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular assay, such as, for example, a 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).
The functional activity of the polypeptides, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide of the present invention for binding to an antibody to the full length polypeptide, 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, E., et al., Microbiol. Rev. 59:94–123 (1995). In another embodiment, physiological correlates polypeptide of the present invention binding to its substrates (signal transduction) can be assayed. 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.
Polynucleotides and Polypeptides of the Invention
Features of Protein Encoded by Gene No: 1
The translation product of this gene shares sequence homology with npdcf-1 which is thought to be important in promoting the survival of bi-potential glial progenitor cells (See Genbank Accession No. gi|456107).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:291)









LRRPSTPLRRPWLHLQLPRISLGDQRLAQSAEMYHYQHQRQQMLSLERHK



 


EPPKELDTALRMRRMRTETSRCTSARAWPRPGKWRCATICSTTPHCPRPC


 


RPPAHRLHCHDLEADRRPLAPR,


 








(SEQ ID NO:292)









RATQGAGHGSSDEENEDGDFTVYECPGMAPTGEMEVRNHLFDHAALSAPL



 


PAPSSPLALP,


 








(SEQ ID NO:293)









KAEYATAKALATPAATPDLAWGPAPGTERGDVPLPAPTATDVVPGAA,



 








(SEQ ID NO:294)









SAEMYHYQHQRQQML,



 








(SEQ ID NO:295)









LERHKEPPKEL,



 








(SEQ ID NO:296)









AKCPPGAHACGP,



 








(SEQ ID NO:297)









PVHMSPLEP,



 








(SEQ ID NO:298)









WCRLQREIRLTQ,



 








(SEQ ID NO:299)









SSDEENEDGDFTVYECPG,



 








(SEQ ID NO:300)









APTGEMEVRN,



 








(SEQ ID NO:301)









CPGSLDCALK,



 








(SEQ ID NO:302)









NEDGDFTVYECPGMAPTGEMEV,



 








(SEQ ID NO:303)









RPTRPSSSCVLPRCLRCSRRGARSPRRAPGLAVPCCPGGGAEGWRRRCLR



 


PPRGTCGCCGCCSPASSSAPPCVEPPPATRNVAACPGSLDCALKKRASVL


 


LVHMPVGLPSALPXGTAKACFAXMRRASXGGRAQPXLEMRLIPGPRELAR


 


KGIWTSIPP,


 








(SEQ ID NO:304)









RCLRCSRRGARSPRRAPGLAVPCCP, and/or



 








(SEQ ID NO:305)










GSLDCALKKRASVLLVHMPVGLPSALPXGTAKAC. 






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 cerebellum, synovial sarcoma, and to a lesser extent, in several other cancer cell lines.
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, neural or skeletal disorders, particularly tumors characterized by cells of a relatively undifferentiated state, including neural tumors. Similarly, polypeptides and antibodies directed to those 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 synovial fluid, prostate, breast and uterus, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, skeletal, 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: 151 as residues: Pro-6 to Arg-11, Glu-52 to Gly-59. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in the cerebellum, combined with the homology to the human npdcf-1 protein indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosing, detecting, preventing and/or treating tumors that contain relatively high numbers of undifferentiated cells. Moreover, this gene would be useful for the detection/treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Alternatively, the expression of this gene product in synovium would indicate a role in the detection, diganosis, prevention and/or treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis, bone cancer, 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 protein may be useful for inducing astroglial proliferation and promoting neuronal survival, in addition to other highly vascular tissues. The protein can also be used to regulate cellular metabolism (e.g., through the modulation of protein expression). 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:11 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 1128 of SEQ ID NO:11, b is an integer of 15 to 1142, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:11, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 2
The translation product of this gene was found to have homology to the RoBo-1 protein from Rattus norvegicus (see, e.g., Genbank Accession No.gi|2895563 (AF041083), all references available through this accession are hereby incorporated in their entirety by reference herein) which is thought to be important as a mediator in bone remodeling.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: DSHQARSRRLEALWSPSLGEVSSST (SEQ ID NO: 306). 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 colon, pituitary, and to a lesser extent in fetal lung and fibrosarcoma.
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, endocrine, gastrointestinal, pulmonary, skeletal, or developmental and proliferative disorders, particularly those effecting the Gut/pituitary/hypothalamic axis. 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 digestive system and regulation of feeding, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., gastrointesinal, endocrine, developmental, skeletal, pulmonary, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amniotic fluid, pulmonary surfactant or sputum, 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: 152 as residues: Asn-26 to Cys-32, Cys-100 to Leu-112, Cys-128 to Ser-135. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in colon and pituitary indicates that polynucleotides and polypeptides corresponding to this gene would be useful for treating disorders related to the intake and utilization of food since this gene is expressed in the digestive tract and a CNS site involved in regulation of weight homeostasis. The polynucleotides and/or polypeptides corresponding to this gene can 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, and as nutritional supplements. It may also have a very wide range of biological activities. 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 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. 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:12 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 1020 of SEQ ID NO:12, b is an integer of 15 to 1034, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:12, and where 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 familial adenomatous polyposis gene which is thought to be important in the tumorigenesis of colon cancer (see, e.g., Fulton, Nature 368, 32–38 (1994); accession no. U28412; Joslyn et al., Cell 66 (3), 601–613 (1991); accession no. M73547; and Spirio et al., Nucleic Acids Res. 19 (22), 6348 (1991), all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: CRWRPESAAPC (SEQ ID NO: 307), TRPGRGAQAPVK (SEQ ID NO:308), MVSWMISRAVVLVFGMLYPAY (SEQ ID NO:309), GMLYPAYYSYKAVKTKN (SEQ ID NO:310), EYVRWMMYWIVFALYTV (SEQ ID NO:311), YPAYYSYKAVKTKNVKE (SEQ ID NO:312), VAWFPLYYELKIA (SEQ ID NO:313), and/or MVSWMISRAVVLVFGMLYPAYYSYKAVKTKNVKEYVRWMMYWIVFALYT VIETVADQTVAWFPLYYELKIAFVIWLLSPYTKGASLIYRKFLHPLLSSKEREI DDYIVQAKERGYETMVNFGRQGLNLAATAAVTAAVKSQGAITERLRSFSMH DLTTIQGDEPVGQRPYQPLPEAKKKSXQPPVN (SEQ ID NO:314), and/or QXVMEFHXKTXMXKQXKKQRGHIQIMRC (SEQ ID NO:507). 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 osteoclastoma, prostate, bone marrow and to a lesser extent in testes and dendritic cells. Northern data has demonstrated that an abundant 1.3 kb band is seen in testes tissues.
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, colon cancer and cancers of various origin, including osteoclastoma and prostate cancer, as well as reproductive 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 tumorigenesis and reproductive disorders, 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., bone, prostate, reproductive, bone marrow, colon and other gastrointestinal tissue, tissue of the nervous system, and testis and other reproductive tissue, 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, two, or all three of the immunogenic epitopes shown in SEQ ID NO: 153 as residues: Ser-59 to lle-64, Ala-71 to Tyr-76, Pro-125 to Ser-141. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in osteoclastoma, prostate, bone marrow and homology to familial adenomatous polyposis gene indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis and intervention of tumors of various origins, including colon cancer, osteoclastoma and prostate cancer. Alternatively, the Northern data demonstrating expression in testes tissues indicates that polynucleotides and/or polypeptides of the invention would be useful for the diagnosis and/or treatment of reproductive disorders and conditions concerning proper testicular function (e.g., endocrine function, sperm maturation), as well as cancer. Therefore, this gene product would be 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) would be useful as male contraceptive agents. Similarly, the protein is believed to be 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.
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: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 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 1260 of SEQ ID NO:13, b is an integer of 15 to 1274, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:13, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 4
The translation product of this gene shares sequence homology with the L6 cell surface antigen, which is highly expressed in lung, breast, colon, and ovarian carcinomas (see e.g., Marken et al., Proc Natl Acad Sci USA Apr. 15, 1992;89(8):3503–3507, all references available through this accession are hereby incorporated in their entirety by reference herein). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: ITLCLVCIVANA (SEQ ID NO: 315). 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 was recently cloned and sequenced by another group, which identified the gene as a putative tetraspan transmembrane (TM4) protein L6H from humans. The transmembrane 4 superfamily (TM4SF) or tetraspan superfamily has at least 16 members (including CD9, CD20, CD37, CD53, CD63, CD81, CD82, A15, CO-029, Sm23, RDS, Uro B, Uro A, SAS, Rom-1, PETA3, and YKK8), is the second biggest subfamily among CD antigen superfamilies, and are activation antigens of T-cells. All TM4SF members contain four putative transmembrane domains, two extracellular loops, and two short cytoplasmic tails. They are variously expressed on immature, early, mature, activated lymphocytes, monocytes, macrophages, granulocytes, platelets, eosinophils, basophils, certain leukemic and lymphoma cells, and a variety of other cells and tissues.
This gene is expressed primarily in fetal liver/spleen tissues.
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, cancers of the liver, immune system 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 hepatic and immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., lung and pulmonary tissue, colon and other gastrointestinal tissue, mammary tissue, ovarian tissue and other tissue of the reproductive system, hepatic tissue, immune system tissues, 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, two or all three of the immunogenic epitopes shown in SEQ ID NO: 154 as residues: Asn-32 to Asn-41, Thr-140 to Ala-147, Asp-188 to His-197. Polynucleotides encoding said polypeptides are encompassed by the invention.
The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is highly expressed in lung, breast, colon, and ovarian carcinomas and is referred to as the L6 antigen. This antigen is an attractive target for therapeutic intervention due to its high level expression on malignant cells. The tissue distribution and homology to L6 antigen indicates that polynucleotides and polypeptides corresponding to this gene would be useful for detection, diganosis, prevention and/or treatment of neoplastic tissues—particularly of the liver. The translation product of this gene is a member of the tetraspan transmembrane superfamily, and therefore, antigenic regions of members of this family could be valuable immunogens or targets to implement active and passive immunotherapy in patients with cancer. 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:14 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 954 of SEQ ID NO:14, b is an integer of 15 to 968, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:14, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 5
The translation product of this gene shares sequence homology with prostaglandin EP3-9 receptor, which is thought to be important in prostaglandin hormonal reaction.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MAIPAFSSCQQISSAAALQI (SEQ ID NO: 316), and/or CNGPFKHFSFTVST (SEQ ID NO: 317). 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 retina.
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, glaucoma or other ocular diseases. 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 ocular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., retinal and other optic tissue, tissue of the nervous 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.
The tissue distribution in retinal tissues and the homology to prostaglandin receptor indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection and intervention of ocular diseases like glaucoma. Specifically, the receptor can be used for the identification of agonists or antagonists, anti-inflammatories for the eyes, and vasoconstrictive agents, etc. Furthermore, the tissue distribution in retina indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis and/or detection of eye disorders including blindness, color blindness, impaired vision, short and long sightedness, retinitis pigmentosa, retinitis proliferans, and retinoblastoma.
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:15 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 787 of SEQ ID NO:15, b is an integer of 15 to 801, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:15, 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 shares sequence homology with Cortical granule lectin which is thought to be important in blocking polyspermy (see, e.g., Genbank Accession No. gnl|PID|e1181610, all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: RSCKEIKD (SEQ ID NO: 318), GGGWTLVASVHEN (SEQ ID NO: 319), ADYPEGDGNWANYNTFGSA (SEQ ID NO: 320), ATSDDYKNPGYYDI (SEQ ID NO: 321), CIGGGGYFPEA (SEQ ID NO: 322), DSDKIT (SEQ ID NO: 324), YQTFCDMT (SEQ ID NO: 325), and/or EITEAAVLLFY (SEQ ID NO: 323). 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 benign and metastatic colon, and to a lesser extent in HEL 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, gastrointestinal, reproductive, or developmental disorders, particularly cancer, or inflammatory conditions. 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 digestive system, expression of this gene at significantly higher or lower levels may be detected in certain tissues or cell types (e.g., gastrointestinal, reproductive, proliferating, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, seminal fluid, 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: 156 as residues: Arg-15 to Ser-33, Pro-35 to Cys-41. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in colon, combined with the homology to cortical granule lectins indicates that polynucleotides and polypeptides corresponding to this gene would be useful for treating, preventing, detecting, and/or diagnosing disorders of the colon. These may include diseases related to damage or chronic inflammation as well as tumors of the colon. The product may also be useful for the identification of colon cancer metastasis and, as a secreted protein, may have diagnostic and prognostic applications. Moreover, the protein would be useful in the treatment, detection, and/or prevention of reproductive disorders, particularly normal testicular function, in addition having utility in the development of contraceptives, or in the treatment of polyspermy. 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.
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:16 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 1184 of SEQ ID NO:16, b is an integer of 15 to 1198, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:16, 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 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: MMATPSTRPPPPAASTTSATAPALPPRPPWPWPPSSWPPSGVSSKAPEADPLKNKAL (SEQ ID NO: 326); LLLTSPLPRCPPACSHDAPAHPDPGGPHGLTSGPGLGLPRVCLQRRQLLQPHA LPGYGCLLHDHAHLLHPHQDEGQ (SEQ ID NO: 327); and/or WLLQARVHHLLLPVRPLQRHRPCHPGHPGPGPHPPGHPLGSPLKPPRQTHSRTKLS (SEQ ID NO: 328). 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: 157 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:17 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:17, 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:17, 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 was shown to have homology to the mammalian notch I protein which has been shown to be important in the regulation of cell-fate during pattern formation and development (see, e.g., Genbank Accession No. gi|57635, all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: KHEXHQVSDGALRCFASLADRFTRRGVDPAPLAKHGLTEELLSRMAAAGGT VSGPSSACKPXRSTTGAPSTTADSKLSNQVSTIVSLLSTLCRGSPVVTHDLLRS ELPDSIESALQGDERCVLDTMRLVDFLLVLLFEGRKALPKSSAGSTGRIPGLRR LDSSGERSHRQLIDCIRSKDTDALIDAIDTGAFEVNFMDDVGQTLLNWASAFG TQEMVEFLCERGADVNRGQRSSSLHYAACFGRPQVAKTLLRHGANPDLRDE DGKTPLDKARERGHSEVVAILQSPGDWMCPVNKGDDK (SEQ ID NO: 329), PLDKARERGHSEVVAIL (SEQ ID NO: 330), AKTLLRHGANPDLRD (SEQ ID NO: 331), GRGRAWLCRRPVGSWIGAVWNDKPDKETFKKPWQMWTQIHCWNGYRWDXXDXKD (SEQ ID NO: 332), SWIGAVWNDKPDKETFKKPWQMW (SEQ ID NO: 333), KTMADVDPDTLLEWLQMGXGRXKGHATN TP (SEQ ID NO: 334), RGVDPAPLAKHGLTEELLSRMAAAGGTVSGPSSA (SEQ ID NO: 335), RSTTGAPSTTADSKLSNQVSTIVSLLSTLCR (SEQ ID NO: 336), FEVNFMDDVGQTLLNWASAFGTQEMVEFLCERGA (SEQ ID NO: 337), and/or EDGKTPLDKARERGHSEVVAILQSPGDW (SEQ ID NO: 338). 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 endothelial 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, vascular disorders, particularly diseases involving angiogenic abnormalities including diabetic retinopathy, macular degeneration, and other diseases including arterioscerosis, stroke, aneurysm, embolism, and cancer. 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 vascular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., endothelial, vascular, 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 from an individual not having the disorder. Preferred polypeptides of the present invention comprise, or alternatively consist of the immunogenic epitopes shown in SEQ ID NO: 158 as residues: Asp-17 to Phe-23. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in endothelial cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for treating, detecting, preventing and/or diagnosing diseases where an increase or decrease in angiogenesis is indicated and as a factor in the wound healing process. Polynucleotides and/or polypeptides corresponding to this gene would be useful in the treatment of cancer cells and tissues, particularly in inhibiting angiogenesis of the invading tumor. 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. Alternatively, considering the homology to the Notch I protein, polynucleotides and/or polypeptides corresponding to this gene may show utility in the detection/treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. 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:18 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 1607 of SEQ ID NO:18, b is an integer of 15 to 1621, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:18, 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 consist of, an amino acid sequence selected from the group: KADVKWHMCLQSPLCGLFCSIEGVLK (SEQ ID NO: 339), AC MNPAMCFVCACPHTGSTPEKAILQGRLISLGTSLSPASNGSGQQSFSICMINPS LPXSTSSHHLFSVLTGDLDSYSQRKLKPTSRKSFLLPKTQTYXVXHPSSPPLVL VQHRSPLSTYPKPVPSCCALDLISVIALETFLVYIHLFPSIDLSYWILSMLQPLL LIKQQSTKTLSLNCMLYSSYYLISFLSFKAKVLRRGGNILHHFFTSYSFFNTY (SEQ ID NO: 340), CPHTGSTPEKAILQGRLISLGTSLSPAS (SEQ ID NO: 341), QHRSPLSTYPKPVPSCCALDLISV (SEQ ID NO: 342), and/or IKQQSTKTLSLNCMLYSSYYLISFLSFKA (SEQ ID NO: 343). 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 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, immune and/or haematological 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, 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 the immunogenic epitopes shown in SEQ ID NO: 159 as residues: Pro-55 to Ser-66. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. 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 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, 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. 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:19 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 1108 of SEQ ID NO:19, b is an integer of 15 to 1122, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:19, 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 retinal epithelial membrane protein (REMP), which is thought to be important in development and maintenance of normal retinal function (see e.g., Philp et al., Exp. Cell Res. 219 (1), 64–73 (1995); and Genbank Accession No.U15685, all references available through this accession are hereby incorporated in their entirety by reference herein). The translation product of this gene also shares homology with monocarboxylate transporter protein (see, e.g., Genbank Accession No.U87627, all references available through this accession are hereby incorporated in their entirety by reference herein). Another group recently cloned and sequenced this gene, describing it as a monocarboxylate transporter protein (Genbank Accession No. gi|2463634, all references available through this accession are hereby incorporated in their entirety by reference herein). In quantitative terms, lactic acid is one of the most important metabolites in the body, substantial amounts being used and/or produced by almost all mammalian cells. As such it must be rapidly transported into and out of cells. Lactic acid transport across the plasma membrane is catalysed by proton-linked monocarboxylate transporters (MCTs), which are also responsible for the transport of pyruvate and the ketone bodies acetoacetate, -hydroxybutyrate and acetate.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: FLCALSPLGQLLQDRYGWRGGFLILGGL (SEQ ID NO: 344), LLNCCVCAALMRPLVVTAQPGXGPPRP (SEQ ID NO: 345), and/or SRRLXDLSVFRDRGFVLYAVAASVM (SEQ ID NO: 346). 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.
The gene encoding the disclosed cDNA is thought to reside on chromosome 17. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 17.
This gene is expressed primarily in neutrophils, and to a lesser extent in a variety of other tissues and cell types, including retina.
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, eye, and metabolic and cellular transport 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 eye and 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., retinal cells, neutrophils and other blood cells, 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.
The tissue distribution in retinal tissue and the homology to REMP indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of eye disorders, including neoplasms, visual impairments and blindness. Alternatively, the homology to monocarboxylate transporter protein indicates that the translation product of this gene would be useful for the diagnosis and/or treatment of disorders involving the cellular transport of lactic acid into and out of the cell.
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 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 1354 of SEQ ID NO:20, b is an integer of 15 to 1368, 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: 11
This gene is expressed primarily in human stomach cancers.
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, gastointestinal disorders, particularly cancer. 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 cancer, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., endothelial, gastrointestinal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, chyme, 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 tumors of the stomach indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of these tumors, in addition to other tumors in other tissues. 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:21 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 1174 of SEQ ID NO:21, b is an integer of 15 to 1188, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, 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 4. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 4.
When tested against a Jurkat T-cell line, supernatants removed from cells containing this gene activated the GAS (gamma activation site) pathway. Thus, it is likely that this gene activates T-cells through the Jaks-STAT signal transduction pathway. The GAS is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a complex, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
This gene is expressed primarily in brain.
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, neurodegenerative conditions and behavioural 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 brain and central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., brain and other tissues of the nervous 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 the immunogenic epitopes shown in SEQ ID NO: 162 as residues: Asp-57 to Gly-64. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution of this gene primarily in brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis and/or detection of neurodegenerative disease states and behavioural disorders such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder and panic disorder. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. 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:22 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 907 of SEQ ID NO:22, b is an integer of 15 to 921, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 13
This gene is expressed primarily in the spongy tissue from Alzheimer's brain.
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, neural disorders, which include, but are not limited to Alzheimer's disease and other neurodegenerative diseases. 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, 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, two, three, or all four of the immunogenic epitopes shown in SEQ ID NO: 163 as residues: Ser-31 to Ala-37, Ala-50 to Tyr-55, Phe-63 to Arg-68, His-83 to Pro-89. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in spongy tissue from Alzheimer's patient indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection/treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo. 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:23 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 1824 of SEQ ID NO:23, b is an integer of 15 to 1838, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:23, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 14
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: 528).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:









(SEQ ID NO:347)












QAQSDCSCSTVSPG,




 









(SEQ ID NO:348)












VLAGIVMGDLVLTVLIALAVYFLG,




 









(SEQ ID NO:349)












VPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDL, and/or




 









(SEQ ID NO:350)












ETESPYQELQGQRSDVYSDLNT.







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: 164 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-supressive 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:24 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:24, 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:24, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 15
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: LVCYCSTKKEKKLHEIAIQQGQNWRWLLFYKEISVPGFQSVWCSYKCLCVV WKAGEGG (SEQ ID NO: 351), RRSCSGPPLVNTAGKILSSSPAKLACKRTDFHIPSI (SEQ ID NO: 352), RASILGIDNERGCHFRHFNPLKEYKRKKKENKSFRIV (SEQ ID NO: 353), SKNKTRGGDWCVTVLRKRRKSFMKSPFSKDRTGDGFSFTKKSLSQAFSLFGV HTSVCVLCGRRGKAGEGGPVQGPLW (SEQ ID NO: 354), and/or MKSPFSKDRTGDGFSFTKKSLSQAFSLFGVHTSVCVLCGRRGKAGEGGPVQGPLW (SEQ ID NO: 355). 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 meningima and neutrophils and to a lesser extent in anergic T cells and CD34 depleted buffy coat.
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, inflammatory, immune and hemopoietic 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 hemopoietic, immune and inflammatory systems, 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 the immunogenic epitopes shown in SEQ ID NO: 165 as residues: Glu-45 to Asn-50. 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 study, diagnosis and treatment of various disorders and diseases of the immune, inflammatory, and hemopoietic systems. Furthermore, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and/or treatment of hematopoietic disorders. This gene product is primarily expressed in hematopoietic cells and tissues, indicateing that it plays a role in the survival, proliferation, and/or differentiation of hematopoieitic lineages. Expression of this gene product in T cells and neutrophils 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:25 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 614 of SEQ ID NO:25, b is an integer of 15 to 628, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:25, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 16
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MGKRAHEVRRPPHSRPLHGTPAGWVLDPSGYKDVTQDAEVMEVLQNLYRT KSFLFVGCGETLRDQIFQALFLYSVPNKVDLEHYMLVLKENEDHFFKHQADM LLHGIKVVSYGDCFDHFPGYVQDLATQICKQQSPGHLYSNSWSATPDGRGGP (SEQ ID NO:356), VLDPSGYKDVTQDAEVMEVLQNLYRT (SEQ ID NO:357), YSVPNKVDLEHY MLVLKENEDHFFKH (SEQ ID NO: 358), DLATQICKQQSPGHLYSNSWSATPD (SEQ ID NO:359), RRMKTISLSIRQICFCTESKLYPTGTVLTTFQDMCKTLPLRSANSKAQDICTRIH GVPLLMGEEAHDSDSHASDRGHHTMLPLPAGSFSESSHQAWEVEMLIAWTA PHYWVMHARTVQRGS (SEQ ID NO: 360), TESKLYPTGTVLTTFQDMCKTLPLRSA (SEQ ID NO: 361), and/or LMGEEAHDSDSHASDRGHHTMLPLPAG (SEQ ID NO: 362). 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 endothelial cells, and to a lesser extent in lymph node, tonsils, heart and spinal cord.
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, vascular diseases, such as restenosis, including disorders of the integumentary system. 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 vascular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., vascular, integumentary, immune, hematopoietic, neural, cardiovascular, 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.
The tissue distribution in endothelial cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for treating, preventing, detecting and/or diagnosing diseases of the vasculature including problems associated with diabetes and restenosis following angioplasty. Moreover, the protein would be useful in the detection, treatment, and/or prevention of a variety of other vascular conditions, which include, but are not limited to, stroke, microvascular disease, aneurysm, vascular leak syndrome, or embolism. 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:26 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 1408 of SEQ ID NO:26, b is an integer of 15 to 1422, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:26, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 17
This gene is expressed primarily in synovial tissue.
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, arthritis, tendonitis and chrondomalacia. 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 synovium, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., synovial tissue, 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.
The tissue distribution in synovial tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, detection and/or diagnosis of connective tissue disorders such as arthritis, tendonitis, chrondomalacia, inflammation and trauma. In addition, the expression of this gene product in synovium indicates a role in the detection, diganosis, prevention and/or 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 (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). 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:27 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 781 of SEQ ID NO:27, b is an integer of 15 to 795, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:27, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 18
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: VDPPGCRNSARGCTRLLRGSSKI (SEQ ID NO: 363). 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 the frontal cortex of the brain.
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, developmental and neurodegenerative diseases of the brain. 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., brain and other tissue of the nervous 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 the immunogenic epitopes shown in SEQ ID NO: 168 as residues: Ser-4 to Tyr-13. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diganosis, prevention and/or treatment of developmental and neurodegenerative diseases of the brain and nervous system, including malignancies as well as behavioral disorders. Examples of such conditions might include: depression, schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mania, dementia, paranoia, addictive behavior and sleep disorders. Furthermore, elevated expression of this gene product within the frontal cortex of 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.
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 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 563 of SEQ ID NO:28, b is an integer of 15 to 577, 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: 19
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:364)









MSTGDGRDAEKGWPVSEEENQRSVYPGYPECDERQAVPQHCAIASPSSLQ



SHHPASACVPRR,


 








(SEQ ID NO:365)









QQMTLGTKIKWGQLQRGQEIPTGDFTVRNFMRFSIIYC, and/or



 








(SEQ ID NO:360)









PFLFCASRIRXQGIGIHGQVACSAVRMYNNR.







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.

The gene encoding the disclosed cDNA is thought to reside on chromosome 10. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 10.
This gene is expressed primarily in neutrophils and activated monocytes.
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, immune and 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 immune and hematopoietic systems, 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 or both of the immunogenic epitopes shown in SEQ ID NO: 169 as residues: Met-1 to Ser-6, Pro-29 to Ser-34. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in monocytes and neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, diagnosis, detection, prevention and/or treatment of diseases of the immune and hematopoietic systems. Expression of this gene product in monocytes and neutrophils 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 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 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. 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:29 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 742 of SEQ ID NO:29, b is an integer of 15 to 756, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:29, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 20
The protein of the invention has sequence identity to the Saccharomyces cerevisiae ankyrin repeat-containing protein (see, e.g., Genbank Accession Nol gi|466522, all references available through this accession are hereby incorporated in their entirety by reference herein). The translation product of this gene also shares homology with C. elegans protein C43H6.7 gene product (see, e.g., Genbank Accession No. gi|1255324, all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: KCIYPKPARTHHCSICNRCVLKMDHHCPWLNNCVGHYNHRYFFSFCFFMTL GCVYCSYGSWDLFREAYAAIEKMKQLDKNKLQAVANQTYHQTPPPTFSFRER (SEQ ID NO: 367), ARGHWNLILIVFHYYQAITTPPGYPPQGRNDIATVSIC (SEQ ID NO: 368), WQCELDCVSHDSSTHSAPYVISRASKGSFSQNP (SEQ ID NO: 369), SKRASGPALGYHAGQFKDQPFYHCRRKTQCGEILGLTSLYSGKQKFQPQTRG QAASYLPCPVLTRTSSRIQHWSWPPPLLLAV (SEQ ID NO: 370), ESLQLRLLGQLEGIPGCGYRKALAYSGALTF (SEQ ID NO: 371), and/or SLAPWEWNELGAPSLGDCSLSLCDGSVSWTVSATTRALILLPMLFQGPPRAAFLRILDQKEPVGLP (SEQ ID NO: 372). 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 endometrial tumor, colon tumor, prostate cancer, and ovarian cancer.
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 a number of types of cancers, particularly endometrial, prostate, ovarian, and colon cancers. 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 endometrium, prostate, colon, and ovary, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., prostate, colon, ovary, endometrium, 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: 170 as residues: Asn-43 to Arg-49, Phe-57 to Cys-65, Pro-93 to Ser-99. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in prostate cancer, ovarian cancer, colon cancer, and endometrial cancer tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention, and/or treatment of diseases and cancers of the prostate, ovaries, colon, and endometrium, as well as cancers of other tissues where expression has been observed. 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:30 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 1282 of SEQ ID NO:30, b is an integer of 15 to 1296, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:30, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 21
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: LKCTIYGGA (SEQ ID NO: 373). 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 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, diseases of the immune system, including inflammatory diseases and allergies. 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, haematopoeitic, 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 the immunogenic epitopes shown in SEQ ID NO: 171 as residues: Gln-36 to Lys-41. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. 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 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, 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. 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:31 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 1546 of SEQ ID NO:31, b is an integer of 15 to 1560, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:31, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 22
The translation product of this gene shares sequence homology with bovine and human uroplakin III which is thought to be important in urothelial differentiation (see, e.g., Genbank Accession Nos. d10226610 and gi|304261|gb|AAC37309.1|; all references available through these accession are hereby incorporated by reference herein). The translation product of this gene also shares sequence homology with the human hPMS2R6 gene (see, e.g., Genbank Accession No. >gi|1061426|gb|AAA97460.1|; all references available through this accession are hereby incorporated by reference herein) which is a member of the hPMS2 family. The hPMS2 gene (HGMW-approved symbol PMS2) encodes a mutL homolog that causes hereditary non-polyposis colon cancer (HNPCC) when inherited in mutant form.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:









(SEQ ID NO:374)












ASIDTWPGRRSGGMIVITSI and/or




 









(SEQ ID NO:375)












GSPQAETRWSDPIALHQGKSPASIDTWPGRRSGGMIVITSI.







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.

The polypeptide encoded by this gene has been determined to have a transmembrane domain at about amino acid position 59 to about 75, and at about 203 to about 219 of the amino acid sequence referenced in Table 1A for this gene. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type IIIa membrane proteins.
This gene is expressed primarily in ovarian tumor.
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, reproductive or endocrine disorders, particularly proliferative conditions such as ovarian cancer. 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 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, endocrine, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, vaginal pool, 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, four, or all five of the immunogenic epitopes shown in SEQ ID NO: 172 as residues: Phe-3 to Gly-9, Ala-80 to Ala-91, Asp-119 to Arg-125, Ala-167 to Asp-179, Trp-195 to Gly-201. Polynucleotides encoding said polypeptides are encompassed by the invention.
FIGS. 1A–C show the nucleotide (SEQ ID NO:32) and deduced amino acid sequence (SEQ ID NO: 172) corresponding to this gene.
Table 6 shows an analysis of the amino acid sequence (SEQ ID NO: 172). 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 columns in Table 6 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 Table 6: “Res”: amino acid residue of SEQ ID NO:172 and FIGS. 1A–C; “Position”: position of the corresponding residue within SEQ ID NO:172 and FIGS. 1A–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 Table 6, 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 6 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 may, as shown in Table 6, be represented or identified by using tabular representations of the data. The DNA*STAR computer algorithm was used to present the data in a tabular format (See Table 6). The tabular format of the data 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:32, 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:32. 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:32. 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 1462, of SEQ ID NO:32, 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−281 where m is an integer from 2 to 276, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:172. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: G-2 to P-281; F-3 to P-281; P-4 to P-281; Q-5 to P-281; R-6 to P-281; Q-7 to P-281; P-8 to P-281; G-9 to P-281; L-10 to P-281; S-11 to P-281; G-12 to P-281; L-13 to P-281; L-14 to P-281; L-15 to P-281; L-16 to P-281; V-17 to P-281; W-18 to P-281; A-19 to P-281; L-20 to P-281; A-21 to P-281; W-22 to P-281; P-23 to P-281; L-24 to P-281; P-25 to P-281; C-26 to P-281; M-27 to P-281; S-28 to P-281; L-29 to P-281; E-30 to P-281; L-31 to P-281; I-32 to P-281; P-33 to P-281; Y-34 to P-281; T-35 to P-281; P-36 to P-281; Q-37 to P-281; I-38 to P-281; T-39 to P-281; A-40 to P-281; W-41 to P-281; D-42 to P-281; L-43 to P-281; E-44 to P-281; G-45 to P-281; K-46 to P-281; V-47 to P-281; T-48 to P-281; A-49 to P-281; T-50 to P-281; T-51 to P-281; F-52 to P-281; S-53 to P-281; L-54 to P-281; E-55 to P-281; Q-56 to P-281; P-57 to P-281; R-58 to P-281; C-59 to P-281; V-60 to P-281; L-61 to P-281; D-62 to P-281; G-63 to P-281; L-64 to P-281; A-65 to P-281; G-66 to P-281; V-67 to P-281; A-68 to P-281; S-69 to P-281; T-70 to P-281; I-71 to P-281; W-72 to P-281; L-73 to P-281; V-74 to P-281; V-75 to P-281; A-76 to P-281; F-77 to P-281; S-78 to P-281; N-79 to P-281; A-80 to P-281; S-81 to P-281; R-82 to P-281; D-83 to P-281; F-84 to P-281; Q-85 to P-281; N-86 to P-281; P-87 to P-281; Q-88 to P-281; T-89 to P-281; R-90 to P-281; A-91 to P-281; E-92 to P-281; I-93 to P-281; P-94 to P-281; A-95 to P-281; F-96 to P-281; P-97 to P-281; R-98 to P-281; L-99 to P-281; L-100 to P-281; T-101 to P-281; E-102 to P-281; G-103 to P-281; H-104 to P-281; Y-105 to P-281; M-106 to P-281; T-107 to P-281; L-108 to P-281; P-109 to P-281; L-110 to P-281; S-111 to P-281; L-112 to P-281; D-113 to P-281; Q-114 to P-281; L-115 to P-281; P-116to P-281; C-117 to P-281; Q-118 to P-281; D-119 to P-281; P-120 to P-281; A-121 to P-281; G-122 to P-281; G-123 to P-281; G-124 to P-281; R-125 to P-281; D-126 to P-281; V-127 to P-281; P-128 to P-281; L-129 to P-281; L-130 to P-281; R-131 to P-281; V-132 to P-281; G-133 to P-281; N-134 to P-281; D-135 to P-281; P-136 to P-281; G-137 to P-281; C-138 to P-281; L-139 to P-281; A-140 to P-281; D-141 to P-281; L-142 to P-281; L-143 to P-281; Q-144 to P-281; P-145 to P-281; P-146 to P-281; Y-147 to P-281; C-148 to P-281; N-149 to P-281; S-150 to P-281; P-151 to P-281; L-152 to P-281; P-153 to P-281; S-154 to P-281; P-155 to P-281; G-156 to P-281; P-157 to P-281; Y-158 to P-281; R-159 to P-281; V-160 to P-281; K-161 to P-281; F-162 to P-281; L-163 to P-281; L-164 to P-281; M-165 to P-281; D-166 to P-281; A-167 to P-281; R-168 to P-281; G-169 to P-281; S-170 to P-281; P-171 to P-281; Q-172 to P-281; A-173 to P-281; E-174 to P-281; T-175 to P-281; R-176 to P-281; W-177 to P-281; S-178 to P-281; D-179 to P-281; P-180 to P-281; I-181 to P-281; A-182 to P-281; L-183 to P-281; H-184 to P-281; Q-185 to P-281; G-186 to P-281; K-187 to P-281; S-188 to P-281; P-189 to P-281; A-190 to P-281; S-191 to P-281; I-192 to P-281; D-193 to P-281; T-194 to P-281; W-195 to P-281; P-196 to P-281; G-197 to P-281; R-198 to P-281; R-199 to P-281; S-200 to P-281; G-201 to P-281; G-202 to P-281; M-203 to P-281; I-204 to P-281; V-205 to P-281; I-206 to P-281; T-207 to P-281; S-208 to P-281; I-209 to P-281; L-210 to P-281; S-211 to P-281; S-212 to P-281; L-213 to P-281; A-214 to P-281; S-215 to P-281; X-216 to P-281; L-217 to P-281; L-218 to P-281; L-219 to P-281; A-220 to P-281; F-221 to P-281; L-222 to P-281; A-223 to P-281; A-224 to P-281; S-225 to P-281; T-226 to P-281; X-227 to P-281; R-228 to P-281; F-229 to P-281; S-230 to P-281; S-231 to P-281; L-232 to P-281; W-233 to P-281; W-234 to P-281; P-235 to P-281; E-236 to P-281; E-237 to P-281; A-238 to P-281; P-239 to P-281; E-240 to P-281; Q-241 to P-281; L-242 to P-281; R-243 to P-281; I-244 to P-281; G-245 to P-281; S-246 to P-281; F-247 to P-281; M-248 to P-281; G-249 to P-281; K-250 to P-281; R-251 to P-281; Y-252 to P-281; M-253 to P-281; T-254 to P-281; H-255 to P-281; H-256 to P-281; I-257 to P-281; P-258 to P-281; P-259 to P-281; S-260 to P-281; E-261 to P-281; A-262 to P-281; A-263 to P-281; T-264 to P-281; L-265 to P-281; P-266 to P-281; V-267 to P-281; X-268 to P-281; C-269 to P-281; E-270 to P-281; P-271 to P-281; G-272 to P-281; L-273 to P-281; D-274 to P-281; P-275 to P-281; and L-276 to P-281 of SEQ ID NO:172. 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, or 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. 1A–C (SEQ ID NO:172), as described by the general formula 1−n, where n is an integer from 6 to 280, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:172. 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 S-280; M-1 to L-279; M-1 to S-278; M-1 to P-277; M-1 to L-276; M-1 to P-275; M-1 to D-274; M-1 to L-273; M-1 to G-272; M-1 to P-271; M-1 to E-270; M-1 to C-269; M-1 to X-268; M-1 to V-267; M-1 to P-266; M-1 to L-265; M-1 to T-264; M-1 to A-263; M-1 to A-262; M-1 to E-261; M-1 to S-260; M-1 to P-259; M-1 to P-258; M-1 to I-257; M-1 to H-256; M-1 to H-255; M-1 to T-254; M-1 to M-253; M-1 to Y-252; M-1 to R-251; M-1 to K-250; M-1 to G-249; M-1 to M-248; M-1 to F-247; M-1 to S-246; M-1 to G-245; M-1 to I-244; M-1 to R-243; M-1 to L-242; M-1 to Q-241; M-1 to E-240; M-1 to P-239; M-1 to A-238; M-1 to E-237; M-1 to E-236; M-1 to P-235; M-1 to W-234; M-1 to W-233; M-1 to L-232; M-1 to S-231; M-1 to S-230; M-1 to F-229; M-1 to R-228; M-1 to X-227; M-1 to T-226; M-1 to S-225; M-1 to A-224; M-1 to A-223; M-1 to L-222; M-1 to F-221; M-1 to A-220; M-1 to L-219; M-1 to L-218; M-1 to L-217; M-1 to X-216; M-1 to S-215; M-1 to A-214; M-1 to L-213; M-1 to S-212; M-1 to S-211; M-1 to L-210; M-1 to I-209; M-1 to S-208; M-1 to T-207; M-1 to I-206; M-1 to V-205; M-1 to I-204; M-1 to M-203; M-1 to G-202; M-1 to G-201; M-1 to S-200; M-1 to R-199; M-1 to R-198; M-1 to G-197; M-1 to P-196; M-1 to W-195; M-1 to T-194; M-1 to D-193; M-1 to I-192; M-1 to S-191; M-1 to A-190; M-1 to P-189; M-1 to S-188; M-1 to K-087; M-1 to G-186; M-1 to Q-185; M-1 to M-184; M-1 to L-183; M-1 to A-182; M-1 to I-181; M-1 to P-180; M-1 to D-179; M-1 to S-178; M-1 to W-177; M-1 to R-176; M-1 to T-175; M-1 to E-174; M-1 to A-173; M-1 to Q-172; M-1 to P-171; M-1 to S-170; M-1 to G-169; M-1 to R-168; M-1 to A-167; M-1 to D-166; M-1 to M-165; M-1 to L-164; M-1 to L-163; M-1 to F-162; M-1 to K-161; M-1 to V-160; M-1 to R-159; M-1 to Y-158; M-1 to P-157; M-1 to G-156; M-1 to P-155; M-1 to S-154; M-1 to P-153; M-1 to L-152; M-1 to P-151; M-1 to M-150; M-1 to N-149; M-1 to C-148; M-1 to Y-147; M-1 to P-146; M-1 to P-145; M-1 to Q-144; M-1 to L-143; M-1 to L-142; M-1 to D-141; M-1 to A-140; M-1 to L-139; M-1 to C-138; M-1 to G-137; M-1 to P-136; M-1 to D-135; M-1 to N-134; M-1 to G-133; M-1 to V-132; M-1 to R-131; M-1 to L-130; M-1 to L-129; M-1 to P-128; M-1 to V-127; M-1 to D-126; M-1 to R-125; M-1 to G-124; M-1 to G-123; M-1 to G-122; M-1 to A-121; M-1 to P-120; M-1 to D-119; M-1 to Q-118; M-1 to C-117; M-1 to P-116; M-1 to L-115; M-1 to Q-114; M-1 to D-113; M-1 to L-112; M-1 to S-111; M-1 to L-110; M-1 to P-109; M-1 to L-108; M-1 to T-107; M-1 to M-106; M-1 to Y-105; M-1 to H-104; M-1 to G-103; M-1 to E-102; M-1 to T-101; M-1 to L-100; M-1 to L-99; M-1 to R-98; M-1 to P-97; M-1 to F-96; M-1 to A-95; M-1 to P-94; M-1 to I-93; M-1 to E-92; M-1 to A-91; M-1 to R-90; M-1 to T-89; M-1 to Q-88; M-1 to P-87; M-1 to N-86; M-1 to Q-85; M-1 to F-84; M-1 to D-83; M-1 to R-82; M-1 to S-81; M-1 to A-80; M-1 to N-79; M-1 to S-78; M-1 to F-77; M-1 to A-76; M-1 to V-75; M-1 to V-74; M-1 to L-73; M-1 to W-72; M-1 to I-71; M-1 to T-70; M-1 to S-69; M-1 to A-68; M-1 to V-67; M-1 to G-66; M-1 to A-65; M-1 to L-64; M-1 to G-63; M-1 to D-62; M-1 to L-61; M-1 to V-60; M-1 to C-59; M-1 to R-58; M-1 to P-57; M-1 to Q-56; M-1 to E-55; M-1 to L-54; M-1 to S-53; M-1 to F-52; M-1 to T-51; M-1 to T-50; M-1 to A-49; M-1 to T-48; M-1 to V-47; M-1 to K-46; M-1 to G-45; M-1 to E-44; M-1 to L-43; M-1 to D-42; M-1 to W-41; M-1 to A-40; M-1 to T-39; M-1 to I-38; M-1 to Q-37; M-1 to P-36; M-1 to T-35; M-1 to Y-34; M-1 to P-33; M-1 to I-32; M-1 to L-31; M-1 to E-30; M-1 to L-29; M-1 to S-28; M-1 to M-27; M-1 to C-26; M-1 to P-25; M-1 to L-24; M-1 to P-23; M-1 to W-22; M-1 to A-21; M-1 to L-20; M-1 to A-19; M-1 to W-18; M-1 to V-17; M-1 to L-16; M-1 to L-15; M-1 to L-14; M-1 to L-13; M-1 to G-12; M-1 to S-11; M-1 to L-10; M-1 to G-9; M-1 to P-8; M-1 to Q-7; and M-1 to R-6 of SEQ ID NO:172. 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:172, 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 No. 209124, where this portion excludes any integer of amino acid residues from 1 to about 276 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 209124, or any integer of amino acid residues from 6 to about 281 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. 209124. 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.
The tissue distribution in ovarian tumor tissue, combined with the homology to uroplakin III indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of reproductive disorders, urogential condtions, or endocrine disorders. Expression in ovarian tissue, indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, detection and diagnosis of conditions concerning proper ovarian function (e.g., egg maturation, endocrine function), as well as cancer. The expression in ovarian tissue may indicate that polynucleotides and/or polypeptides of the invention would be useful for treatment, preventing, detecting and/or diagnosing disorders of the ovary, including inflammatory disorders, such as oophoritis (e.g., caused by viral or bacterial infection), ovarian cysts, amenorrhea, infertility, hirsutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, endometrioid carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, Ovarian Krukenberg tumor). Polynucleotides polypeptides and/or antibodies of the invention would be useful for the diagnosis and imaging of ovarian tissues as described below in the sections entitled “Diagnosis and Imaging” and “Kits.” In addition, polynucleotides polypeptides and/or agonists or antagonists thereof (including antibodies of the invention) would be useful for the treatment, detection, diagnosis and/or prevention of hyperproliferative disorders, specifically ovarian neoplasms, as described below in the section entitled “Hyperproliferative Disorders.” Moreover, the protein would be useful for the detection, treatment, and/or prevention of a variety of reproductive disorders such as infertility. In addition, the protein may also be useful in the development of novel or improved contraceptives. 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 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:32 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 1448 of SEQ ID NO:32, b is an integer of 15 to 1462, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:32, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 23
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: GSKGQERKWRVRMGYLN (SEQ ID NO: 376), QRYRLLPLFCYVCSRKIKLNENLFVFSAYSLATLPHTYLFSIVEC SSFCLSGTRN (SEQ ID NO: 377), and/or FSAYSLATLPHTYLFSIVEC SSFCLSG (SEQ ID NO: 378). 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.
The gene encoding the disclosed cDNA is believed to reside on chromosome 7. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 7.
This gene is expressed primarily in human placenta.
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, developmental, vascular, and/or reproductive 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 embryonic and reproductive systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developmental, vascular, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amniotic fluid, 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 human placenta tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, detection and/or diagnosis of the disorders of embryonic and reproductive systems. Moreover, the protein would be useful for the detection, treatment, and/or prevention of a variety of vascular disorders, which include, but are not limited to, microvascular disease, aneurysm, arteriosclerosis, atherosclerosis, stroke, or embolism. 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:33 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 1258 of SEQ ID NO:33, b is an integer of 15 to 1272, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:33, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 24
This gene is expressed primarily in anergic 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, immune or hematopoietic disorders, particularly inflammatory conditions and immunodeficiencies such as AIDS. 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, 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.
The tissue distribution in anergic T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, detection, and/or diagnosis of T cell related disorders. Moreover, 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. Polynucleotides and/or polypeptides corresponding to this gene 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 polynucleotides and/or polypeptides of the invention 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. 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:34 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 759 of SEQ ID NO:34, b is an integer of 15 to 773, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:34, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 25
The translation product of this gene shares sequence homology with a rat secretory carrier membrane protein which is believed to play a role in cell surface recycling (see e.g., Brand et al., EMBO J 1993 October; 12(10):3753–3761, all references available through this accession are hereby incorporated in their entirety by reference herein). Secretory carrier membrane proteins (SCAMPs) are widely distributed as components of post-Golgi membranes that function as recycling carriers to the cell surface. In fibroblasts, SCAMPs are concentrated in compartments involved in the endocytosis and recycling of cell surface receptors while in neurons and other cell types having regulated transport pathways, SCAMPs are also components of regulated carriers (synaptic vesicles, secretion granules and transporter vesicles). Their presence in multiple pathways distinguishes them from proteins (e.g., recycling cell surface receptors and synaptic vesicle proteins) which are concentrated in selected pathways. The SCAMPs also do not appear to reside beyond the boundaries of these pathways. This distribution indicates that SCAMPs are general markers of membranes that function in cell surface recycling. Accordingly, polpeptides of the invention and antibodies thereto, may be used to identify membranes that function in cell surface recycling.
The gene encoding the disclosed cDNA is thought to reside on chromosome 15. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 15.
This gene is expressed primarily in hematopoietic cell types.
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, immune and hematopoetic 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 and hematopoetic systems, 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., hematopoietic cells, 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: 175 as residues: Ser-25 to Gly-31, Gln-149 to Ser-155. Polynucleotides encoding said polypeptides are encompassed by the invention.
The hematopoetic tissue distribution and homology to a cell surface molecule indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention and/or treatment of immune or hematopoietic disorders including arthritis, asthma and immunodeficiency diseases. 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:35 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 2441 of SEQ ID NO:35, b is an integer of 15 to 2455, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:35, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 26
The translation product of this gene shares sequence homology with intestinal epithelium proliferating cell-associated mRNA sequence, which is thought to be important in the growth and development of epithelial cells.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:









(SEQ ID NO:379)










MTLDEWKNLQEQTRPKPEFNIRKPESTVPSKAVVIRESKYRDDMVKDDY



EDDSHVFRKPANDITSQLEINFGNLPRPGRGARGGTRGGRGRIRRAENY


GPRAEVVMQDVAPNPDDPEDFPALS,


 









(SEQ ID NO:380)










CKMLPPTQMTRKISLRCLERALFPSTAELHCTPVGRLFQLGQGSQTLRT



IDVAFPVSCKFVALFWAELLEGLLQRLESRPFPKKMKNGDCVFIEGISN


EE,


 









(SEQ ID NO:381)










PPSSWAWSQRRHPGRPGKDQEGRELWTQSRSGDARCCPQPR, and/or



 









(SEQ ID NO:382)










CLKCVYRDSIDSSAEAWRERRL.







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 brain and central nervous system tissues, such as the frontal cortex, amygdala, and hypothalmus, and to a lesser extent in testis.
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, diseases of the neural system. 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 neural and reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, reproductive, 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: 176 as residues: Glu-20 to Glu-27, Glu-30 to Trp-44. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution and homology to intestinal epithelium proliferating cell-associated mRNA sequence indicates that polynucleotides and polypeptides corresponding to this gene would be useful for growth and developmental diseases of the brain, central nervous system and reproductive system. The tissue distribution in neural tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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 bahaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. Elevated expression of this gene product within the frontal cortex of 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. 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:36 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 900 of SEQ ID NO:36, b is an integer of 15 to 914, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:36, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 27
The translation product of this gene was shown to have homology to the highly conserved ras gene which is known to be important in the regulation of cell growth, and thus has been shown to serve as an inducible oncogene in eukaryotic tissues (see, e.g., Genbank Accession No. gb|Z11804|DDRASX, all references available through this accession are hereby incorporated in their entirety by reference herein).
When tested against PC12 (rat pheocbromocytoma cells) and NIH3T3 cell lines, supernatants removed from cells containing this gene activated the EGR1 (early growth response gene 1) promoter element. Thus, it is likely that this gene activates sensory neuron cells and fibroblasts, in addition to other tissues or cell types, through the EGR1 signal transduction pathway. The EGR1 (early growth response gene 1) is a separate signal transduction pathway from Jaks-STAT, genes containing the EGR1 promoter are induced in various tissues and cell types upon activation, leading the cells to undergo differentiation and proliferation. Moreover, contact of cells with supernatant expressing the product of this gene has been shown to increase the permeability of the plasma membrane of monocytes to calcium. Thus it is likely that the product of this gene is involved in a signal transduction pathway that is initiated when the product binds a receptor on the surface of the plasma membrane of monocytes, in addition to other cell-lines or tissue cell types, such as immune or hematopoietic cells. Thus, polynucleotides and polypeptides have uses which include, but are not limited to, activating monocytes.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: ARAGQMQNLESARAGRSVSTQTGS (SEQ ID NO: 383). 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.
The gene encoding the disclosed cDNA is believed to reside on chromosome 13. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 13.
This gene is expressed primarily in 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, diseases involving immune regulation, which include, but are not limited to autoimmune diseases such as rheumatoid arthritis, lupus, and leukemia. 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, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., hematopoietic, immune, 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: 177 as residues: Ala-28 to His-41, Pro-43 to Gln-64. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in T-cells, combined with the detected EGR1 and calcium flux activities, indicates polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of a variety of immune system disorders, particularly those dependent upon signalling aberrations. Expression of this gene product in T-cells indicates a role in regulating 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—particularly considering the homology to a conserved ras gene, and the detected EGR1 biological activity. Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore polynucleotides and/or polypeptides of the invention 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:37 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 1541 of SEQ ID NO:37, b is an integer of 15 to 1555, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:37, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 28
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: TVWGILPRKR (SEQ ID NO: 384). 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 ovarian tumor.
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, reproductive or endocrine disorders, particularly proliferative conditions such as ovarian cancer. 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 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, endocrine, 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.
The tissue distribution in ovarian tumor tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis and treatment of ovarian cancer. Moreover, the protein would be useful for the detection, treatment, and/or prevention of a variety of reproductive disorders such as infertility. In addition, the protein may also be useful in the development of novel or improved contraceptives. 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 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: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 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 1753 of SEQ ID NO:38, b is an integer of 15 to 1767, 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: 29
The translation product of this gene was found to have homology to the cell division control protein 48 (cdc48) of Methanococcus jannaschii (see, e.g., Genbank Accession No.gi|1591785, all references available through this accession are hereby incorporated in their entirety by reference herein) which is thought to play a key role in the regulation of cellular division.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: HEAAQGAVCRGQGAPATNPQAPVAAAARVARRVN (SEQ ID NO:385), KIPSANRRATRCLGCDHQNFVKVRNKHKGKPTFMEEVLEHLPGKTQDEVQQ HEKWYQKFLALEERKKESIQIWKTKKQQKREEIFKLKEKADNTPVLFHNKQE DNQKQKEEQRKKQKLAVEAWKKQKSIEMSMKCASQLKKKKKKKKKNQKE RQRQFKLKLLLESYTQQKKEQEEFLRLEKEIREKAEKAEKRKNAADEISRFQE RDLHKLELKILDRQAKEDEKSQKQRRLAKLKEKVENNVSRDPSRLYKPTK (SEQ ID NO:386), VKVRNKHKGKPTFMEEVLEHLPGK (SEQ ID NO:387), QHEKWYQKFLALEERKKESIQIW (SEQ ID NO:388), FKLKEKADNTPVLFHNKQEDNQKQKEEQRKK (SEQ ID NO:389), FIRLEKEIREKAEKAEKRKNAADEISRFQERDLHKL (SEQ ID NO:390), and/or KQRRLAKLKEKVENNVSRDPSRLY (SEQ ID NO: 391). 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 pancreas, and to a lesser extent in kidney and bone marrow.
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, pancreas, urogenital, developmental, metabolic, immune, and/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 pancreas, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., endocrine, developmental, metabolic, immune, hematopoietic, gastrointestinal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, bile, 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, four, five, six, seven, eight, or all nine of the immunogenic epitopes shown in SEQ ID NO: 179 as residues: Pro-35 to Cys-43, Gln-56 to Lys-67, Thr-73 to Lys-78, Tyr-93 to Asp-98, Ser-116 to Gln-125, Leu-142 to Phe-151, Phe-169 to Arg-174, Ile-181 to Glu-190, Thr-243 to Gly-248. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in pancreas indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, treatment, and/or prevention of various endocrine disorders and cancers, particularly Addison's disease, Cushing's Syndrome, and disorders and/or cancers of the pancreas (e.g., diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-, hypoparathyroidism), hypothallamus, and testes. Alternatively, the expression within bone marrow indicates that polynucleotides and polypeptides corresponding to this gene would be 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, the protein product of this gene could be used in the treatment, prevention, diagnosis 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. Considering the homology to a conserved cell division control protein indicates that the protein may show utility in the diagnosis, prevention, and/or treatment of developmental disorders, and may even serve as a suppressor in tumorigenesis. 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:39 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 1565 of SEQ ID NO:39, b is an integer of 15 to 1579, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:39, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 30
This gene is expressed primarily in kidney cortex.
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, diseases of the kidney including cancer and renal dysfunction, in addition to, endocrine disorders, particularly of the adrenal glands. 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 renal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., urogenital, renal, endocrine, and cancerous and wounded tissues) or bodily fluids (e.g., urine, 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 kidney cortex indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, diagnosis, and/or prevention of diseases of the kidney 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. 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. 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:40 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 1529 of SEQ ID NO:40, b is an integer of 15 to 1543, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:40, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 31
The translation product of this gene was shown to have homology to the retinoic acid receptor gamma-2 which is thought to be important in the development of, and may be a key determinant for, human breast cancer during aberrant activation (see, e.g., Genbank Accession No. AA176435, all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: LPPCLAQIFPFFSSGTNLTFCFFVFVFVFVFAELDYRNSYEIEY (SEQ ID NO: 392). 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 ovary, and to a lesser extent, in meningioma.
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, reproductive or neural disorders, particularly ovarian cancer, as well as, other cancers of the reproductive system, meninges, and endothelial tissue in general. 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 female reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., ovarian, reproductive, neural, endothelial, endocrine, and cancerous and wounded tissues) or bodily fluids (e.g., amniotic fluid, 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, four, or all five of the immunogenic epitopes shown in SEQ ID NO: 181 as residues: Leu-8 to Gln-18, Thr-26 to Lys-33, Met-39 to Cys-46, Ala-62 to Pro-69, Pro-83 to Glu-90. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in ovary tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis and intervention of tumors within these tissues, in addition to other tumors where expression has been indicated. The protein may also show utility in the treatment, detection, prevention, and/or amelioration of degenerative conditions or congenital disorders of the meninges, and the brain and spinal cord, in general. 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 tumors and 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:41 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 2081 of SEQ ID NO:41, b is an integer of 15 to 2095, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:41, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 32
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: HVLWSLLSACWTQFLVYFCCLMILQRTFPPRALRTSPWLSNPMGVKGKKKKGTFME (SEQ ID NO: 393), and/or FLVYFCCLMIILQRTFPPRALRTSPWLSNPM (SEQ ID NO: 394). 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 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, immune or hematopoietic disorders, including inflammatory diseases and allergies. 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, haematopoeitic, 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.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of a variety of immune system disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. 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:42 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 1078 of SEQ ID NO:42, b is an integer of 15 to 1092, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:42, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 33
When tested against a K562 kidney cell line, supernatants removed from cells containing this gene activated the ISRE (interferon-sensitive responsive element) pathway. Thus, it is likely that this gene activates kidney cells through the Jaks-STAT signal transduction pathway. The ISRE is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a complex, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: IRHERLWAELALLTGRNE (SEQ ID NO: 395). 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.
The gene encoding the disclosed cDNA is thought to reside on chromosome 3. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 3.
This gene is expressed primarily in activated T-cells and osteoarthritis, and to a lesser extent in aortic endothelium and placenta.
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, inflammatory conditions, vascular 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 and vascular tissues, 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., T-cells and other cells and tissue of the immune system, bone tissue, endothelium and placenta, vascular tissue, 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, two or all three of the immunogenic epitopes shown in SEQ ID NO: 183 as residues: Gln-36 to Glu-49, Glu-51 to Leu-66, Asp-68 to Ser-73. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in activated T-cells and under inflammatory conditions like osteoarthritis indicate that the protein product of this gene is involved in the inflammatory reactions. Therefore it may be useful in the diagnosis or intervention in the inflammatory diseases with the involvement of T-cells, including osteoarthritis. Furthermore, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and/or treatment of disorders of the placenta. Specific expression within the placenta indicates that polynucleotides and/or polypeptides of the invention may play a role in the proper establishment and maintenance of placental function. Alternately, this gene product may be 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 polynucleotides and/or polypeptides of the invention may be 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.
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:43 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 399 of SEQ ID NO:43, b is an integer of 15 to 413, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:43, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 34
This gene is expressed primarily in 12 week old early stage human.
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, developmental abnormalities. 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 developmental system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developing and differentiating tissue, 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 the immunogenic epitopes shown in SEQ ID NO: 184 as residues: Thr-14 to Thr-19. Polynucleotides encoding said polypeptides are encompassed by the invention.
The expression of this gene primarily in the embryo indicates a key role in embryonic development, and could be used in the treatment and or detection of developmental disorders. Expression within embryonic tissue and other 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, 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. 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:44 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 721 of SEQ ID NO:44, b is an integer of 15 to 735, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:44, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 35
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: LISSVNKTKQKRSDATLSHKHDRLLNHFVFFGNSYNY (SEQ ID NO: 396), SSKFPSDMLLRIQQIIYCHKLTIILTKWRNTARHKSKKKEDELILKHELQLKKW KNRLILKRAAAEESNFPERSSSEVFLVDETLKCDISLLPEXAILQVCMNSVYIIY YNLPSVVVHACNPSCLGG (SEQ ID NO: 397), SLESTNAIKSN (SEQ ID NO: 398), IRPNKNDQMRHCLINMIDY (SEQ ID NO: 399), ITLCFLETAITINIYSNLVNFLQICYCGYNRSSIVTS (SEQ ID NO: 400), and/or ISFRYAIADTTDHLLSQANHYPNKMAEYSKT (SEQ ID NO: 401). 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 T-cells, tonsils, and heart tissue.
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 immune system and vascular tissue 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 and vascular tissue, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, vascular, 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 T-cells, tonsils and heart indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention, and/or treatment of disorders of the immune system and vascular tissues. Expression of this gene product in tonsils 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 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 polynucleotides and/or polypeptides of the invention 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. Expression of this gene product in T cells also strongly indicates a role for this protein in immune function and immune surveillance. The tissue distribution in heart muscle tissue indicates that the protein product of this gene would be useful for the diagnosis and treatment of conditions and pathologies of the cardiovascular system, such as heart disease, restenosis, atherosclerosis, stoke, angina, thrombosis, and wound healing. 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:45 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 761 of SEQ ID NO:45, b is an integer of 15 to 775, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:45, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 36
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: TLVAGSPCSLSRWIMAGFCHGELVQSDMESQEWERGQVVLSHTSLPWCYVSPR (SEQ ID NO: 402), MAGFCHGELVQSDMESQEWERGQVVLSHTSLPWCYVSPR (SEQ ID NO: 403), MAVWISGSYSSFCSRSNWDVFSPNIVLASLPFSFRSVSKAAKPWWLALPALFP DGLWLDSAMGSLYSQTWKARNGKEVRWFSPTPHCLGAMSHL (SEQ ID NO: 404), and/or GWLYGSVGLIPHSAAEATG (SEQ ID NO: 405). 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 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 immune disorders and diseases. 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 the immunogenic epitopes shown in SEQ ID NO: 186 as residues: Pro-54 to Gly-62. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, detection, diagnosis, prevention and/or treatment of immune disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. Expression of this gene product in neutrophils 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: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 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 492 of SEQ ID NO:46, b is an integer of 15 to 506, 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: 37
This gene is expressed primarily in eight week human embryos.
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, fetal and/or developmental abnormalities. 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 developing fetus, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developing, differentiating, and cancerous and wounded tissues) or bodily fluids (e.g., amniotic fluid, serum, plasma, lymph, 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 eight week old tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for detecting, diagnosing, preventing, and/or treating embryonic abnormalities, in particular congenital abnormalities, which include, but are not limited to Tay-Sachs disease, phenylkenonuria, galactosemia, hyperlipidemias, porphyrias, and Hurler's syndrome. Expression within embryonic tissue and other cellular sources marked by proliferating cells indicates that polynucleotides and/or polypeptides of the invention may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention 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:47 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 1433 of SEQ ID NO:47, b is an integer of 15 to 1447, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:47, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 38
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: VCIPGAAGLSVLLG (SEQ ID NO: 406). 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 fetal kidney.
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, urogenital, renal, or developmental disorders, particularly renal failure, tumors of the kidney, and/or developmental abnormalities associated with the kidney. 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 renal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., urological, renal, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., amniotic fluid, 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 the immunogenic epitopes shown in SEQ ID NO: 188 as residues: Gln-26 to Gln-34. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in fetal kidney indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of cancer and other proliferative disorders, particularly renal disorders. Expression within embryonic tissue and other cellular sources marked by proliferating cells indicates that polynucleotides and/or polypeptides of the invention 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. Moreover, the protein product of this gene could be used in the treatment, prevention, diagnosis and/or detection of kidney diseases including 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. 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:48 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 1406 of SEQ ID NO:48, b is an integer of 15 to 1420, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:48, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 39
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: IAWSGNIPSLLCLFEHDMSFQDE (SEQ ID NO: 407). 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 tonsil.
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, inflammatory conditions, infections, or immunodeficiencies, and immune or hematopoietic diseases and/or 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 diseases, 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, two, three or all four of the immunogenic epitopes shown in SEQ ID NO: 189 as residues: Ile-2 to Lys-9, Gln-43 to Phe-49, Asn-59 to His-69, Gly-87 to Asp-93. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in tonsils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of a variety of immune system disorders. Expression of this 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 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:49 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 1206 of SEQ ID NO:49, b is an integer of 15 to 1220, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:49, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 40
The translation product of this gene shares sequence homology with a human apoptosis regulating protein which is thought to be important in regulating cell death.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: IRHEGQSSSRGSSHCDSPSPQEDGQIMFDVEMHTSRDHSSQSEEEVVEGEKEV EALKKSADWVSDWSSRPENIPPKEFHFRHPKRSVSLS (SEQ ID NO: 408), GHLLTLYPFWPEDILEFPNRVYCCLEICKGFFSANATSRL (SEQ ID NO: 409), EFGTRDRVVPEAVLTVTALRHKKMGRSCLMWKCTPAGTIALSQKKKL (SEQ ID NO: 410), AHPLPAPTEGKEKPLEMRVTCEVVYCHSSLFELETIVSMTQPTTLFLHIQFQ (SEQ ID NO: 411), TFCVFKHEEKWSHEERGYFLRRISEGVHSISLPFSCFGFGARHLYWKATTEHTL CQHLLRERKSPWKCV (SEQ ID NO: 412), and/or QSLLLFRNLQGLLFRKCHQQIIILSAMLLSLISATRLDLYHSWYKFYSCNITTIS LLKRDQVSK (SEQ ID NO: 413). 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 Jurkat cell lines, supernatants removed from cells containing this gene activated the NF-kB transcription factor. Thus, it is likely that this gene activates Jurkat cells by activating a transcriptional factor found within these cells. Nuclear factor kB is a transcription factor activated by a wide variety of agents, leading to cell activation, differentiation, or apoptosis. Reporter constructs utilizing the NF-kB promoter element are used to screen supernatants for such activity.
This gene is expressed primarily in muscle, fibroblast cells, haemopoietic cells, and 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, diseases of the haemopoietic, muscular and developing systems. 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 and muscular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, muscular, 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 the immunogenic epitopes shown in SEQ ID NO: 190 as residues: Met-1 to Ala-6. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution and homology to apoptosis regulating protein indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis and treatment of diseases of the haemopoietic, muscular and developing systems. Expression within embryonic tissue and other cellular sources marked by proliferating cells indicates that this protein may play a role in the regulation of cellular division. Additionally, the expression in hematopoietic cells and tissues indicates that polynucleotides and/or polypeptides of the invention may play a role in the proliferation, differentiation, and/or survival of hematopoietic cell lineages. In such an event, this gene may be useful in the treatment of lymphoproliferative disorders, and in the maintenance and differentiation of various hematopoietic lineages from early hematopoietic stem and committed progenitor cells. 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. 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:50 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 1034 of SEQ ID NO:50, b is an integer of 15 to 1048, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:50, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 41
The translation product of this gene shares sequence homology with Tbx, which is thought to be important in developmental regulation (see, e.g., Knezevic et al., Development 124, 411–419 (1997) hereby incorporated in its entirety by reference herein; and Genbank Accession No. U80951, all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: VTAYQNQQITRLKIDRNPFAKGFR (SEQ ID NO: 414), GTATVTAYQNQQITRL (SEQ ID NO: 415), KIDRNPFAKGFRDSGRNRMGLEAL (SEQ ID NO: 416), STLLQVLGMAFLPLTLTFCLA (SEQ ID NO: 417), and/or VESYAFWRPSLRTLTFEDIPGIPKQGNASS (SEQ ID NO: 418). 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 synovial sarcoma and to a lesser extent in osteoclastoma, osteoblastoma, and hemangiopericytoma.
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, osteosarcoma, osteoclastoma, and chondrosarcoma, and diseases of the skeletal system, such as osteoporosis. 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 skeletal 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., bone cells and tissue, synovial cells and tissue, cartilage, 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: 191 as residues: Ala-45 to Asp-50, Arg-57 to Pro-63. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in skeletal tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment and diagnosis of osteoperosis, fracture, osteosarcoma, osteoclastoma, chondrosarcoma, ossification and osteonecrosis, arthritis, tendonitis, chrondomalacia, and inflammation. Elevated levels of expression of this gene product in osteoclastoma and osteoblastoma indicates thatpolynucleotides and/or polypeptides of the invention may play a role in the survival, proliferation, and/or growth of these cells. Therefore, it may be useful in influencing bone mass in such conditions as osteoporosis. 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:51 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 954 of SEQ ID NO:51, b is an integer of 15 to 968, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:51, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 42
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: HGDWIYVHIVEQLNQANNKSVTSHTYFVVKTCKIHSLSNFQASNTLLXTVVT MLYNRSLELILPV (SEQ ID NO: 419), TYSSCLTKILYSLINIYPIPHCSPAXITTILLSASMNLTFFHFHICEIAQYLSFC AWLISLNIKSL (SEQ ID NO: 420), and/or MNLTFFFFRFHICEIAQYLSFCAWLISLNIKSL (SEQ ID NO: 421). 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 brain medulloblastoma.
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 cancer and disorders of the CNS. 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., brain, 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 brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of cancers and other disorders and diseases of the CNS. The tissue distribution further indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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 bahaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. 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:52 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 572 of SEQ ID NO:52, b is an integer of 15 to 586, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:52, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 43
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: RSKRQSQGSRCSVPLLAQQSRSPPVPLQAQPAWLLGSETIAWSGGGSGWEGP RDPGTSTAAGNSGPGIGMGHRTPPPSHTGR (SEQ ID NO: 422), RWDPAWGLDIPESSCPVTMGELRSGDGIVL (SEQ ID NO: 423), GALLWDNSMISAPRGSHREAGALFPSWLSNPAVLPSRSRPSQPGCLDPRQ (SEQ ID NO: 424), NSAREPRRWIRPTRGSGETTAPCCFEPLNGGTLVHAAAMARASEAAGTG (SEQ ID NO: 425), MARASEAAGTG (SEQ ID NO: 426), CFHTAFQKALRDPRPTLPDTHGSLRNAPLKSLTLPAAFVVSFFFLSLLQDGIKE RSQTQNATFFFHDRSDIEGLSEEPCSGTTP (SEQ ID NO: 427), LALQEAVTGKQVLCSPPGSAIPQSSRPAPGPASLAAWIRDNSLVWRRLRVGG TQGPGHQYSSWEFRPRDRDGAQDTTPISHREMKVGSSMGTGHP (SEQ ID NO: 428), and/or MAGRLFTLLLWQELARRLVPGDASPRLSRKRSVTPGPPFPTLTVPSE (SEQ ID NO: 429). 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 sensory neuron cell lines, supernatants removed from cells containing this gene activated the EGR1 assay. Thus, it is likely that this gene activates sensory neuron cells through a signal transduction pathway. Early growth response 1 (EGR1) is a promoter associated with certain genes that induces various tissues and cell types upon activation, leading the cells to undergo differentiation and proliferation.
This gene is expressed primarily in 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 immune disorders and diseases. 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 the immunogenic epitopes shown in SEQ ID NO: 193 as residues: Met-25 to Gly-30. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study and treatment of immune disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. Expression of this gene product in neutrophils 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:53 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 737 of SEQ ID NO:53, b is an integer of 15 to 751, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:53, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 44
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:430)









VWLLSSILLRVLWNRYTLQELSFWLPWFASRATSLVLQHGDNYLLFLFCF



VCFVLAMPF,


 








(SEQ ID NO:431)









IRHEVSMAFVFHLAQGTLEPLYIAGA,



 








(SEQ ID NO:432)









NSARGEYGFCLPSCSGYFGTAIHCRSLASGYHGLLPEQQA, and/or



 








(SEQ ID NO:433)









HELTVPSRMGSKGKPYPCGFYSSLIP.







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 U937 Myeloid cell lines, supernatants removed from cells containing this gene activated the GAS assay. Thus, it is likely that this gene activates myeloid 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.
This gene is expressed primarily in rejected kidney, stromal cells, and infant brain.
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, diseases of the renal, central nervous and immune systems. 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, renal and central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, renal, nervous, 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 the immunogenic epitopes shown in SEQ ID NO: 194 as residues: Ser-6 to Arg-15. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution rejected kidney, stromal cells, and infant brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of diseases of the renal, central nervous, and immune systems. The tissue distribution in infant brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection/treatment of neurodegenerative disease states and behavioural disorders such as 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 bahaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. Alternatively, 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 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 polynucleotides and/or polypeptides of the inventionmay 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. The tissue distribution in kidney indicates that this gene or gene product would be useful in the treatment, prevention, diagnosis 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. 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:54 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 463 of SEQ ID NO:54, b is an integer of 15 to 477, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:54, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 45
This gene is expressed primarily in 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, immune or hematopoietic disorders, including inflammatory conditions and allergies. 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, 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: 195 as residues: Lys-9 to Leu-16, Ser-33 to Met-43. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of a variety of immune system disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. 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:55 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 1139 of SEQ ID NO:55, b is an integer of 15 to 1153, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:55, and where b is greater than or equal to a+14. FEATURES OF PROTEIN ENCODED BY GENE NO: 46
The translation product of this gene was shown to have homology to the chicken LRP/alpha-2-macroglobulin receptor which is thought to play a pivitol role on the metabolism of alpha-2-macroglubulins, as well as, complexes between plasminogen activators and their endogenous inhibitors (see, e.g., Genbank Accession No. gb|X74904|GGLRPA2MR, all references available through this accession are hereby incorporated in their entirety by reference herein).
This gene is expressed primarily in neuronal tissues, and to a lesser extent in uterine cancer.
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, neuronal disorders and uterine cancer. 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 central neuron system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, reproductive, cancerous and wounded tissues) or bodily fluids (e.g., 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 neuronal tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. 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:56 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 1227 of SEQ ID NO:56, b is an integer of 15 to 1241, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:56, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 47
The translation product of this gene shares weak sequence homology with Hemophilus influenzae outmembrane protein P6 which is thought to be important in host cell interaction.
This gene is expressed primarily in human adrenal gland tumor.
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, adrenal insufficiency or hyperfunction, adrenal gland tumors. 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 endocrine systems and cancers thereof, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., adrenal gland, 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.
The tissue distribution in adrenal gland tumor and homology to Haemophilus influenzae outer membrane protein indicate that polynucleotides and polypeptides corresponding to this gene would be useful for adrenal insufficiencies or hyperfunction, because a secretory protein from an endocrine organ may function as a hormone. The protein product of this gene is also useful as a diagnostic and/or treatment for adrenal gland tumors, as well as tumors of other tissues where expression has been observed. 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:57 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 894 of SEQ ID NO:57, b is an integer of 15 to 908, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:57, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 48
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: GTESPMVMCCREVSQSENCLFLDTTFRFIFGKTFTNHDYISIHFYFLKAFLFSFFYSNV (SEQ ID NO: 434). 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 breast lymph nodes, B-cell lymphoma, and to a lesser extent in neutrophils and bone marrow 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, inflammation, immunodeficiency, allergy. 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, hematopoietic cells, and cells and tissue of the immune system, 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 the cells of immunological functions indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis or intervention of immunologically mediated disorders, such as allergy, immunodeficiency, immune surveillance, etc. 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:58 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 835 of SEQ ID NO:58, b is an integer of 15 to 849, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:58, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 49
This gene is expressed primarily in hematopoietic progenitor cells (CD34+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, autoimmune and immunodeficiency disease states. 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 hematopoietic 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., hematopoietic cells, 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.
The tissue distribution of this gene predominantly in hematopoietic progenitor cell types indicates that polynucleotides and/or polypeptides corresponding to this gene could be important for the treatment, prevention, diagnosis and/or detection of immune or hematopoietic disorders including arthritis, asthma, immunodeficiency diseases, leukemia, hypersensitivity and transplant rejection. Additionally, expression of this gene product in CD34+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 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 polynucleotides and/or polypeptides of the invention 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.
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:59 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 664 of SEQ ID NO:59, b is an integer of 15 to 678, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:59, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 50
This gene is expressed primarily in hematopoietic progenitor cells (CD34+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, auto-immune and immunodeficiency disease states. 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 hematopoietic 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., hematopoietic cells, 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.
The tissue distribution of this gene predominantly in hematopoietic progenitor cell types indicates that polynucleotides and/or polypeptides corresponding to this gene may be important for the treatment, prevention, diagnosis, and/or detection of immune or hematopoietic disorders including arthritis, asthma, immunodeficiency diseases, leukemia, and transplant rejection. Expression of this gene product in CD34+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 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 polynucleotides and/or polypeptides of the invention 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.
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:60 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 843 of SEQ ID NO:60, b is an integer of 15 to 857, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:60, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 51
When tested against a K562 kidney cell line, supernatants removed from cells containing this gene activated the ISRE (interferon-sensitive responsive element) pathway. Thus, it is likely that this gene activates kidney cells through the Jaks-STAT signal transduction pathway. The ISRE is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a complex, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
This gene is expressed primarily in bone marrow and spleen.
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 diseases, transplant rejection and immundeficiency disease states. 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 hematopoietic and immune systems, 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 or both of the immunogenic epitopes shown in SEQ ID NO: 201 as residues: Pro-22 to His-33, Ser-42 to Trp-48. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution of this gene predominantly in hematopoietic cell types indicates that polynucleotides and/or polypeptides corresponding to this gene may be important for the treatment, prevention, diagnosis and/or detection of immune or hematopoietic disorders including arthritis, asthma, immunodeficiency diseases, leukemia, and transplant rejection. Furthermore, the polypeptides or polynucleotides are also useful to enhance or protect the proliferation, differentiation, and functional activation of hematopoietic progenitor cells (e.g., bone marrow cells), useful in treating cancer patients undergoing chemotherapy or patients undergoing bone marrow transplantation. The polypeptides or polynucleotides are also useful to increase the proliferation of peripheral blood leukocytes, which can be used in the combat of a range of hematopoietic disorders, including immmunodeficiency diseases, leukemia, and septicemia.
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 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 753 of SEQ ID NO:61, b is an integer of 15 to 767, 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: 52 In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:









(SEQ ID NO:435)












SLQYRIRIPGRPT,




 









(SEQ ID NO:436)











DLVTYTSSLQYRIRIPGRPTRP, 



 









(SEQ ID NO:438)












VKTAECYSIPLGSCPVNIQRVR, and/or




 









(SEQ ID NO:437)












LGNKKYINIRCLEMQVTLKILCEIEKKERRGTHCLV.







Moreover, fragments and variants of these polypepti des (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 polynucleoti des encoding these polypeptides are also encompassed by the invention.

Contact of cells with supernatant expressing the product of this gene increases the permeability of U937 monocyte cells to calcium. Thus, it is likely that the product of this gene is involved in a signal transduction pathway that is initiated when the product of this gene binds a receptor on the surface of the monocyte cell. Thus, polynucleotides and polypeptides have uses which include, but are not limited to, activating monocyte cells.
This gene is expressed in primary dendritic 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, auto-immune disorders such as asthma and arthritis, in transplant rejection, leukemia and immunodeficiency disease states. 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., primary dendritic cells and other 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: 202 as residues: Gly-2 to Glu-7, Arg-27 to Gly-34. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution of this gene predominantly in hematopoietic cell types indicates that polynucleotides and/or polypeptides corresponding to this gene may be important for the treatment, prevention, diagnosis, and/or detection of immune or hematopoietic disorders including arthritis, asthma, immunodeficiency diseases, leukemia, hypersensitivity and graft rejection. Expression of this gene product in 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:62 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 714 of SEQ ID NO:62, b is an integer of 15 to 728, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:62, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 53
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: HKCFQCFILANGFLKVIKPFQRNWSDKTFFLVCLNKAISEALLSKMTFLSFPKT NLLLLETFCTI (SEQ ID NO: 439), LLGVLKPLYFSVEPVLGERSVAFEEVREKNHGTSGFLSLYSLAAIVCGHLMFF HTLLGRGGNDHPGQSPLPGMRPLRGGLAGQAPSGHPWMQPLDTCLL (SEQ ID NO: 440), RPTRPPTRPDRPSLELAPGLCADFLGSSNHClFLLSLYLGRDQ (SEQ ID NO: 441), and/or EKRIMVPQGFFPFIRWQPLSVGTSCFSTLYWAVEVTITQASLLCLGCAL (SEQ ID NO: 442). 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 haemopoietic and neural tissues, and to a lesser extent in a number of cancers and other tissues.
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, diseases of the haemopoietic and neural systems. 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 and neural system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, neural, 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 and neural tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of diseases of the haemopoietic and neural systems, including several cancers. 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 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 polynucleotides and/or polypeptides of the invention 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. Furthermore, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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 bahaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. 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:63 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 930 of SEQ ID NO:63, b is an integer of 15 to 944, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:63, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 54
This gene is expressed primarily in bone marrow 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, immune or hematological 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 hematological and immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, haematopoeitic, 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, two or all three of the immunogenic epitopes shown in SEQ ID NO: 204 as residues: Glu-22 to Ser-33, Leu-47 to Ser-55, Thr-87 to Arg-104. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in bone marrow cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis, and/or detection of hematopoetic related disorders, which include, but are not limited to 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:64 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 768 of SEQ ID NO:64, b is an integer of 15 to 782, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:64, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 55
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:443)











NSARVTQKGESVGSVGCMRAIAGFDNYPLF,




 








(SEQ ID NO:444)











GTIGIFWPLPVAILSSGDYLQTQIHRPLLHRGT,




 








(SEQ ID NO:445)











LPLPLSSLLHIATCNPFPKT,




 








(SEQ ID NO:446)











SYFFVYNLILKIIQGDHASIILLATIPIFGDIYYVKGQLASFGPYL,




 








(SEQ ID NO:447)











LFYHLEIISRHKSIAHCSIEA,




 








(SEQ ID NO:448)











CSCHCPSRAFST, and/or




 








(SEQ ID NO:449)











PHAIHSQKPSSIFLITDVFPDPPVGIYLL.







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 chronic synovitis.
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, inflammatory diseases and disorders of the musculo-skeletal system. 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 inflammatory and musculo-skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., musculo-skeletal, 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 the immunogenic epitopes shown in SEQ ID NO: 205 as residues: Ser-39 to Pro-44. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in chronic synovitis tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis, and/or detection of disorders and diseases of the inflammatory and musculo-skeletal system. In addition, the expression of this gene product in synovium indicates a role in the detection, diganosis, prevention and/or 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 (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). 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:65 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 428 of SEQ ID NO:65, b is an integer of 15 to 442, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:65, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 56
The gene encoding the disclosed cDNA is believed to reside on chromosome 1. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 1.
This gene is expressed primarily in ovary, and to a lesser extent, in infant brain.
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, reproductive, neural, or developmental disorders, particularly cancers and other diseases of the reproductive system including ovarian cysts and hormonal 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 female 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, neural, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., amniotic fluid, lymph, 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. Preferred polypeptides of the present invention comprise, or alternatively consist of the immunogenic epitopes shown in SEQ ID NO: 206 as residues: Ser-32 to Glu-37. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in ovarian tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis, detection, prevention, and/or intervention of ovarian tumors, in addition to other tumors where expression has been indicated. Alternatively, expression within the fetal brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo. Moreover, the expression within fetal tissue indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, detection, prevention and/or 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:66 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 819 of SEQ ID NO:66, b is an integer of 15 to 833, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:66, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 57
This gene is expressed primarily in T-cell lymphoma, and to a lesser extent, in bone marrow stromal 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, immune or hematopoietic disorders, particularly cancers, such as lymphomas and leukemias. 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.
The tissue distribution in bone marrow stromal cells and T-cell lymphoma indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of a variety of immune or hematopoietic disorders. Expression of this gene product in T-cells indicates a role in regulating 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 polynucleotides and/or polypeptides of the invention 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. Expression in bone marrow cells indicate that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis, and/or detection 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.
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:67 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 1248 of SEQ ID NO:67, b is an integer of 15 to 1262, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:67, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 58
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: TRPTMPNFLWFPKCA (SEQ ID NO: 450). 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 meningioma tissues.
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, neural or central nervous system disorders, particularly cancers of the central nervous system and endothelium. 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, endothelial, CNS, 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.
The tissue distribution in meningioma tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Moreover, the protein would be useful in inhibiting or ameliorating infections of the meninges, particular viral infections. In addition, the protein may show utility in the treatment, detection, and/or prevention of such infections and disorders, in addition to degenerative conditions or congenital defects of the meninges. 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:68 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 907 of SEQ ID NO:68, b is an integer of 15 to 921, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:68, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 59
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: RNSLHCYNEQPPNASGLIQWSSD LIPISLQCGCSW (SEQ ID NO: 451). 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 synovial membrane.
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, diseases of synovial membrane, skeletal and/or musculoskeletal 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 synovial membrane system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., skeletal, muscular, rheumatiod, synovial, 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 the immunogenic epitopes shown in SEQ ID NO: 209 as residues: Pro-10 to Ser-20. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution within synovial tissue indicates the product of this gene may play a role in the detection, treatment, and/or prevention of disorders and conditions affecting the skeletal systemskeletal system, in particular osteoporosis, bone cancer, 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). 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:69 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 464 of SEQ ID NO:69, b is an integer of 15 to 478, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:69, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 60
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: IRHEEKGGKAQRWAE (SEQ ID NO: 452). 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 bone marrow.
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, hemapoiesis 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 hemapoietic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., bone marrow, 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 the immunogenic epitopes shown in SEQ ID NO: 210 as residues: Thr-45 to Tyr-50. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in bone marrow indicates that polynucleotides and polypeptides corresponding to this gene would be useful for hemapoiesis disorders. The gene product may function as a growth factor or mobilization agent for the cells of myeloid or lymphoid lineages. Furthermore, the polypeptides or polynucleotides are also useful to enhance or protect proliferation, differentiation, and functional activation of hematopoietic progenitor cells (e.g., bone marrow cells), useful in treating cancer patients undergoing chemotherapy or patients undergoing bone marrow transplantation. 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 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 705 of SEQ ID NO:70, b is an integer of 15 to 719, 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: 61
The translation product of this gene shares regional and weak sequence homology with neu differentiation factor and a serine protease N-terminal fragment which contains a EGF-like domain and is thought to be important in the growth and differentiation of several cell types, including colon epithelial cells and Schwann cells.
This gene is expressed primarily in fetal lung, bone marrow, fetal liver, and to a lesser extent in brain.
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, tissue injuries or diseases in lung, bone marrow, or liver. 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 liver and lung, 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., lung and pulmonary tissue, bone marrow, hepatic tissue, 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.
The tissue distribution and homology to neu differentiation factor indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis or intervention of liver or lung injuries, including hepatic failure, recovery from hepatitis, cirrhosis, hepatoblastoma, jaundice, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells, and complications from liver transplantation. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and immunotherapy targets for the above listed tumors and 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:71 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 505 of SEQ ID NO:71, b is an integer of 15 to 519, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:71, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 62
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:453)









VDPRVRLPLFWWQPSCAVYLFPRVYNNMCTRVLGTLPHCWDLATLLQPSS



RIWGNVSEAPGM,


 








(SEQ ID NO:454)









VPYHIAGTLPHCCSLPVGYGGMSVRLQGCRYVGNVGPQGNMQSGRSWALK



MVLLCNSCLGLGVGSVGPSMSSLFGAVLSETPGSSVY, and/or


 








(SEQ ID NO:455)









MLDPRATCNLVGVGLSKWCCCVTAAWVLG.







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 chronic lymphocytic leukemia.
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 of immune system including cancer. 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.
The tissue distribution in chronic lymphocytic leukemia indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, diagnosis, detection, prevention and/or treatment of disorders of the immune system including cancers. Therefore polynucleotides and/or polypeptides of the invention 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. 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:72 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 812 of SEQ ID NO:72, b is an integer of 15 to 826, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:72, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 63
An embodiment of the invention is directed to polypeptides comprising those which exhibit sequence homology with honeybee venom sacepin.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: PQIKLLNSDALGMRTTSXDLVPCNQCHIPLPPSCNRIASRKAVNWKQQRLPAV RGLLNNAPHRRPPTPRTPCVFPSEGPKGYGFHV (SEQ ID NO: 456), EQLAXISCRVINVSFRCLHHVIESLPERQLTGSSRGSQP (SEQ ID NO: 457), EDCSTMPPIAAPPPLAPLVFSPLRGPRVMAFMSRCGDRGGRGRSXAGRGWP WSESGVINAHPKKRPCPGPMLS (SEQ ID NO: 458), and/or EFGTRRQWGTRCFPPLVGRKQSALRRREGKARAGRCCGKRSVKAGFDA (SEQ ID NO: 459). 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 activated and control neutrophils, and to a lesser extent in fetal liver and spleen.
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 disorders of the immune and endocrine systems. 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, inflammatory and hormonal systems, 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 neutrophils and fetal liver and spleen indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, diagnosis, detection, prevention and/or treatment of inflammation and various disorders of the immune and endocrine systems. 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 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 polynucleotides and/or polypeptides of the invention 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. Expression of this gene product in neutrophils 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:73 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 897 of SEQ ID NO:73, b is an integer of 15 to 911, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:73, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 64
This gene is expressed primarily in 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, immune conditions. 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 the immunogenic epitopes shown in SEQ ID NO: 214 as residues: Lys-33 to Lys-41. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for study, detection, diagnosis, prevention and/or treatment of immune disorders. 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 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 polynucleotides and/or polypeptides of the invention 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.
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:74 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 708 of SEQ ID NO:74, b is an integer of 15 to 722, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:74, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 65
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: PKVLAVLKKKNHVALSIFELLSNDICSFISFFMS (SEQ ID NO: 460), EGPDINSNLKFLLCLKKKIMWPFQYLNC (SEQ ID NO: 461), and/or LLSLILLRIWYDFSKQTVFWFFLNVFNFFSSCNNDGACSYKYRKVQI (SEQ ID NO: 462). 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 osteoblasts, and to a lesser extent in bone marrow and bladder.
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, skeletal and hematopoietic diseases and 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 skeletal and vascular systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., skeletal, vascular, 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 the immunogenic epitopes shown in SEQ ID NO: 215 as residues: Gly-33 to Lys-38. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in bone marrow and osteoblasts indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, diagnosis, and treatment of bone and hematopoietic disorders. Elevated levels of expression of this gene product in osteoblasts indicates that polynucleotides and/or polypeptides of the invention may play a role in the survival, proliferation, and/or growth of osteoblasts. Therefore, it may be useful in influencing bone mass in such conditions as osteoporosis. More generally, as evidenced by expression in bone marrow, this gene may play a role in the survival, proliferation, and/or differentiation of hematopoietic cells in general, and may be of use in augmentation of the numbers of stem cells and committed progenitors. 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:75 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 831 of SEQ ID NO:75, b is an integer of 15 to 845, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:75, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 66
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:463)









RKLFHKINSKSFHLSGMHILISVWIVRSRIIKVKYELLLCFFDVIFYV,



 








(SEQ ID NO:464)









NSARDVFFTQKILYSQTCIFFPCLVPFSFLFSFFFFLSFVG,



 








(SEQ ID NO:465)









MFSSLKKFYILKHVYSFPVLFHFLFFFLFSFSFLSWAEKGAGKMKLATEN



CKMVKS, and/or


 








(SEQ ID NO:466)









IQLLYLKGAAMKYLSYVARLLFLKALDLFAPKMVQIDSF.







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 kidney and infant brain.
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, diseases of the renal and central nervous systems. 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 neural and renal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, renal, 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 the immunogenic epitopes shown in SEQ ID NO: 216 as residues: Gly-24 to Lys-31. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in kidney and infant brain tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of diseases of the neural and renal systems. The tissue distribution in infant brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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 bahaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. The tissue distribution in kidney indicates that this gene or gene product would be useful in the treatment, prevention, diagnosis 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. 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: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 868 of SEQ ID NO:76, b is an integer of 15 to 882, 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: 67
The translation product of this gene was found to have homology to the human env endogenous retrovirus protein (see, e.g., Genbank Accession No. gi|757872, all references available through this accession are hereby incorporated in their entirety by reference herein), which is thought to play a contributing role in the events leading up to the onset of cancer or of proliferative disorders in teratocarcinoma cell lines.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: VDPRVRRFWEDPEYPPVAVMSRLMLRRIPTVMSNTHRTQPSTWEQIKKLSQ MVGENLRKAGQPVT (SEQ ID NO:467), VRRFWEDPEYPPVAVMSRLMLRRIP (SEQ ID NO:468), SNTHRTQPSTWEQIKKLSQMVGENLRK (SEQ ID NO:469), SACHSHTVFNWSEQNGQMVQMVRRMARVPIIWNHGSIGAPQPQMIWPIVGA KHKDLWQLLIALNKIKIWERIKKHLEGHSANLSLDIAKYIYIFKASQAHLTLM PELECSKELQTD (SEQ ID NO:470), MARVPIIWNHGSIGAPQPQMIWPIV (SEQ ID NO:471), RIKKHLEGHSANLSLDIAKYIYIFKASQAHLT (SEQ ID NO:472), VFLQQGLTQRSVHLIGHICQFWLAIMPGYNHFMTQLHMLSGLNIYHNKSAPIIE AYHP QKSICKQN (SEQ ID NO:473), IGHICQFWLAIMPGYNHFMTQLHMLSGL (SEQ ID NO:474), SIPGTPDLNARTGVLEGAADRLAASNPLKWIKTLRSSVISMMIVLLICVVCLYI VCRC (SEQ ID NO:475), VLEGAADRLAASNPLKWIKTLRSSVIS (SEQ ID NO:476), LTVTKLPWLFIALQNKRMGTSWEQAPKSGHKLAPKLVINKISAALSHACDSL TPTLEGCRFTGMRARNNWPTQGG (SEQ ID NO:477), and/or MGTSWEQAPKSGHKLAPKLVINKISAALS (SEQ ID NO:478). 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 PHA stimulated 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 the following diseases and conditions which include, but are not limited to, immune disorders, particularly autoimmune, inflammatory, or immunodeficiency diseases, in addition to, proliferative conditions such as cancers. 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, hematopoietic, teratocarcinoma, 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.
The tissue distribution in T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of a variety of immune system disorders. The expression of this 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 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 polynucleotides and/or polypeptides of the invention may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, and leukemia. 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 protein may also show utility in the development of novel inhibitors to viral infections, or the protein may be useful in the development of novel vectors, such as those used in gene therapy, and/or immuno-therapy which could lead to the amelioration of disease of disease states. 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.
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:77 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 1576 of SEQ ID NO:77, 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:77, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 68
The translation product of this gene was shown to have homology to the human retrovirus-related reverse transcriptase pseudogene (see, e.g., Genbank Accession No. pir|A253131|GNHUL1, all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: STHASVQKKDLTKFSAHSWLKKKKTFRKMIMEEIFLNLIKNIYKSPYSQCNT (SEQ ID NO:479), VRSEKGFDKIQCPFMVK (SEQ ID NO:480), FSKPSSYKTYIPKINLHFYILLMNIWETIKIVPLNNCFTKMNYLGI (SEQ ID NO: 481), KKETKLSLFANDMI (SEQ ID NO:482), and/or SPLLFNILLEVLSSAVRKEKELK (SEQ ID NO:483). 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 PHA 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: immune or hematopoietic disorders, particularly inflammation, immunodeficiencies, and autoimmune diseases. 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, 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 the immunogenic epitopes shown in SEQ ID NO: 218 as residues: Ile-14 to Thr-24. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. The expression of this 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. Polynucleotides and/or polypeptides corresponding to this gene 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 polynucleotides and/or polypeptides of the invention may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, and leukemia. 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. Alternatively, the homology to a reverse transcriptase human gene may implicate this gene as providing utility in the understanding of host-viral interactions, particularly those involving retroviruses and other integration-dependent viruses. Moreover, the protein may show utility in the development of novel inhibitors to viral infection, and thus to the amelioration of human diseases and conditions. 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:78 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 1359 of SEQ ID NO:78, b is an integer of 15 to 1373, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:78, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 69
The translation product of this gene was shown to have homology to the Gcapl gene product of Mus musculus, which is specifically expressed in cerebellum and appears to be developmentally regulated (see, e.g., Genbank Accession No. gi|862343, all references available through this accession are hereby incorporated in their entirety by reference herein). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: LCAVEKTRTHTRGDCGPNRHHKHVLKAKDNNHIQRHQFSSTLEFSSNSTDGL KYICVYLYVCTHPCIYIYLSAHTLHMYTHYLCKI (SEQ ID NO: 484), SSTLEFSSNSTDGLKYICVYLYVCTHPCIY (SEQ ID NO: 485), STSVCICTCAHTHVYIFIYLHTHYICIHTIYVKYNICMHNSNKClCVIFKIEQLY LEVVNAENWFYC (SEQ ID NO: 486), IHTIYVKYNICIMIHIN SNKClCVIFKIEQLY (SEQ ID NO: 487), and/or NSAVTVQMA (SEQ ID NO: 488). 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 fetal lung, endothelial cells and to a lesser extent, in astrocytes and fetal brain.
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, vasdcular, developmental, neural, or proliferative conditions, particularly endothelial cell proliferation, such as occurs in restenosis. 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 vascular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developmental, neural, vascular, and cancerous and wounded tissues) or bodily fluids (e.g., 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 fetal brain, in addition to the homology to a brain-specific regulatory protein indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, treatment, or prevention of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Alternatively, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for treating, preventing, detecting, and/or diagnosing abnormal proliferation of endothelial cells such as occurs upon injury to the lung or arteries. Moreover, this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, detection, prevention and/or 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:79 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 1093 of SEQ ID NO:79, b is an integer of 15 to 1107, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:79, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 70
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: KYLVSSVLPTISMARSLISALRSG (SEQ ID NO: 489). 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.
The gene encoding the disclosed cDNA is believed to reside on chromosome 7. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 7.
This gene is expressed primarily in ovarian cancer.
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, reproductive disorders, particularly ovarian cancer. 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 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, ovarian, 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.
The tissue distribution in ovarian tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis, detection, prevention and/or treatment of ovarian cancer. Moreover, the protein would be useful for the treatment, detection, and/or prevention of endocrine disorders, particularly those related to the reproductive system. 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:80 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 1115 of SEQ ID NO:80, b is an integer of 15 to 1129, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:80, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 71
The translation product of this gene shares sequence homology with estrogen-responsive finger protein which is thought to be important in uterine implantation. (see, e.g., Genbank Accession No. 1088467, all references available through this accession are hereby incorporated in their entirety by reference herein; and J. Biol. Chem. 270 (41), 24406–24413 (1995), herein incorporated by reference in its entirety.) Moreover, the protein product of this gene was also shown to homology to the human rfp transforming protein (see, e.g., Genbank Accession No. gi|337372, all references available through this accession are hereby incorporated in their entirety by reference herein) which is thought to play a role in in male germ cell development.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:









VXDITFDPDTAHKYLRLQEENRKVTNTTPWEHPYPDLPSRFLH;
(SEQ ID NO:490)



 


LYLHRYYFEVEIFGAGTYV;
(SEQ ID NO:491)


 


SCISGNNFSWSLQWNGKEFTAW;
(SEQ ID NO:492)


 


TPLKAGPFWSSGSILTS;
(SEQ ID NO:493)


 


SVSEVKAVAEMQFGELLAAVRKAQANVMLFLXEKEQAAL;
(SEQ ID NO:494)


 


EKSKQELETMAAISNTVQFLEEYCKFKNTEDITFPSVYIGLKD;
(SEQ ID NO:495)


 


LENYKKKLQEFSKEEEYDIRTQVSAXVQR;
(SEQ ID NO:496)


 


GTVSRERRAG,
(SEQ ID NO:498)


 


HGDPTQSWPFLELGVYIDFPGGILSFYGVEYDSMTLVHKFACKFS
(SEQ ID NO:499)


EPVYAAFWLSKKENAIRIVDLGEEPEKPAPSLVGTAP,


 


SFYGVEYDSMTLVHKFACKFSEPVYAAFWL,
(SEQ ID NO:500)


 


AELQCTQLDLERKLKLNENAISRLQANQKSVLVSVSEVKAVAEMQFGELLAA
(SEQ ID NO:501)


VRKAQANVMLFLXEKEQAALSQANGIKAHLEYKSAEMEKSKQELETMAAIS


NTVQFLEEYCKFKNTEDITFPSVYIGLKDKLSGIRKVITESTVHLIXXLENYKK


KLQEFSKEEEYDIRTQVSAXVQRKYWTSKPEPSTREQFLQYVXDITFDPDTAH


KYLRLQEENRKVTNTTPWEHPYPDLPSRFLHWRQVLSQQSLYLHRYYFEVEI


FGAGTYVGLTCKGIDXKGEERXSCISGNNFSWSLQWNGKEFTAWYSDMETP


LKAGPFWSSGSILTSQEGSFPSMA,


 


RTAPYGAKESSWRMLFSFRDPIGFQKPATISSYFC
(SEQ ID NO:502)


PQITLKCKSHHCSWQRSGIWLLESREQSPPRTVLASRVPLPDLQSGWRFPSWK


ARRQHRLVLKTCRQTCEPESWNHTLRHRRKGSLLGSQYRPRAPERASFEWG


LHVTVPGRELLPVPLEAPGEVVSGNATXALLPFXVDAFAGQANIGACPEDLH


LKIVPVQVQTLLGQHLPPVQEPAGEVRVGMLPGRGVGDLAVLLLQPEWVCC


VRVERDVXHILEELFPGAGLRFGSPIFALNNGRHLSSDVILLFLGKLLELFLIVL


QXXD, and or


 


GVYIDFPGGILSFYGVEYDSMTLVHKFACKESEPVYAA.
(SEQ ID NO: 497)






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 ovarian cancer.
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, ovarian cancer and other disorders of the reproductive system. 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 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, developmental, ovarian, testicular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, 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 ovarian tumors, combined with the homology to estrogen-responsive finger protein, in addition, to the conserved rfp transforming protein indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis, detection, prevention and/or treatment of ovarian cancer and other disorders of the reproductive system. Moreover, 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, detection, prevention and/or treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus polynucleotides and/or polypeptides of the inventionmay also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. The protein may also show utility in the development of novel contraceptives. 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:81 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 1973 of SEQ ID NO:81, b is an integer of 15 to 1987, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:81, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 72
This gene shows sequence homology to a Caenorhabditis elegans gene, called D1054.3, in addition, to the Sgt1p protein of Saccharomyces cerevisiae which are thought to play a role in the regulation of cellular division and developmental precesses (see, e.g., Genbank Accession Nos. gnl|PID|e348554 and gi|1870791, respectively, all references available through these accessions are hereby incorporated in their entirety by reference herein)
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: SKIKYDWYQTESQVVITLMIKNVQKNDVNVEFSEKELSALVKLPSGEDYNLK LELLHPIIPEQSTFKVLSTKIEIKLKKPEAVRWEKLEGQGDVPTPKQFVADVKN LYPSSSPYTRNWDKLVGEIKEEEKNEKLEGDAALNRLFQQIYSDGSDEVKRA MNKSFMESGGTVLSTNWSDVGKRKVEINPPDDMEWKKY (SEQ ID NO: 503); GDAALNRLFQQIYSDGSDEVKRAMNKSFMESGGTVLSTN (SEQ ID NO: 504); MAAAAAGTXXSQRFFQSFSDALIDEDPQAALEELTKALEQKPDDAQYYCQR AYCHILLGNYCVAVADAKKSLELNPNNSTAMLRKGICEYHEKNYAAALETF TEGQKLDSADANFSVWIKRCQEAQNGSESEVVSPKFSFFMFLLF (SEQ ID NO: 506), LEELTKALEQKPDDAQYYCQRAYCHILLGNYCVAVADA (SEQ ID NO: 507), AMLRKGICEYHEKNYAAALETFTEGQKLDSA (SEQ ID NO: 508), LRLWNRNQMMHSIHVKELIVTFLGITVLLLLMQRSL (SEQ ID NO: 509), NSIQIIPLLC (SEQ ID NO: 510), YMHFNNTVAKLTCKNLSLSTYQNQSASQWTHQSKIKYDWYQTESQVVITLM IKNVQKNDVNVEFSEKELSALVKLPSGEDYNLKLELLHPIIPEQSTFKVLSTKI EIKLKKPEAVRWEKLEGQGDVPTPKQFVADVKNLYPSSSPYTRNWDKLVGEI KEEEKNEKLEGDAALNRLFQQIYSDGSDEVKRAMNKSFMESGGTVLSTNWS DVGKRKVEINPPDDMEWKKY (SEQ ID NO: 511), TCKNLSLSTYQNQSASQWTHQSKIKYDWY (SEQ ID NO: 512), EKELSALVKLPSGEDYNLKLELLH (SEQ ID NO: 513), LHPIIPEQSTFKVLSTKIEIKLKKPEAVR (SEQ ID NO: 514), KQFVADVKNLYPSSSPYTRNWDKL (SEQ ID NO: 515), and/or DWYQTESQVVITLMIKNVQKNDV (SEQ ID NO: 505). 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 osteoclastoma.
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, skeletal or developmental disorders, particularly osteoclastoma and other forms of cancer. 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 skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., skeletal, 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.
The tissue distribution in osteoclastoma, combined with the homology to the D1054.3 and Sgt1p proteins indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis, detection, prevention and/or treatment of osteoclastoma and other forms of cancers. Moreover, the expression within embryonic tissue 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, detection, prevention and/or 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 may also play a role as a tumor supressor, or in the development of tumor progression inhibitors. 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:82 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 2039 of SEQ ID NO:82, b is an integer of 15 to 2053, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:82, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 73
The translation product of this gene was shown to have homology to the MEK kinase 3 of Mus musculus, mutations of which and/or aberrant regulation of, may provide a predisposition to cancer.
The gene encoding the disclosed cDNA is thought to reside on chromosome 17. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 17.
This gene is expressed primarily in pituitary, and to a lesser extent in ulcerative colitis and hematopoietic 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, immune, gastrointestinal, hematopoietic diseases. 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 neuronal and immune tissues, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neuronal, 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 hematopoietic tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. Expression of this gene product in ulcerative colitis 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.
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 1179 of SEQ ID NO:83, b is an integer of 15 to 1193, 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: 74
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence:









(SEQ ID NO:516)










SILPVEMAAAVAGMLRGGLLPQAGRLPTLQTVRYGSKAVTRHRRV,



and or


 









(SEQ ID NO:517)










AGMLRGGLLPQAGRLPTLQTVRYGSK.







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 the frontal cortex of the brain.
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, neural disorders, particularly neurodegenerative disorders, ischemia, Alzheimer's, or Parkinson's. 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, 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, or alternatively consist of the immunogenic epitopes shown in SEQ ID NO: 224 as residues: Glu-31 to Gly-37. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in frontal cortex indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo. 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.
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:84 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 527 of SEQ ID NO:84, b is an integer of 15 to 541, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:84, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 75
The translation product of this gene was shown to have homology to the intrinsic factor-B12 receptor precursor of Rattus norvegicus which is thought to be important in development (see, e.g., Genbank Accession No. gi|2961490 (AF022247), all references available through this accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: DCNRDYHKAFGNLRSPGWPDNYDNDXDCXVTLTAPQNHHSGIVENAETISWR (SEQ ID NO: 518), FGNLRSPGWPDNYDN (SEQ ID NO: 519), ASFYRTS (SEQ ID NO: 521), and/or APQNHXLKCRNDFLEV (SEQ ID NO: 520). 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 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, immune or hematopoietic disorders, including inflammatory disorders, and allergies. 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, haematopoetic, 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.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. 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:85 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 971 of SEQ ID NO:85, b is an integer of 15 to 985, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:85, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 76
This gene is expressed primarily in T-cells, and to a lesser extent in cord blood and osteosarcoma.
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, auto-immune diseases, immunodeficiency diseases and host-graft rejection. 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., cells and tissues of the immune system, bone, 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 the immunogenic epitopes shown in SEQ ID NO: 226 as residues: Pro-36 to Ala-41. Polynucleotides encoding said polypeptides are encompassed by the invention.
The expression of this gene in T-cells indicates a potential role in the treament, prevention, diagnosis, and/or detection of immune disorders such as arthritis, asthma, immune deficiency diseases such as AIDS, leukemia and transplant rejection. Expression of this gene product in T 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:86 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 875 of SEQ ID NO:86, b is an integer of 15 to 889, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:86, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 77
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MGESECYRRLSGASCTWTVHVDFA (SEQ ID NO: 522); MHCGTRVWKTMKHDYFLLACLSMTSTGGILCTL SEQ ID NO: 5239SEQ ID NO: 555); STLSLIPTSSSLSFWPWCTAIIGSIFTYCVCVCVCFVVMNRTCYLPNSIIYHNSK LATIIDKSMTLS (SEQ ID NO: 524); and/or MWILPKVSLICIVELGYGKP (SEQ ID NO: 525). 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 polypepti des of the invention and polynucleotides encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in human meningima.
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, meningitis and other inflammatory conditions. 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 cerebrospinal membranes, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., 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 the immunogenic epitopes shown in SEQ ID NO: 227 as residues: Ser-35 to Phe-41. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for study, treatment, prevention, detection and/or diagnosis of disorders of the meningima.
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:87 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 544 of SEQ ID NO: 87, b is an integer of 15 to 558, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:87, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 78
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.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:









(SEQ ID NO:526)










MCVTRMHVKCPPPSASVTAVKWPLSWSSSSFCISLHIAGRH,



and or


 









(SEQ ID NO:527)










EERNKNHLSCQGLSTICCSYLSSKGEHLRNLSPYSF.







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 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, immune conditions. 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.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study and treatment of immune disorders. 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 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. Expression of this gene product in neutrophils 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:88 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 917 of SEQ ID NO:88, b is an integer of 15 to 931, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:88, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 79
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: GLCMVHSLLTSSLGGRCCNYPYIADKDIETEVKPPSQGHTWHLHCS (SEQ ID NO: 528), QLWCITALPSTRHCSKGFAWFTHSLRHPSVAGAVIILILQTRTLRQRSSHLPKG THGICTAPDRPTERAAVTILK (SEQ ID NO:529), SFDNNNSYGVSQLYQVPDTVLRALHGSLTPYVIPRWQVL (SEQ ID NO:530), and/or DRGQATFPRAHMASALLLTDRQRELLSRSSNELCMSKV (SEQ ID NO:531). 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 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 immune diseases and 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.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, detection, diagnosis, prevention and/or treatment of immune disorders. 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 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. Expression of this gene product in neutrophils 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:89 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 574 of SEQ ID NO:89, b is an integer of 15 to 588, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:89, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 80
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:532)









LLLILRPFLNSQFKLQLPLVLFHSSCTYICLLYNYELFHIVALTGKLMNL



GLHLFAHHLILAVAHXGCSIPIY, and or


 








(SEQ ID NO:533)









THNSNYSSLWFSSTAVVLTYVYYIIMNCFILSPLQVN.







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 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 immune disorders and diseases. 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.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, detection, diagnosis, prevention and/or treatment of inflammatory and immune disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. Expression of this gene product in neutrophils 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: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 798 of SEQ ID NO:90, b is an integer of 15 to 812, 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: 81
This gene is expressed primarily in cerebellum.
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, neural disorders, particularly damage to the cerebellum or additional CNS tissues caused by insults, which include, but are not limited to, trauma or ischemia. 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 central nervous system (CNS), expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, 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: 231 as residues: Pro-7 to Cys-21, Leu-25 to Ser-30. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in cerebellum indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions which include, but are not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. 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. 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.
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:91 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 1868 of SEQ ID NO:91, b is an integer of 15 to 1882, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:91, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 82
The translation product of this gene shares sequence homology with the hunan M phase phosphoprotein 10 as well as ORF YJR002w of Saccharomyces cerevisiae (see, e.g., Genbank Accession No.gnl|PID|e266673, all references available through this accession are hereby incorporated in their entirety by reference herein) which are thought to play important roles in the regulation of cellular division.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: AKNSQKEENPEHVEIQKMMDSLFLKLDALSNFHIPKPPVPEIKVVSNLPAIT MEEVAPVSVSDAALLAPEEIKEKNKAGDIKTAAEKTATDKKRERRKKKYQK RMKIKEKEKRRKLLEKSSVDQAGKYSKTVASEKLKQLTKTGKASFIKVRTRE RKLLKGTFVGEVDSKCWVTGMSEPADSPPVG (SEQ ID NO: 534), LQDEGKDKALKSSQAFFSKLQDQVKMQINDAKKTEKKKKKRQDISVHKLKL (SEQ ID NO: 535), DEGKDKALKSSQAFFSKLQDQVKMQINDA (SEQ ID NO: 536), EENPEHVEIQKMMDSLFLKLDALSNFHF (SEQ ID NO: 537), SSVDQAGKYSKTVASEKLKQLTKTGKASFIK (SEQ ID NO: 539), VSVSDAALLAPEEIKEKNKAGDI (SEQ ID NO: 540), VLEVMVTVAPK (SEQ ID NO: 541), LQDEGKDKALKSSQAFFSKLQDQVKMQINDAKKTE (SEQ ID NO: 542), and/or SNLPAITMEEVAP (SEQ ID NO: 538). 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 thyroid.
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, endocrine, proliferative, or developmental disorders, particularly diseases relating to the thyroid gland, particularly hyper- and hypothyroidism. 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 endocrine system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., endocrine, developmental, metabolic, 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 human thyroid indicates that polynucleotides and polypeptides corresponding to this gene would be useful for treating, detecting, diagnosing, and/or preventing metabolic disorders, particularly hyper-, hypothyroidism, Graves' disease, Hashimoto's thyroiditis, and/or cancer or neoplasias of the thyroid, and/or other endocrine organs and immune system. Moreover, the protein may show utility in the diagnosis, prevention, and/or treatment of developmental disorders. In addition, the homology to an M phase phosphoprotein indicates it may be a key player in the proliferation, maintenance, and/or differentiation of various cell types during development. It may also act as a morphogen to control cell and tissue type specification. 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. 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:92 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 1377 of SEQ ID NO:92, b is an integer of 15 to 1391, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:92, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 83
This gene is expressed primarily in uterine cancer.
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, uterine cancer, and other reproductive 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 uterine cancer, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, cancerous and wounded tissues) or bodily fluids (e.g., 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 tumors of the uterus indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis, detection, prevention, treatment and/or intervention of these tumors, in addition to other tumors where expression has been indicated. 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:93 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 916 of SEQ ID NO:93, b is an integer of 15 to 930, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:93, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 84
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: VKPPDQSCNHWRDEQCLV (SEQ ID NO: 543). 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 wilm's tumor.
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, Wilm's tumor, and other urogenital 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 Wilm's tumor, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., urogenital, 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.
The tissue distribution in Wilm's tumor indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of Wilm's tumor. 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. Furthermore, this gene or gene product would be useful in the treatment, prevention, diagnosis 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 congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. 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:94 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 984 of SEQ ID NO:94, b is an integer of 15 to 998, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:94, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 85
When tested against Jurkat T-cell cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activation site) pathway. Thus, it is likely that this gene activates T-cells through the Jaks-STAT signal transduction pathway. GAS (gamma activation site) 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-STATs pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
This gene is expressed primarily in fetal spleen and adipose tissues.
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, immune, metabolic, and developmental 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 fetal spleen and adipose tissues, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, developing, 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 the immunogenic epitopes shown in SEQ ID NO: 235 as residues: Tyr-41 to Phe-47. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in fetal liver/spleen, combined with the detection of GAS promoter activation activity, indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, treatment, and/or prevention of a variety of immune system disorders. Expression of this gene product in fetal spleen 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:95 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 816 of SEQ ID NO:95, b is an integer of 15 to 830, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:95, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 86
This gene is expressed primarily in IL-1/TNF stimulated synovial and human adipose tissues.
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, rheumatoid arthritis or obessity, and disorders of the musculo-skeletal system. 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 and musculo-skeletal systems, 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., synovial and adipose cells and tissues, musculo-skeletal, 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: 236 as residues: Leu-37 to Arg-45, Ser-60 to Ser-65. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in synovial tissue and adipose tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis or treatment of rheumatoid arthritis or other immune diseases. In addition, the expression of this gene product in synovium indicates a role in the detection, diganosis, prevention and/or 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). The tissue distribution in adipose tissue indicates that polynucleotides and polypeptides corresponding to this gene would be 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. 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:96 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 853 of SEQ ID NO:96, b is an integer of 15 to 867, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:96, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 87
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MLYLILISLSSLSFSFSLPPFSIII (SEQ ID NO: 544), SSYFLFRIYHTCPKYFSMNIIN (SEQ ID NO: 545), KLTLTKGNKSWSSTAVAAALELVDPPGCRNSARDSLPNSTM MFYYACFILYSSLSPLSLSLSPSLLSLL (SEQ ID NO: 546), and/or QFHTGNSYDHDYAK (SEQ ID NO: 547). 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 U937 Myeloid cell lines, supernatants removed from cells containing this gene activated the GAS assay. Thus, it is likely that this gene activates myeloid 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.
This gene is expressed primarily in striatum.
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 a number of diseases of the neural system. 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 neural diseases, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., striatum, 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 striatum indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of diseases of the neural system. Furthermore, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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 bahaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. 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:97 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 531 of SEQ ID NO:97, b is an integer of 15 to 545, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:97, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 88
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: IRHEESFNPLTCGFSLFFSLFS (SEQ ID NO: 548), METLLLLLFFLSLLIFRFRILVSQCIN (SEQ ID NO: 549), FLLTTVLLFSSKVRDPRANFDQSLRVLKHAKKVQPDVISKTSIMLGLGENDEQ VYATMKGKEIEK (SEQ ID NO: 550), and/or QQSCCFPVRFVILGPILISPYVY (SEQ ID NO: 551). 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 synovium.
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, arthritis and other diseases of the musculo-skeletal system. 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 musculo-skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., musculo-skeletal, 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 synovium indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis, and/or detection of diseases of the muscular-skeletal system. Furthermore, the expression of this gene product in synovium indicates a role in the detection, diganosis, prevention and/or 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). 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:98 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 708 of SEQ ID NO:98, b is an integer of 15 to 722, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:98, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 89
This gene is expressed primarily in medulloblastoma.
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, disorders of the central nervous system, including cancers. 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, 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, or alternatively consist of the immunogenic epitopes shown in SEQ ID NO: 239 as residues: Phe-22 to Leu-28. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, treatment, and/or prevention of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo. 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:99 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 739 of SEQ ID NO:99, b is an integer of 15 to 753, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:99, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 90
When tested against a Jurkat T-cell line, supernatants removed from cells containing this gene activated the GAS (gamma activation site) pathway. Thus, it is likely that this gene activates T-cells through the Jaks-STAT signal transduction pathway. The GAS is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a complex, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
This gene is expressed primarily in human kidney pyramid, and to a lesser extent in human brain.
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, nephrotic, nephritic syndromes, renal failure, hypertensive nephrosclerosis. 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 renal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., renal, 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 kidney indicates that polynucleotides and polypeptides corresponding to this gene would be useful for treatment, prevention, detection and/or diagnosis of renal 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. Additionally, the gene product may have endocrine functions related to renal function, metabolism and homeostasis. 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: 100 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 682 of SEQ ID NO: 100, b is an integer of 15 to 696, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 100, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 91
This gene is expressed primarily in both normal or cancerous human breast tissue.
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, Non-neoplastic breast diseases or breast cancers. 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 breast, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., mammary tissue, 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 the immunogenic epitopes shown in SEQ ID NO: 241 as residues: Pro-20 to Ser-28. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in breast indicates that polynucleotides and polypeptides corresponding to this gene would be useful for either non-neoplastic breast diseases, such as congentital anomalities, gynecomastia, mastitis and abscess, duct ectasia and fat necrosis, or neoplasia in the breast. 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: 101 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 441 of SEQ ID NO: 101, b is an integer of 15 to 455, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 101, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 92
This gene is expressed primarily in 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, arthritis, asthma, auto-immune and immunodeficiency diseases. 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., T-cells and other 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.
The expression of this gene in T-cells indicates a potential role in the treament, prevention, diagnosis, and/or detection of immune disorders such as arthritis, asthma, hypersensitivity reactions and transplant rejection, and also in 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 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: 102 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 375 of SEQ ID NO:102, b is an integer of 15 to 389, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:102, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 93
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MFYSKIFYFLLLNSDTSNNVTSKTLVSSISSSNNRLAVSIVF (SEQ ID NO: 552), SRQKNLLKLHSNPNCDNFCFIFNYKPKYICIFKLICLKILLYIFGSG (SEQ ID NO: 553), and/or MLLSLLMVFTSELYVKRHISFKSXDKPHCHKNQDIDVLFRKLLEKHFKVINMICFP (SEQ ID NO: 554). 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 fetal liver, and to a lesser extent in bone and breast cancer cell lines.
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, cancer and metabolic 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 digestive and skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., digestive, skeletal, 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 fetal liver/spleen, as well as bone and breast cancer, indicates that polynucleotides and polypeptides corresponding to this gene would be useful for study, detection, diagnosis, prevention and/or treatment of growth and metabolic disorders and neoplasias (e.g., hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells). Furthermore, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and/or treatment of bone and breast cancer, as well as cancers of other tissues where expression has been observed.
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:103 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 946 of SEQ ID NO:103, b is an integer of 15 to 960, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:103, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 94
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: FREYGFYNLHFC (SEQ ID NO: 555), LVTTDYYDGCNEDYEYNWSYMFLNSEQLFIKFYPTFFC (SEQ ID NO: 556), and/or NVIAPGLESSCANSLFLLFVCLPVAHIRHNFLFIKHSLYNHLRDYESDFDKI (SEQ ID NO: 557). 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 T cells, fetal heart and infant brain, and to a lesser extent in some transformed cell lines.
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 growth and immune disorders and diseases. 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 and cardiovascular systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cardiovascular, 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, heart tissue, and brain tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, detection, diagnosis, prevention and/or treatment of developmental and immune disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. Expression of this gene product in T cells also strongly indicates a role for this protein in immune function and immune surveillance. The tissue distribution in heart muscle tissue indicates that polynucleotides and/or polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of conditions and pathologies of the cardiovascular system, such as heart disease, restenosis, atherosclerosis, stoke, angina, thrombosis, and wound healing. The tissue distribution in brain tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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 bahaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. 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:104 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 1428 of SEQ ID NO:104, b is an integer of 15 to 1442, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:104, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 95
This gene is expressed primarily in 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, immune and haematological 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, 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.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. 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 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 polynucleotides and/or polypeptides of the invention 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. 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:105 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 584 of SEQ ID NO:105, b is an integer of 15 to 598, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:105, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 96
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 aortic endothelium, and to a lesser extent in melanocyte.
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, cardiovascular diseases. 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 cardiovascular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., vascular, 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: 246 as residues: Met-1 to Trp-12, Arg-33 to Ser-53. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in human aortic endothelial cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection or intervention of cardiovascular diseases, such as hypertension, cadiovascular injuries, congenital heart diseases, ischemic heart diseases, rheumatic and other hypersensitivity diseases, cardiomyopathy, restenosis, atherosclerosis, stoke, angina, thrombosis, and wound healing. 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:106 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 671 of SEQ ID NO:106, b is an integer of 15 to 685, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:106, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 97
The translation product of this gene shares weak sequence homology with Interferon induced 1–8 gene encoded polypeptide, which is thought to be important in retroviral REV responsive element binding and thus viral replication.
This gene is expressed primarily in B-cell lymphoma.
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, immune response to viral infections and other immunologically related 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., T-cells and other 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 polypepti des of the present invention comprise, or alternatively consist of the immunogenic epitopes shown in SEQ ID NO: 247 as residues: Pro-47 to Asn-53. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in B-cell lymphoma and homology to interferon induced 1–8 gene indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the intervention, prevention, treatment, detection, and/or diagnosis of viral infection and other immunologically related disorders. The homology with interferon induced 1–8 REV response element binding gene indicates the gene product may bind to viral components to interfere with the entry, packaging, replication, or induce the host cell anti-viral response by intereferon mediated pathways.
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:107 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 491 of SEQ ID NO:107, b is an integer of 15 to 505, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:107, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 98
The gene encoding the disclosed cDNA is thought to reside on chromosome 12. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 12.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: LNIDSFDYGKFESLLAKQHYKFSFLLPLAAGTERCKWWLKIEEASSDQCGCWFLVKCVPKPPSPCRQPPTQVSKIGHAPFFL (SEQ ID NO: 558). 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 brain, and to a lesser extent in placenta and spleen.
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, behavioural disorders and neurodegenerative disease states. 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 brain and central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., brain and other tissue of the nervous system, spleen and other cells and tissue of the immune system, placenta, 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.
The tissue distribution in brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder and panic disorder. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. 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:108 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 1135 of SEQ ID NO:108, b is an integer of 15 to 1149, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:108, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 99
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: QEFQTGLGNMVKPCLYEKYRNISWLWWHTFPVVPATWEAEVGGSLEPGRLRLQ (SEQ ID NO: 559), and/or ILGGESILILSWVFSYIFFRIALEITIYILNVSPFCLGRWLMPVIPALWEAEVGGL PELRSSRPA (SEQ ID NO: 560). 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 adult lymph node tissue.
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, immune disorders and lymphomas. 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 and metabolic systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, metabolic, 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 lymph nodes indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, diagnosis, detection, prevention and/or treatment of immune and lymph diseases and disorders such as lymphomas. 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:109 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 671 of SEQ ID NO:109, b is an integer of 15 to 685, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:109, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 100
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: VLCEEAGQKVPSTPSWSSWTLQKRLRGSPAEANCSPSFPAPPGKE (SEQ ID NO: 561), MSLSALACDFTPIQPWEWEEYEQITLGLTAPSNLLESNYLGQASECFVRKLVR RFPQLLPGPPGHCRKDLGDPQQRPIALLPSLPHQERNNVHRLEADSEVDL (SEQ ID NO: 562), CVDFDEYFSSWEPLLKMMFKGVVGGKMKAWRRKKRRKPLPYKIHAD (SEQ ID NO: 563), and/or MMFKGVVGGKMKAWRRKKRRKPLPYKIHAD (SEQ ID NO: 564). 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 bone marrow, and to a lesser extent in testes.
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, hematopoietic and reproductive 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 hematopoietic and 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, 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 bone marrow and testes indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, diagnosis, detection, prevention and/or treatment of various disorders involving the hematopoietic and reproductive systems. 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. Furthermore, polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis, and/or detection of conditions concerning proper testicular function (e.g. endocrine function, sperm maturation), as well as cancer. Therefore, this gene product would be 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) would be useful as male contraceptive agents. Similarly, the protein is believed to be 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, polynucleotides and/or polypeptides corresponding to this gene 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 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:110 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 1132 of SEQ ID NO:110, b is an integer of 15 to 1146, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:110, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 101
The translation product of this gene shares sequence homology with adipose specific collagen-like factor as well as the human adipocyte complement related protein Acrp30, the latter of which is known to be important in energy balance and homeostasis involving food intake, particularly in carbohydrate and lipid catabolism/anabolism (see, e.g., Genbank Accession Nos.gnl|PID|d1008822 and W09108, respectively, all references available through these accession are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: XLWDPGLPGVCRCGSIVLKSAFSVGITTSYPEXRLPIHFNKVLLPRGXALQPCH RGSSSVLSQGIYYFSYDITLANKHLAIGLVHNGQYRIKTFDANTGNHDVASGS TVIYLQPEDE VWLEIFFTDQNGLFSDPGWADSLFSGFLLYVDTDYLDSISEDDEL (SEQ ID NO: 565), GSIVLKSAFSVGITT (SEQ ID NO: 566), GIYYFSYDITLANK (SEQ ID NO: 567), DSLFSGFLLYVDT (SEQ ID NO: 568), and/or NHDVASGSTVIYL (SEQ ID NO: 569). 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 schwanoma.
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, neural or integumentary disorders, particularly neurofibroma. 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 diseases relating to peripheral or sympathetic nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, integumentary, extracellular matrix, 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, four, five, or all six of the immunogenic epitopes shown in SEQ ID NO: 251 as residues: Gly-16 to Pro-30, Pro-42 to Gly-56, Gly-62 to Gly-77, Glu-93 to Gly-104, Glu-109 to Glu-114, Pro-121 to Asp-126. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in schwanoma cells combined with the homology to a conserved human adipose specific collagen-like factor as well as to the human adipocyte complement related protein Acrp30, indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders particularly neuroschwannoma, and including 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Moreover, polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, detection, diagnosis, and/or prevention of various 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. In addition, 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, althlete's foot, and ringworm). Moreover, polynucleotides and/or polypeptides corresponding to this gene may also be useful for the treatment or diagnosis of various connective tissue disorders such as arthritis, trauma, tendonitis, chrondomalacia and inflammation, 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). Alternatively, considering the homology to a conserved adipose specific collagen-like factor, would indicate that polynucleotides and/or polypeptides of the invention may also be important 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 ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid. 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:111 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 1319 of SEQ ID NO:111, b is an integer of 15 to 1333, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:111, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 102
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: 570), TLRVLGKVPAVCPWCALWRKAGMDMTYSWLSRGDSTYTFHEG PVLSTSWRPGDSALSYTCRANNPISNVSSCPIPDGPFYADPNYASEKPSTAFCL LAKGLLIFLLLVILAM GLWVIRVQKRHKMPRMKKLMRNRMKLRKEAKPGSSPA (SEQ ID NO: 571), AVCPWCALWRKAGMDMTYSWL (SEQ ID NO: 572), PGDSALSYTCRANNPISNVSSCPI (SEQ ID NO: 573), YASEKPSTAFCLLAKGLLIFLLLV (SEQ ID NO: 574), and/or QKRHKMPRMKKLMRNRMKLRKEAKPG (SEQ ID NO: 575). 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: 252 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:112 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:112, 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:112, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 103
The gene encoding the disclosed cDNA is believed to reside on chromosome 17. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 17.
This gene is expressed primarily in breast, fetal kidney, and 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, reproductive, immune, developmental, or renal disorders, particularly autoimmune diseases, chronic inflammatory conditions, or urogenital 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, reproductive, cancerous and wounded tissues) or bodily fluids (e.g., breast milk, 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: 253 as residues: His-2 to Lys-7, Ser-28 to Glu-35. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in breast and T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. Moreover, the expression of this gene product indicates a role in regulating 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 polynucleotides and/or polypeptides of the invention 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. Alternatively, the expression within fetal tissue indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, detection, prevention and/or 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:113 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 1561 of SEQ ID NO:113, b is an integer of 15 to 1575, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:113, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 104
This gene is expressed primarily in early stage brain.
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, developmental and neurodegenerative diseases of the brain and nervous system. 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 brain, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., brain and other tissue of the nervous 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.
The tissue distribution in brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis, and/or detection of developmental and neurodegenerative diseases, as well as behavioral or nervous system disorders. Examples of such conditions would include: depression, schizophrenia, mania, dementia, paranoia, addictive behavior and sleep disorders. In addition a brain-specific gene product may be useful in the diagnosis of specific brain tumors. 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:114 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 320 of SEQ ID NO:114, b is an integer of 15 to 334, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:114, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 105
This gene is expressed primarily in prostate cancer.
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, prostate cancer. 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 prostate, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., prostate, 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 prostate cancerous tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diganosis, prevention and/or treatment of prostate cancer and other prostate disorders, as well as cancers in other tissues where expression has been indicated. 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:115 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 852 of SEQ ID NO:115, b is an integer of 15 to 866, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:115, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 106
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: LSYSVLLILPLFHSLPTLKDTHTHNKWVE (SEQ ID NO: 576), EVNGVGYKHSCFSDISSVLENKDSRMRAPHYASFQHFFSVLLKLSPQACLTES QCIPLTFY (SEQ ID NO: 577), KTHTHTISGWSKKSTELDISIPAFLTSPVSWRTRILE (SEQ ID NO: 578), and/or IRHELGSSDPPAEASQIAGTAAVSHHAQP (SEQ ID NO: 579). 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 spinal cord.
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, spinal cord injuries and diseases of the neural system. 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 neural system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, 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 the immunogenic epitopes shown in SEQ ID NO: 256 as residues: Pro-45 to Gln-52. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in spinal cord indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of spinal cord injuries and diseases of the neural system, such as spinal deformation, spinocerebullar ataxia types I and III, dentatorubropallidoluysian and spinal bulbar muscular atrophy. 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 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 448 of SEQ ID NO:116, b is an integer of 15 to 462, 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: 107
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: LKGREAGAGPGTAGAPGREDANGXXRGRGGXHQLYLWVDNIPLSRPKRNLS RDFSDGVLVAEVIKFYFPKMVEMHNYVGTSSLQQKLSNWGHILNRKVLKRLN FSVPDDV (SEQ ID NO: 580), WVDNIPLSRPKRNLSRDFSDGVLVA (SEQ ID NO: 581), YVGTSSLQQKLSNWGHLNRKVLKRL (SEQ ID NO: 582), GSAWRRGRGAG SRAPAPYRSWLPRMAVATWMWVYPRRPEVKVSRTPREGVSSAGTGRRRLG LQRITGRCRATPASSSRSLKRSRSCWPLKRPCRSCR (SEQ ID NO: 583), WLPRMAVATWMWVYPRRPEVK (SEQ ID NO: 584), CRATPASSSRSLK RSRSCWPLKR (SEQ ID NO: 585), EHNTFNGAALSRNLQTFRLSTPCARREGRLLRAHRRCPPYSWRSHASPLPL QLLRSPSPRWVPGKLPGGAGEPLSGPGQIPPWLRAWGTSLJDGDAAVLGAGR GPDSGGVDRAKGPPPKAQRREMQGRAQGVGHCFGGQARSLHVASGLWKAV HSPDPDLRSGRRRLSPGPALLEFLSHLLHAHPSQGRRALGPQQARES SGLRPP NGLSIGGWVRRGVGALAGTRASPRGPGRRSPLLTXRXLEPPGEVFDPHIELE QVLQAPYLHLQDLHGLLRGQQLLLLFSDLEDEAGVALQRPVIRWRPRRRRPV PAELTPSLGVRDTFTSGLLGYTHIHVATAHLGSQLL (SEQ ID NO: 586), TDFNGAALSRNLQTFRLSTPCARREG (SEQ ID NO: 587), RCPPYSWRSHASPLPLQLLRSPSPR (SEQ ID NO: 588), GAGEPLSGPGQIPPWLRAWGTSLD (SEQ ID NO: 589), LGAGRGPDSGGVDRAKGPPPKAQRREMQGR (SEQ ID NO: 590), QARSLHVASGLWKAVHSPDPDLR (SEQ ID NO: 591), HPSQGRRALGPQQARESSGL (SEQ ID NO: 592), IGGWVRRGVGALAGTRASPRGPGRRSP (SEQ ID NO: 593), EPPGEVFDPHILELEQVLQAPYLHL (SEQ ID NO: 594), and/or VPAELTPSLGVRDTFTSGLLGYTHIHVA (SEQ ID NO: 595). 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 adult testis.
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, reproductive and/or endocrine 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 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, endocrine, cancerous and wounded tissues) or bodily fluids (e.g., lymph, 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. Preferred polypeptides of the present invention comprise, or alternatively consist of one or both of the immunogenic epitopes shown in SEQ ID NO: 257 as residues: Gln-21 to Gly-33, Gln-55 to Glu-60. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in testicular tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of reproductive system disorders, and may be indicative of a role for this gene product in normal testicular function, male fertility, and/or as a male contraceptive. 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:117 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 1486 of SEQ ID NO:117, b is an integer of 15 to 1500, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:117, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 108
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: HTLFISFLWAEG (SEQ ID NO: 596), MLPVFVLFFCFIYSARKQSVFKKGNVFE (SEQ ID NO: 597), and/or SPCSAAECHNLSLLSSCSLVSSNILFSFPFFGQKARCCLFLFYFSAHIAHESRV YSKKEMCL (SEQ ID NO: 598). 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 prostate cancer.
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, prostate and other cancers. 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 endocrine system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., endocrine, 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 prostate cancer indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, detection, diagnosis, prevention and/or treatment of prostate cancer, as well as cancers of other tissues where expression has been observed. 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:118 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 346 of SEQ ID NO:118, b is an integer of 15 to 360, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:118, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 109
The gene encoding the disclosed cDNA is thought to reside on chromosome 7. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 7.
This gene is expressed primarily in brain, and to a lesser extent in breast.
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, neurodegenerative conditions and behavioural 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., brain and other tissue of the nervous system, mammary tissue, 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 the immunogenic epitopes shown in SEQ ID NO: 259 as residues: Leu-40 to His-46. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in brain indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder and panic disorder. In addition, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. 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:119 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 809 of SEQ ID NO:119, b is an integer of 15 to 823, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:119, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 110
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: AFFLLQALEIQSQLATPASSTARNPAPDLHHPHQPTIERFCRHSSSWERIEY (SEQ ID NO: 599). 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 Jurkat T-cell lines, 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. Furthermore, contact of cells with supernatant expressing the product of this gene increases the permeability of Brian microvascular pericyte cells to calcium. Thus, it is likely that the product of this gene is involved in a signal transduction pathway that is initiated when the product of this gene binds a receptor on the surface of the pericyte cell. Thus, polynucleotides and polypeptides have uses which include, but are not limited to, activating pericyte (endothelial) cells.
This gene is expressed primarily in activated 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, immune disorders, inflammation. 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, 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 neutrophils and the biological activity data indicate that polynucleotides and polypeptides corresponding to this gene would be useful for the study, detection, diagnosis, prevention and/or treatment of inflammatory and immune conditions. 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 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 polynucleotides and/or polypeptides of the invention 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. Expression of this gene product in neutrophils 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:120 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 442 of SEQ ID NO:120, b is an integer of 15 to 456, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:120, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 111
This gene is expressed primarily in glioblastoma.
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, glioblastoma. 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 central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., tissue of the nervous 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.
The tissue distribution in glioblastoma indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diganosis, prevention and/or treatment of malignancies, as well as developmental and neurodegenerative diseases of the brain and nervous system. 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:121 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 539 of SEQ ID NO:121, b is an integer of 15 to 553, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:121, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 112
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MRTLFGAVRAPFSSLTLLLITPSPSPL (SEQ ID NO: 600), MAYAFHRTST (SEQ ID NO: 601), LKSTYTLLSILWFLVLIPVEGN (SEQ ID NO: 602), and/or GPLLASHATLCFSLGSKF (SEQ ID NO: 603). 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 ovarian cancer.
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, reproductive, or endocrine disorders, particularly proliferative condtions such as ovarian cancer. 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 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, endocrine, 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.
The tissue distribution in ovarian tumor tissue indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis, detection, prevention and/or treatment of ovarian cancer. Moreover, the protein would be useful for the detection, treatment, and/or prevention of a variety of reproductive disorders such as infertility. In addition, the protein may also be useful in the development of novel or improved contraceptives. 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, detection, prevention and/or 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:122 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 1144 of SEQ ID NO:122, b is an integer of 15 to 1158, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:122, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 113
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: ATVPGSIYNYFYHYNAGALKPEHASESPRGLCAQTAGPFPSF (SEQ ID NO: 604), IRPPPPRFKRFSCLSLLSSWDYRRAPPHIVAIFCTLSRDGVLPHWPGWSQTPDLK (SEQ ID NO: 605), STHLGLPRCWDYRHEPLCLAPFTTISIIIMQGLSNLSMPQNPPEGCAIHRLLDLS PASDSVPPEWGSKIAEEV (SEQ ID NO: 606), and/or LRVGGTSENCCRGECCGSVCIPPGRL (SEQ ID NO: 607). Moreover, fragments and variants of these polypepti des (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 Jurkat T-cell lines, 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.
This gene is expressed primarily in 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, immune 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.
The tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, detection, diagnosis, prevention and/or treatment of immune disorders. Expression of this gene product in neutrophils 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 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 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.
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 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 540 of SEQ ID NO:123, b is an integer of 15 to 554, 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: 114
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: SNSHTHTHVKSFLR (SEQ ID NO: 608). 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, 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.
The tissue distribution in T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, 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 polynucleotides and/or polypeptides of the invention 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:124 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 1241 of SEQ ID NO:124, b is an integer of 15 to 1255, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:124, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 115
The translation product of this gene shares sequence homology with rat synaptogyrin which is thought to be important in membrane trafficking (see, e.g., Stenius et al., J. Cell Biol. 131 (6 Pt 2), 1801–1809 (1995), incorporated in its entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: QPYQVLPSRQVFALI (SEQ ID NO: 609), VFSCIYGEGYSNAIHESKQMYCVFN (SEQ ID NO: 610), RNEDACRYGSAIGVLAFL (SEQ ID NO: 611), LVVDAYFPQISNATDRK (SEQ ID NO: 612), and/or SALWTFLWFVGFCFLTNQWAVTNPK (SEQ ID NO: 613). 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 breast and ovary, and to a lesser extent in most hematopoietic tissue types.
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, female infertility and female reproductive abnormalities. 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 reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., mammary tissue, and ovary and other reproductive tissue, 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: 265 as residues: Pro-9 to Trp-18, Thr-20 to Ala-27. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in ovary and breast and homology to a protein involved in membrane trafficking indicates that this protein may play a role in the detection, diagnosis, prevention, and/or treatment of female fertility disorders, endocrine disorders, ovarian failure, amenorrhea, ovarian cancer, and also potentially in both non-neoplastic breast diseases such as congenital abnormalities and neoplasia in the breast. 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:125 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 1963 of SEQ ID NO:125, b is an integer of 15 to 1977, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:125, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 116
The translation product of this gene shares sequence homology with adrenalin receptor (Patent serial No. J08 126491-A, all references available through this Patent are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:614)









TATLNSFFGGWGLALLLRLECSDTIMDHCSLDLLGSSNPPASASQVVGTT



GARHHAQLIFCFFVQTRSHSVA,


 








(SEQ ID NO:615)









MDHCSLDLLGSSNPPASASQVVGTTGARHHAQLIFCFFVQTRSHSVA,



 








(SEQ ID NO:616)









GVLKQSSHLVLSKG,



 








(SEQ ID NO:617)









DYSCESLCPALLSIAPDIVLN,



 








(SEQ ID NO:618)









TTIHKTQLGSYKILWEPKEGYHNSTWI,



 








(SEQ ID NO:619)









IREIFLRRP, and or



 








(SEQ ID NO:620)









LKFQKPGKIQMRGGGRVFWYKNCK.







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 polypepti des are also encompassed by the invention.

This gene is expressed primarily in synovial sarcoma.
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, arthritis and other diseases of the synovium including cancers. 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 and muscular-skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, musculo-skeletal, 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 synovial sarcoma tissue and the homology to adrenalin receptor indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of diseases of the synovium, immune system and musculo-skeletal system including cancers of these tissues and systems. It may also be useful for identifying and therapeutically using antagonists and agonists for this receptor family. In addition, the expression of this gene product in synovium indicates a role in the detection, diganosis, prevention and/or 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). 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:126 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 724 of SEQ ID NO:126, b is an integer of 15 to 738, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:126, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 117
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: RhEPDPM (SEQ ID NO: 621). 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 testes.
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, male reproductive or endocrine 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 reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., endocrine, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, 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. Preferred polypeptides of the present invention comprise, or alternatively consist of one, two, three, four, or all five of the immunogenic epitopes shown in SEQ ID NO: 267 as residues: Pro-20 to Trp-25, Arg-33 to Thr-38, Asn-51 to Ile-56, Gly-82 to Ser-91, Lys-151 to Arg-156. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in human testicular tissues and cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of various endocrine disorders and cancers, particularly Addison's disease, Cushing's Syndrome, and disorders and/or cancers of the pancrease (e.g., diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-, hypoparathyroidism), hypothallamus, and testes. Alternatively, expression within the human testis may be indicative for a role in normal testicular function, and may implicate this gene product in male fertility, and could even indicate its use as a novel contraceptive. 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 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 974 of SEQ ID NO:127, b is an integer of 15 to 988, 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: 118
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: AVCTGGYCESCRCEHCVCVCVDLCVLFSGKELRVR (SEQ ID NO: 622), VSFFFVFKWSFAEIKSREEEHWASLTPKPTLLSALLTCDVLKSSIIFKCCESTEDK GFDSFFQASKDGSSSRI (SEQ ID NO: 623), RSWGSQRSLCLLFIPFAAESYSVVWMGHLFVVCLLSSWWTFRPFALAVTVNH VAVNIVCVSAWTCVSCSLGRSCGLEGSFLFPLETLWFPHMVVLCLTF (SEQ ID NO: 624), MGHLFVVCLLSSWWTFRPFALAVTVNHVAVNIVCVSAWTCVSCSLGRSCGL EGSFLFPLETLWFPHMVVLCLTF (SEQ ID NO: 625), HDVLGARNAACVCCSFLLQQNRILLFGWATCLLSVYSPAGGHLGRLHWRLL (SEQ ID NO: 626), MLDFKTSQVSKALKRVGFGVRLAQCSSLDLISAKLHLKTKKKETYITSTVMT AASLFLSYVTSEFIRSIMATFYCFVLKLHIGEMGTLQTAGGSKMTWPLQKAI WQFLKRLSIKLPYVETRESPGETKNY (SEQ ID NO: 627), and/or LTRNSFPENRTHKSTQTHTQCSQRHDSQ (SEQ ID NO: 628). 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 intestine and cancer 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, diseases of the gastrointestinal tract and cancer. 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 digestive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., digestive, 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 gastrointestinal tissues and cancerous tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the treatment, prevention, diagnosis, and/or detection of diseases of the digestive system and cancer. Furthermore, the tissue distribution in gastrointestinal tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, prevention, and/or treatment of various metabolic disorders such as Tay-Sachs disease, phenylkenonuria, galactosemia, porphyrias, and Hurler's syndrome. 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:128 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 898 of SEQ ID NO:128, b is an integer of 15 to 912, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:128, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 119
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: LFYLLTCSCAPGHLAFVCSQCLPFDMGKELWPKSPSSCTSTSVAQGWGGRGR PSPYICVV (SEQ ID NO: 629), IQGSRLPPLPAPLHPLPLIYLLLGSPAQSWLLVPSWGHPSTLTLTMAAEHQAW PSGFHGDH (SEQ ID NO: 630). 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 placenta and 9 week old embryo, and to a lesser extent in fetal spleen.
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, developmental 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 developmental system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developing and differentiating tissues, and spleen and other 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.
The expression of this gene primarily in the embryo indicates a key role in embryonic development, and could be used in the treatment and or detection of developmental disorders. The tissue distribution in placenta also indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, detection, prevention and/or treatment of disorders of the placenta. 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 may be 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 within embryonic 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. 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:129 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 555 of SEQ ID NO:129, b is an integer of 15 to 569, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:129, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 120
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MPKQLAQLLYRLPRG (SEQ ID NO: 631), LFQAISVSGSHRQGSRTWNTLTEGNAEAACTVALQTSKRLILASRW (SEQ ID NO: 632), TLSFMNSHCVPIKALFFLSVVSYIFIMPHIFFTVKILKSCFQVGQLMKL (SEQ ID NO: 633), and/or RPTRPITFSSNISEWVPSTGFQDLEHFNRRKCRSSLHSCFTDFQEADSGFKMEP WSWFFFFFFFFPQRTCGCALCVLFLFSIWGPHGKELLNSFLYELPLCSYKGPFLS (SEQ ID NO: 634). 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 placenta and synovium.
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 of the synovium and placenta. 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 placenta and synovium, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., placenta, synovium, 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 placenta and synovium indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the study, diagnosis, detection, prevention and/or treatment of growth and developmental disorders and arthritic and inflammatory conditions. Expression within embryonic tissue and other 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, detection, prevention and/or treatment of cancer and other proliferative disorders. 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. 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 may be 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. 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:130 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 632 of SEQ ID NO:130, b is an integer of 15 to 646, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:130, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 121
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: TKTSTPLR (SEQ ID NO: 635). 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.
The gene encoding the disclosed cDNA is believed to reside on chromosome 12. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 12.
This gene is expressed primarily in infant brain and fetal tissues.
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, developmental abnormalities or neural disorders, particularly gestational conditions, such as spina bifida. 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 Central nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developing, neural, differentiating, and cancerous and wounded tissues) or bodily fluids (e.g., amniotic fluid, serum, lymph, 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 infant brain and fetal tissues indicates that polynucleotides and/or polypeptides corresponding to this gene would be useful for the detection, diagnosis, prevention, and/or treatment of neurodegenerative disease states and behavioural disorders such as 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, the gene or gene product may also play a role in the treatment, prevention, diagnosis and/or detection of developmental disorders associated with the developing embryo, sexually-linked disorders, or disorders of the cardiovascular system. Alternatively, the tissue distribution indicates that polynucleotides and/or polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of cancer and other proliferative disorders. Moreover, the expression within fetal 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. 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:131 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 1169 of SEQ ID NO:131, b is an integer of 15 to 1183, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:131, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 122
The translation product of this gene shares sequence homology with a murine bone-related sulphatase (see, e.g., Genbank Accession No. 3046314, and Genseq Accession No. R51355, all references available through these accessions are hereby incorporated in their entirety by reference herein) which is thought to be involved in proteoglycan metabolism.
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: ASFGSCSLSLPCSARERTPEGGGWPGGRLSEPLPA (SEQ ID NO: 636), APNVVLV (SEQ ID NO: 637), DGRLTF (SEQ ID NO: 638), PGSQVVKLPFINFM (SEQ ID NO: 639), FLNAYTNSP (SEQ ID NO: 640), ICCPSRAAMWSGLFTHLTESWNNFKGLDPNYTTWMD (SEQ ID NO: 641), TQKFGK (SEQ ID NO: 642), DYTSGHHSI (SEQ ID NO: 643), SNRVEAWTRDVAFLLRQEGRP (SEQ ID NO: 644), DWQNTDKA (SEQ ID NO: 645), YLGLNLPIPYPSPSSGENFGSSTFHTSLYWLEKV (SEQ ID NO: 646), DAIKIPKW (SEQ ID NO: 647), YTKNCTG (SEQ ID NO: 648), NIRAFYYAMCAETDAMLGEIILALH (SEQ ID NO: 649), LDLLQKTIVIY (SEQ ID NO: 650), MEHRQFYKMSMYEAS (SEQ ID NO: 651), HVPLLMMGPGIKA (SEQ ID NO: 652), VVSLVDIYPTMLDIAGI (SEQ ID NO: 653), DPDELTN (SEQ ID NO: 654), WKYIAY (SEQ ID NO: 655), NFPEITYSLDQKLHSIINYPKVSASVHQYNKEQFIKWKQSIGQNYSNVIANFR WHQDWQKEPRKYENAID QWLKTHMNPRAV (SEQ ID NO: 656), FPEITYSLDQKL (SEQ ID NO: 657), NYPKVSASVHQYNKEQFI (SEQ ID NO: 658), GQNYSNVIA (SEQ ID NO: 659), RWHQDWQ (SEQ ID NO: 660), and/or PRKYENAI (SEQ ID NO: 661). 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 retina.
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, visual, skeletal, or metabolic disorders, particularly eye dieases and bone metabolic 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 eye, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., visual, skeletal, metabolic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, vitreous humor, aqueous humor, 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, four, five, six, or all seven of the immunogenic epitopes shown in SEQ ID NO: 272 as residues: Ala-21 to Arg-27, Asp-40 to Arg-45, Glu-97 to Thr-110, Glu-117 to Lys-128, Arg-175 to Lys-182, Pro-207 to Gly-220, Val-253 to Ile-272. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in retina, combined with the homology to sulphatases indicates that polynucleotides and polypeptides corresponding to this gene would be useful for diagnosis, detection, prevention and/or treatment of eye disorders. Moreover, this gene may be useful in the detection, treatment, and/or prevention of bone-related disorders, osteoporosis, Paget's disease, osteomalacia, in addition to bone metabolic disorders, particularly those involving proteoglycans. Polynucleotides and/or polypeptides corresponding to this gene would also be useful in the disorders involving aberrant proteoglycan metabolism or related conditions, which may include arthritis, immune cell migration, cellular proliferation, vascular disorders, hematopoietic disorders, in addition to showing utility in the detection, treatment, and/or prevention of the disorders mentioned above. 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:132 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 2105 of SEQ ID NO:132, b is an integer of 15 to 2119, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:132, and where b is greater than or equal to a+14.
Features of Protein Encoded by Gene No: 123
The translation product of this gene shares sequence homology with a novel human G52-24 secreted protein as well as the early lymphocyte activation antigen CD69, the latter of which has been shown to be important in lymphocyte proliferation and functions as a signal trasmitting receptor in lymphocytes, natural killer cells, and platelets (see, e.g., Genseq and Genbank Accession Nos. W27288 and gi|558352, respectively, all references available through these accessions are hereby incorporated in their entirety by reference herein).
In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group:








(SEQ ID NO:662)









ENFLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQLVMKEDGANLYVAK



VSQVPRMNPXLSWVLLCYPGWSAVXTIVAHCSLDFPGSK,


 








(SEQ ID NO:663)









ELTAIKSHQYVLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDAD



LAQVESFQELVRK,


 








(SEQ ID NO:664)









WIGLSREQGQPWKWING,



 








(SEQ ID NO:665)









CPESWIGFQRKC,



 








(SEQ ID NO:666)









NFLLRYKGPSDHWIGL



 








(SEQ ID NO:667)









ASHLLRLLSSWDYRFPILGAGECAYLNDKGASSARHYTERKWICSKSDIH



V,


 








(SEQ ID NO:669)









EQLEELELKKKDFIKILESVQGNWRQNEDSGKGPQRSCL,



 








(SEQ ID NO:670)









FWPESKIQPYKDMFSCEII,



 








(SEQ ID NO:668)









SWTSSLLNXCLHSKEHSIKATIWRLFFXILTIILCGMVAALSAIRANCHQ



EPSVCSSSCMPRKLDWFSKKVFLFF,


 








(SEQ ID NO:671)









EQLEELELKKKDFIKILESVQGNWRQNEDSGKGPQRSCLHSKEHSIKATL



IWR, and or


 








(SEQ ID NO:672)









ENFLLRYKGPSDHWIGLXXEQGQPWKWINGTEWTRQ.







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 testes.
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, reproductive, endocrine, and/or 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 reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, reproductive, endcrine, cancerous and wounded tissues) or bodily fluids (e.g., lymph, 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. Preferred polypeptides of the present invention comprise, or alternatively consist of one or both of the immunogenic epitopes shown in SEQ ID NO: 273 as residues: Asn-20 to Pro-25, Ser-48 to Asp-65. Polynucleotides encoding said polypeptides are encompassed by the invention.
The tissue distribution in human testes indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of a variety of reproductive disorders, particularly autoimmune disorders, infertility, or the protein may even be useful as a novel contraceptive. Homology of this gene product to the early lymphocyte activation antigen CD69 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 polynucleotides and/or polypeptides of the invention may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, and leukemia. 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:133 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 680 of SEQ ID NO:133, b is an integer of 15 to 694, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:133, and where b is greater than or equal to a+14.





















TABLE 1A














5′ NT
















of

First
Last




ATCC

NT

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





Deposit

SEQ
Total
of
of
of
AA of
SEQ
of
of
AA of
Last


Gene

Nr and

ID
NT
Clone
Clone
Start
Signal
ID
Sig
Sig
Secreted
AA of


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





























1
HCHAA63
209119
pSport1
11
1142
1
1142
130
130
151
1
37
38
264




Jun. 05, 1997 


2
HCNSP40
209119
pBluescript
12
1034
1
1034
106
106
152
1
19
20
237




Jun. 05, 1997 


2
HCNSP40
209119
pBluescript
134
1032
1
1032

111
274
1


14




Jun. 05, 1997 


3
HBMXN79
209125
Uni-ZAP XR
13
1274
141
974
192
192
153
1
44
45
175




Jun. 19, 1997 


4
HGBGV89
209125
Uni-ZAP XR
14
968
1
968
55
55
154
1
26
27
197




Jun. 19, 1997 


5
HARAA15
209125
pBluescript
15
801
1
801
185
185
155
1
34
35
43




Jun. 19, 1997 
SK-


6
HDAAC10
209119
pSport1
16
1198
1
1198
117
117
156
1
21
22
313




Jun. 05, 1997 


7
HLHFP03
209126
Uni-ZAP XR
17
613
1
613
224
224
157
1
20
21
116




Jun. 19, 1997 


8
HMELR03
209119
Lambda ZAP
18
1621
8
1535
200
200
158
1
25
26
173




Jun. 05, 1997 
II


9
HNGJJ65
209124
Uni-ZAP XR
19
1122
1
1122
84
84
159
1
22
23
67




Jun. 19, 1997 


10
HKAFB88
209125
pCMVSport
20
1368
219
795
238
238
160
1
45
46
228




Jun. 19, 1997 
2.0


11
HSOAH66
209124
Uni-ZAP XR
21
1188
7
1188
196
196
161
1
27
28
36




Jun. 19, 1997 


11
HSOAH66
209124
Uni-ZAP XR
135
537
1
537
136
136
275
1
21
22
47




Jun. 19, 1997 


12
HCRBR74
209125
Uni-ZAP XR
22
921
365
911
415
415
162
1
20
21
98




Jun. 19, 1997 


13
HMSJW18
209119
Uni-ZAP XR
23
1838
1
1838
28
28
163
1
23
24
89




Jun. 05, 1997 


14
HHTLF25
209125
ZAP Express
24
697
1
661
142
142
164
1
26
27
111




Jun. 19, 1997 


15
HMKDD07
209126
pSport1
25
628
43
628
267
267
165
1
29
30
63




Jun. 19, 1997 


16
HELDY05
209119
Uni-ZAP XR
26
1422
1
1375
79
79
166
1
34
35
36




Jun. 05, 1997 


17
HFIVB57
209125
pSport1
27
795
92
795
286
286
167
1
35
36
38




Jun. 19, 1997 


18
HFPDE69
209125
Uni-ZAP XR
28
577
1
577
72
72
168
1
33
34
61




Jun. 19, 1997 


19
HMSHM14
209126
Uni-ZAP XR
29
756
1
756
103
103
169
1
29
30
45




Jun. 19, 1997 


20
HSRDE35
209126
Uni-ZAP XR
30
1296
232
804
428
428
170
1
21
22
116




Jun. 19, 1997 


21
HNFID82
209119
pBluescript
31
1560
1
1560
161
161
171
1
17
18
41




Jun. 05, 1997 


22
HOFNY71
209124
pCMVSport
32
1462
690
1462
200
200
172
1
28
29
281




Jun. 19, 1997 
2.0


22
HOFNY71
209124
pCMVSport
136
917
1
917
114
114
276
1
31
32
35




Jun. 19, 1997 
2.0


22
HOFMS34
Jan. 01, 2000
pCMVSport
137
1384
1
1384
123
123
277
1
28
29
281





2.0


23
HPMBR15
209124
Uni-ZAP XR
33
1272
25
1272
262
262
173
1


5




Jun. 19, 1997 


24
HSAVD46
209124
Uni-ZAP XR
34
773
2
767
155
155
174
1
20
21
58




Jun. 19, 1997 


25
HCNSB01
209125
pBluescript
35
2455
533
1308
552
552
175
1
22
23
179




Jun. 19, 1997 


26
HSDGR57
209126
Uni-ZAP XR
36
914
115
914
195
195
176
1
21
22
44




Jun. 19, 1997 


27
HJABB94
209119
pBluescript
37
1555
1
1555
74
74
177
1
28
29
77




Jun. 05, 1997 
SK-


28
HOFAF39
209124
pSport1
38
1767
1
1767
57
57
178
1
22
23
31




Jun. 19, 1997 


29
HTPCS60
209124
Uni-ZAP XR
39
1579
7
1259
105
105
179
1
19
20
257




Jun. 19, 1997 


30
HKIYO61
209119
pBluescript
40
1543
1
1543
181
181
180
1
19
20
37




Jun. 05, 1997 


31
HMKCW19
209119
pSport1
41
2095
473
1934
529
529
181
1
30
31
344




Jun. 05, 1997 


31
HMKCW19
209119
pSport1
138
1720
103
1692
188
188
278
1
27
28
45




Jun. 05, 1997 


32
HNFIG36
209119
pBluescript
42
1092
1
1082
171
171
182
1
18
19
46




Jun. 05, 1997 


33
HBIAS26
209125
Uni-ZAP XR
43
413
1
372
57
57
183
1
27
28
73




Jun. 19, 1997 


34
HE2EC79
209125
Uni-ZAP XR
44
735
1
735
151
151
184
1
21
22
30




Jun. 19, 1997 


35
HNDAH54
209126
pCMVSport
45
775
1
775
26
26
185
1
20
21
31




Jun. 19, 1997 
2.0


36
HNHFU59
209126
Uni-ZAP XR
46
506
1
506
278
278
186
1
16
17
76




Jun. 19, 1997 


37
HE8CV18
209119
Uni-ZAP XR
47
1447
1
1447
176
176
187
1
29
30
98




Jun. 05, 1997 


38
HFKEB72
209119
Uni-ZAP XR
48
1420
1
1420
43
43
188
1
29
30
65




Jun. 05, 1997 


39
HTDAD22
209124
pSport1
49
1220
1
1220
193
193
189
1
37
38
109




Jun. 19, 1997 


40
HSKYU29
209126
pBluescript
50
1048
1
1047
290
290
190
1
36
37
51




Jun. 19, 1997 


41
HHGDU58
209125
Lambda ZAP
51
968
70
898
235
235
191
1
46
47
80




Jun. 19, 1997 
II


42
HMDAA66
209126
Uni-ZAP XR
52
586
1
586
106
106
192
1
23
24
31




Jun. 19, 1997 


43
HNHFW22
209126
Uni-ZAP XR
53
751
1
751
228
228
193
1
26
27
60




Jun. 19, 1997 


44
HSQFP66
209126
Uni-ZAP XR
54
477
1
477
96
96
194
1
32
33
78




Jun. 19, 1997 


45
HNGEV29
209119
Uni-ZAP XR
55
1153
1
1153
173
173
195
1
30
31
73




Jun. 05, 1997 


45
HNGEV29
209119
Uni-ZAP XR
139
1566
1
1566
79
79
279
1


10




Jun. 05, 1997 


46
HUKBH05
209124
Lambda ZAP
56
1241
1
1215
151
151
196
1
18
19
58




Jun. 19, 1997 
II


47
HATDL27
209125
Uni-ZAP XR
57
908
1
908
82
82
197
1
28
29
31




Jun. 19, 1997 


48
HBJFU48
209125
Uni-ZAP XR
58
849
1
849
20
20
198
1
39
40
40




Jun. 19, 1997 


49
HCUBN59
209125
ZAP Express
59
678
1
678
96
96
199
1
39
40
43




Jun. 19, 1997 


50
HCUDB38
209125
ZAP Express
60
857
1
857
221
221
200
1
17
18
41




Jun. 19, 1997 


51
HDPCO25
209125
pCMVSport
61
767
76
767
182
182
201
1
20
21
53




Jun. 19, 1997 
3.0


52
HDPHI51
209125
pCMVSport
62
728
1
728
245
245
202
1
30
31
40




Jun. 19, 1997 
3.0


53
HSDAG05
209126
Uni-ZAP XR
63
944
231
848
419
419
203
1
37
38
75




Jun. 19, 1997 


54
HMWGY01
209119
Uni-ZAP XR
64
782
1
782
423
423
204
1
30
31
104




Jun. 05, 1997 


54
HMWGY01
209119
Uni-ZAP XR
140
774
1
774
17
17
280
1
31
32
39




Jun. 05, 1997 


55
HSVAQ28
209126
Uni-ZAP XR
65
442
1
442
149
149
205
1
24
25
98




Jun. 19, 1997 


56
HGLAM53
209119
Uni-ZAP XR
66
833
219
833
359
359
206
1
27
28
74




Jun. 05, 1997 


56
HGLAM53
209119
Uni-ZAP XR
141
1294
226
1288

369
281
1
26
27
67




Jun. 05, 1997 


57
HLTAI94
209119
Uni-ZAP XR
67
1262
1
1262
47
47
207
1
18
19
44




Jun. 05, 1997 


58
HMKAH10
209119
pSport1
68
921
1
921
48
48
208
1
43
44
54




Jun. 05, 1997 


59
HSVBH58
209124
Uni-ZAP XR
69
478
24
155
249
249
209
1
40
41
57




Jun. 19, 1997 


59
HSVBH58
209124
Uni-ZAP XR
142
680
1
680
168
168
282
1
20
21
22




Jun. 19, 1997 


60
HBMWB01
209125
Uni-ZAP XR
70
719
1
719
48
48
210
1
17
18
62




Jun. 19, 1997 


61
HBMXP84
209125
Uni-ZAP XR
71
519
1
519
161
161
211
1
31
32
39




Jun. 19, 1997 


62
HLYAF80
209126
pSport1
72
826
1
826
222
222
212
1
24
25
47




Jun. 19, 1997 


63
HNFDS53
209126
Uni-ZAP XR
73
911
1
911
200
200
213
1
22
23
23




Jun. 19, 1997 


64
HNGAX58
209126
Uni-ZAP XR
74
722
1
722
100
100
214
1
16
17
46




Jun. 19, 1997 


65
HOECO90
209126
Uni-ZAP XR
75
845
215
845
299
299
215
1
24
25
38




Jun. 19, 1997 


66
HSVAY16
209126
Uni-ZAP XR
76
882
1
790
52
52
216
1
30
31
31




Jun. 19, 1997 


67
HCFCE10
209119
pSport1
77
1590
18
1590
198
198
217
1
19
20
45




Jun. 05, 1997 


68
HCFNC26
209119
pSport1
78
1373
6
1373
85
85
218
1
18
19
24




Jun. 05, 1997 


69
HELDZ32
209119
Uni-ZAP XR
79
1107
12
1107
148
148
219
1
15
16
22




Jun. 05, 1997 


70
HODAZ26
209124
Uni-ZAP XR
80
1129
8
1129
133
133
220
1


30




Jun. 19, 1997 


71
HORBI81
209124
Uni-ZAP XR
81
1987
8
1965
31
31
221
1


34




Jun. 19, 1997 


72
HOSCY73
209124
Uni-ZAP XR
82
2053
196
2048
209
209
222
1


28




Jun. 19, 1997 


73
HADFF38
209124
pSport1
83
1193
1
1034
64
64
223
1
19
20
33




Jun. 19, 1997 


74
HFXHN31
209119
Lambda ZAP
84
541
1
541
172
172
224
1
30
31
91




Jun. 05, 1997 
II


74
HFXHN31
209119
Lambda ZAP
143
1168
1
1168
293
293
283
1
22
23
26




Jun. 05, 1997 
II


75
HNGIK21
209119
Uni-ZAP XR
85
985
1
985
152
152
225
1
25
26
28




Jun. 05, 1997 


76
HE9FE83
209125
Uni-ZAP XR
86
889
332
889
351
351
226
1
21
22
59




Jun. 19, 1997 


77
HMKDS08
209126
pSport1
87
558
1
558
230
230
227
1
30
31
67




Jun. 19, 1997 


78
HNGEM24
209126
Uni-ZAP XR
88
931
1
931
239
239
228
1
30
31
31




Jun. 19, 1997 


79
HNGFT78
209126
Uni-ZAP XR
89
588
1
588
20
20
229
1
29
30
35




Jun. 19, 1997 


80
HNHDL85
209126
Uni-ZAP XR
90
812
1
812
194
194
230
1
22
23
50




Jun. 19, 1997 


81
HCEIA77
209119
Uni-ZAP XR
91
1882
676
1882
785
785
231
1
37
38
53




Jun. 05, 1997 


82
HTNBR95
209124
pBluescript
92
1391
1
1386
70
70
232
1
28
29
35




Jun. 19, 1997 
SK-


83
HUKEX85
209124
Lambda ZAP
93
930
7
925
35
35
233
1
18
19
33




Jun. 19, 1997 
II


83
HUKEX85
209124
Lambda ZAP
144
930
6
917
83
83
284
1
30
31
122




Jun. 19, 1997 
II


84
HWTBM45
209124
Uni-ZAP XR
94
998
1
998
69
69
234
1
19
20
25




Jun. 19, 1997 


85
HADFK68
209124
pSport1
95
830
1
830
91
91
235
1
24
25
58




Jun. 19, 1997 


85
HADFK68
209124
pSport1
145
830
1
830
45
45
285
1


26




Jun. 19, 1997 


86
HADGG19
209125
pSport1
96
867
1
867
262
262
236
1
30
31
75




Jun. 19, 1997 


86
HADGG19
209125
pSport1
146
865
1
865
281
281
286
1


7




Jun. 19, 1997 


87
HSDZM95
209126
pBluescript
97
545
1
545
223
223
237
1
23
24
42




Jun. 19, 1997 


88
HSNAA55
209126
Uni-ZAP XR
98
722
1
722
35
35
238
1
15
16
40




Jun. 19, 1997 


89
HMDAI51
209119
Uni-ZAP XR
99
753
1
753
12
12
239
1
21
22
38




Jun. 05, 1997 


90
HBAFQ54
209125
pSport1
100
696
209
696
229
229
240
1
20
21
47




Jun. 19, 1997 


91
HBGBA14
209125
Uni-ZAP XR
101
455
1
452
32
32
241
1
24
25
36




Jun. 19, 1997 


92
HCFMM26
209125
pSport1
102
389
1
389
178
178
242
1
27
28
54




Jun. 19, 1997 


93
HOAAF80
209126
Uni-ZAP XR
103
960
131
960
303
303
243
1
33
34
36




Jun. 19, 1997 


94
HODCJ90
209126
Uni-ZAP XR
104
1442
326
1133
344
344
244
1
18
19
42




Jun. 19, 1997 


95
HNGJU42
209124
Uni-ZAP XR
105
598
6
598
273
273
245
1
17
18
23




Jun. 19, 1997 


96
HAEAV45
209125
pBluescript
106
685
46
647
487
487
246
1
34
35
66




Jun. 19, 1997 
SK-


96
HAEAV45
209125
pBluescript
147
545
1
545
24
24
287
1
25
26
28




Jun. 19, 1997 
SK-


97
HBJFV28
209125
Uni-ZAP XR
107
505
1
505
306
306
247
1
21
22
53




Jun. 19, 1997 


98
HDHMB42
209125
pCMVSport
108
1149
427
1149
592
592
248
1
26
27
31




Jun. 19, 1997 
2.0


99
HLNAB07
209126
Lambda ZAP
109
685
1
685
187
187
249
1
32
33
36




Jun. 19, 1997 
II


100
HMWDC28
209126
Uni-ZAP XR
110
1146
105
754
124
124
250
1
30
31
42




Jun. 19, 1997 


101
HSZAF47
209124
Uni-ZAP XR
111
1333
2
1333
107
107
251
1
18
19
127




Jun. 19, 1997 


102
HTADX17
209124
Uni-ZAP XR
112
1140
22
1140
84
84
252
1
24
25
142




Jun. 19, 1997 


103
HFTCU19
209119
Uni-ZAP XR
113
1575
1266
1575
137
137
253
1
30
31
222




Jun. 05, 1997 


103
HFTCU19
209119
Uni-ZAP XR
148
470
1
470
157
157
288
1
24
25
56




Jun. 05, 1997 


104
HEBFL88
209125
Uni-ZAP XR
114
334
2
334
76
76
254
1
22
23
38




Jun. 19, 1997 


105
HJMAV91
209125
pCMVSport
115
866
74
866
251
251
255
1
16
17
32




Jun. 19, 1997 
3.0


106
HSDJJ82
209126
Uni-ZAP XR
116
462
1
462
79
79
256
1
32
33
52




Jun. 19, 1997 


107
HTLDP69
209124
Uni-ZAP XR
117
1500
237
1500
330
330
257
1
29
30
148




Jun. 19, 1997 


108
HPEBT80
209126
Uni-ZAP XR
118
360
1
360
21
21
258
1
40
41
50




Jun. 19, 1997 


109
HCNAV36
209125
Lambda ZAP
119
823
411
823
505
505
259
1
15
16
46




Jun. 19, 1997 
II


110
HNFIU96
209126
pBluescript
120
456
1
456
170
170
260
1
33
34
79




Jun. 19, 1997 


111
HGLDE38
209125
Uni-ZAP XR
121
553
1
553
31
31
261
1
19
20
61




Jun. 19, 1997 


112
HODDB05
209124
Uni-ZAP XR
122
1158
22
1158
244
244
262
1


10




Jun. 19, 1997 


113
HNGAC63
209126
Uni-ZAP XR
123
554
1
554
214
214
263
1


15




Jun. 19, 1997 


114
HTADV27
209124
Uni-ZAP XR
124
1255
14
1255
69
69
264
1


20




Jun. 19, 1997 


115
HCUFZ62
209125
ZAP Express
125
1977
28
661
233
233
265
1
28
29
51




Jun. 19, 1997 


116
HSSJN64
209126
Uni-ZAP XR
126
738
1
738
70
70
266
1
33
34
61




Jun. 19, 1997 


117
HTEHH53
209124
Uni-ZAP XR
127
988
1
980
22
22
267
1
24
25
209




Jun. 19, 1997 


118
HSIDI15
209126
Uni-ZAP XR
128
912
1
873
273
273
268
1
22
23
74




Jun. 19, 1997 


119
HE9HW52
209125
Uni-ZAP XR
129
569
73
569
122
122
269
1
25
26
34




Jun. 19, 1997 


120
HLWCF05
209126
pCMVSport
130
646
1
646
155
155
270
1
36
37
58




Jun. 19, 1997 
3.0


121
HFGAL10
209119
Uni-ZAP XR
131
1183
1
1183
179
179
271
1
20
21
96




Jun. 05, 1997 


121
HFGAL10
209119
Uni-ZAP XR
149
1766
3
1765
179
179
289
1
17
18
36




Jun. 05, 1997 


122
HSLBF69
209124
Uni-ZAP XR
132
2119
1
2119
107
107
272
1
19
20
405




Jun. 19, 1997 


123
HTEDS39
209124
Uni-ZAP XR
133
694
198
694
205
205
273
1
21
22
80




Jun. 19, 1997 


123
HTEDS39
209124
Uni-ZAP XR
150
1048
1
1048

227
290
1


20




Jun. 19, 1997 

























TABLE 1B












Tissue Distribution









AA SEQ

Library code: count

OMIM


Gene

Contig
SEQ ID
ORF
ID

(see Table IV for
Cytologic 
Disease


No:
Clone ID:
ID:
NO: X
(From–To)
NO: Y
Predicted Epitopes
Library Codes)
Band
Reference(s):
























1
HCHAA63
456591
11
130–924
151
Pro-6 to Arg-11,
AR216: 60, AR214:
9q34.3
120215,








Glu-52 to Gly-59.
57, AR215: 56, AR223:

120215,









47, AR224: 47, AR222:

190198









45, AR169: 44, AR168:









42, AR225: 42, AR311:









40, AR217: 40, AR171:









39, AR272: 38, AR199:









37, AR210: 36, AR170:









35, AR221: 34, AR161:









32, AR172: 32, AR274:









31, AR162: 31, AR163:









30, AR189: 29, AR264:









28, AR165: 27, AR211:









27, AR164: 26, AR188:









26, AR190: 26, AR166:









23, AR236: 23, AR308:









22, AR174: 22, AR178:









20, AR212: 20, AR288:









20, AR173: 19, AR262:









19, AR180: 19, AR219:









19, AR200: 19, AR191:









18, AR313: 18, AR297:









17, AR247: 17, AR218:









16, AR261: 16, AR275:









15, AR255: 15, AR179:









14, AR181: 14, AR257:









14, AR230: 14, AR263:









14, AR312: 13, AR196:









13, AR203: 13, AR183:









12, AR287: 12, AR096:









12, AR213: 12, AR053:









11, AR299: 11, AR175:









11, AR256: 11, AR260:









10, AR176: 10, AR239:









10, AR185: 10, AR291:









10, AR245: 10, AR267:









10, AR296: 10, AR289:









9, AR240: 9, AR290:









9, AR266: 9, AR300:









9, AR258: 9, AR282:









9, AR295: 9, AR228:









9, AR309: 8, AR268:









8, AR089: 8, AR233:









8, AR234: 8, AR294:









7, AR177: 7, AR269:









7, AR285: 7, AR316:









7, AR270: 7, AR293:









7, AR104: 7, AR231:









6, AR195: 6, AR182:









6, AR237: 6, AR277:









6, AR205: 6, AR226:









6, AR286: 6, AR235:









6, AR197: 6, AR060:









6, AR232: 5, AR246:









5, AR201: 5, AR229:









5, AR238: 4, AR033:









4, AR039: 4, AR242:









4, AR227: 4, AR254:









4, AR055: 4, AR193:









4, AR204: 3, AR061:









3, AR271: 3, AR283:









3, AR207: 3, AR243:









3, AR198: 3, AR253:









2, AR252: 2, AR310:









1, AR250: 1, AR292: 1









L0662: 9, L0769: 8,









L0659: 7, L0666: 7,









H0052: 6, L0646: 5,









L0747: 5, L0783: 4,









L0665: 4, L0731: 4,









S0358: 3, H0550: 3,









L0772: 3, L0779: 3,









L0758: 3, H0483: 2,









S0360: 2, S0046: 2,









H0486: 2, H0617: 2,









L0770: 2, L0636: 2,









L0526: 2, L0809: 2,









L0664: 2, H0521: 2,









H0696: 2, L0756: 2,









L0753: 2, S0276: 2,









L0411: 1, H0664: 1,









S0376: 1, S0045: 1,









S0222: 1, H0587: 1,









S0280: 1, H0618: 1,









H0253: 1, H0204: 1,









H0594: 1, H0424: 1,









H0606: 1, H0032: 1,









H0212: 1, H0135: 1,









H0090: 1, H0551: 1,









H0623: 1, H0059: 1,









H0100: 1, H0359: 1,









H0625: 1, H0131: 1,









H0538: 1, H0529: 1,









L0369: 1, L0372: 1,









L0800: 1, L0645: 1,









L0764: 1, L0773: 1,









L0768: 1, L0775: 1,









L0656: 1, L0663: 1,









S0053: 1, S0374: 1,









L0438: 1, H0683: 1,









H0658: 1, H0672: 1,









S0404: 1, S0406: 1,









H0555: 1, S0432: 1,









S3014: 1, L0742: 1,









L0439: 1, L0751: 1,









L0608: 1 and H0423: 1.


2
HCNSP40
711868
12
106–816
152
Asn-26 to Cys-32,
AR216: 13, AR215:








Cys-100 to Leu-
10, AR214: 10, AR169:








112,
9, AR170: 8, AR168:








Cys-128 to Ser-135.
8, AR171: 8, AR172:









8, AR217: 8, AR161:









7, AR162: 7, AR163:









7, AR225: 7, AR224:









7, AR221: 7, AR223:









7, AR165: 6, AR222:









6, AR164: 6, AR166:









6, AR252: 6, AR176:









6, AR060: 5, AR236:









5, AR300: 5, AR245:









5, AR052: 5, AR228:









5, AR235: 5, AR239:









4, AR266: 4, AR261:









4, AR178: 4, AR197:









4, AR272: 4, AR089:









4, AR053: 4, AR264:









4, AR210: 4, AR244:









4, AR241: 4, AR282:









4, AR298: 4, AR269:









4, AR308: 4, AR309:









4, AR193: 4, AR182:









4, AR061: 4, AR262:









4, AR246: 4, AR257:









4, AR263: 4, AR316:









4, AR255: 4, AR299:









4, AR267: 4, AR237:









4, AR230: 3, AR240:









3, AR247: 3, AR283:









3, AR288: 3, AR207:









3, AR313: 3, AR250:









3, AR285: 3, AR183:









3, AR251: 3, AR233:









3, AR055: 3, AR231:









3, AR213: 3, AR291:









3, AR270: 3, AR238:









3, AR289: 3, AR229:









3, AR181: 3, AR268:









3, AR202: 3, AR293:









3, AR104: 3, AR287:









3, AR211: 3, AR173:









3, AR275: 3, AR310:









3, AR096: 3, AR206:









3, AR188: 3, AR296:









3, AR290: 3, AR179:









3, AR196: 3, AR274:









3, AR277: 3, AR286:









3, AR184: 3, AR311:









3, AR191: 3, AR295:









3, AR175: 3, AR185:









3, AR198: 3, AR205:









3, AR200: 3, AR201:









3, AR232: 3, AR227:









3, AR033: 2, AR199:









2, AR204: 2, AR177:









2, AR226: 2, AR174:









2, AR297: 2, AR192:









2, AR312: 2, AR190:









2, AR271: 2, AR195:









2, AR189: 2, AR294:









2, AR203: 2, AR284:









2, AR212: 2, AR273:









2, AR234: 2, AR039:









2, AR292: 2, AR260:









2, AR248: 2, AR258:









2, AR254: 2, AR219:









2, AR186: 1, AR256:









1, AR259: 1, AR253: 1









L0805: 51, L0776: 30,









S0358: 12 , L0789: 8,









L0754: 8, L0777: 8,









L0439: 5, L0750: 5,









S0408: 4, S0444: 3,









L0769: 3, L0794: 3,









L0809: 3, S0152: 3,









L0758: 3, S0354: 2,









S0376: 2, S0410: 2,









H0597: 2, H0231: 2,









H0266: 2, H0424: 2,









L0764: 2, L0771: 2,









L0659: 2, L0438: 2,









H0672: 2, L0745: 2,









L0731: 2, S0436: 2,









H0650: 1, S0212: 1,









S0045: 1, S0046: 1,









H0486: 1, L0563: 1,









H0085: 1, H0596: 1,









H0154: 1, H0051: 1,









H0213: 1, H0418: 1,









H0644: 1, H0673: 1,









H0551: 1, H0269: 1,









S0370: 1, S0344: 1,









S0208: 1, L0768: 1,









L0766: 1, L0803: 1,









L0788: 1, S0374: 1,









H0547: 1, H0519: 1,









S0126: 1, H0711: 1,









H0528: 1, S0404: 1,









S0028: 1, L0779: 1,









L0780: 1, L0757: 1,









L0588: 1, L0591: 1,









H0665: 1, S0192: 1,









H0423: 1 and H0293: 1.



HCNSP40
456440
134
111–155
274


3
HBMXN79
460351
13
192–719
153
Ser-59 to Ile-64,
AR252: 9, AR253: 8,








Ala-71 to Tyr-76,
AR161: 7, AR162: 7,








Pro-125 to Ser-141.
AR163: 7, AR245: 6,









AR176: 6, AR246: 6,









AR271: 6, AR201: 6,









AR272: 6, AR183: 6,









AR182: 6, AR203: 6,









AR089: 5, AR197: 5,









AR188: 5, AR175: 5,









AR282: 5, AR178: 5,









AR207: 5, AR263: 5,









AR238: 5, AR165: 5,









AR199: 5, AR210: 5,









AR181: 5, AR190: 5,









AR164: 4, AR270: 4,









AR250: 4, AR200: 4,









AR309: 4, AR166: 4,









AR269: 4, AR039: 4,









AR191: 4, AR096: 4,









AR204: 4, AR275: 4,









AR268: 4, AR291: 4,









AR104: 4, AR189: 4,









AR274: 4, AR313: 4,









AR195: 4, AR180: 4,









AR173: 4, AR060: 4,









AR174: 4, AR205: 4,









AR290: 3, AR218: 3,









AR266: 3, AR212: 3,









AR267: 3, AR296: 3,









AR240: 3, AR179: 3,









AR235: 3, AR229: 3,









AR316: 3, AR237: 3,









AR264: 3, AR213: 3,









AR239: 3, AR226: 3,









AR172: 3, AR193: 3,









AR231: 3, AR255: 3,









AR211: 3, AR295: 3,









AR177: 3, AR299: 3,









AR234: 3, AR300: 3,









AR242: 3, AR297: 3,









AR311: 3, AR254: 3,









AR198: 3, AR257: 3,









AR171: 3, AR232: 3,









AR308: 3, AR233: 2,









AR236: 2, AR261: 2,









AR185: 2, AR247: 2,









AR293: 2, AR196: 2,









AR288: 2, AR230: 2,









AR228: 2, AR294: 2,









AR277: 2, AR285: 2,









AR227: 2, AR287: 2,









AR061: 2, AR312: 2,









AR262: 2, AR219: 2,









AR225: 2, AR053: 2,









AR289: 2, AR217: 2,









AR286: 2, AR224: 2,









AR243: 2, AR033: 1,









AR168: 1, AR055: 1,









AR258: 1, AR216: 1









L0758: 3, S0116: 2,









S0420: 2, S0152: 2,









L0756: 2, L0777: 2,









L0731: 2, S0444: 1,









H0580: 1, H0549: 1,









H0586: 1, H0581: 1,









S0003: 1, S0214: 1,









H0032: 1, H0674: 1,









H0591: 1, H0038: 1,









S0386: 1, L0650: 1,









L0775: 1, L0783: 1,









H0521: 1, H0631: 1,









L0591: 1 and H0422: 1,


4
HGBGV89
460353
14
 55–648
154
Asn-32 to Asn-41,
AR174: 27, AR191:








Thr-140 to Ala-147,
24, AR178: 23, AR189:








Asp-188 to His-
21, AR173: 20, AR210:








197.
20, AR190: 16, AR176:









15, AR264: 15, AR181:









15, AR188: 14, AR211:









14, AR165: 13, AR164:









13, AR161: 12, AR166:









12, AR162: 12, AR163:









12, AR197: 12, AR180:









12, AR269: 11, AR201:









11, AR270: 11, AR175:









10, AR195: 10, AR254:









10, AR308: 9, AR250:









9, AR203: 9, AR182:









8, AR245: 8, AR199:









8, AR177: 8, AR196:









8, AR272: 8, AR271:









8, AR268: 8, AR290:









8, AR288: 8, AR183:









8, AR309: 8, AR312:









7, AR255: 7, AR039:









7, AR193: 7, AR235:









7, AR200: 7, AR261:









7, AR179: 7, AR202:









7, AR207: 7, AR311:









7, AR246: 6, AR238:









6, AR267: 6, AR239:









6, AR205: 6, AR204:









6, AR297: 6, AR096:









6, AR257: 6, AR240:









6, AR287: 6, AR253:









6, AR275: 5, AR212:









5, AR218: 5, AR089:









5, AR215: 5, AR300:









5, AR192: 5, AR236:









5, AR274: 5, AR230:









5, AR252: 5, AR247:









5, AR229: 5, AR295:









5, AR262: 5, AR234:









5, AR296: 4, AR219:









4, AR293: 4, AR299:









4, AR228: 4, AR263:









4, AR060: 4, AR231:









4, AR285: 4, AR233:









4, AR237: 4, AR198:









4, AR260: 4, AR266:









4, AR289: 4, AR206:









4, AR033: 4, AR185:









4, AR313: 4, AR222:









4, AR053: 3, AR291:









3, AR169: 3, AR316:









3, AR282: 3, AR286:









3, AR232: 3, AR243:









3, AR226: 3, AR217:









3, AR061: 3, AR172:









3, AR258: 3, AR265:









3, AR104: 2, AR277:









2, AR055: 2, AR283:









2, AR213: 2, AR294:









2, AR214: 2, AR224:









2, AR216: 2, AR227:









2, AR168: 2, AR256:









2, AR273: 2, AR225:









2, AR221: 2, AR186:









2, AR184: 1, AR310:









1, AR284: 1, AR244:









1, AR052: 1, AR241: 1









L0749: 5, L0748: 3,









H0509: 2, L0774: 2,









S0376: 1, H0357: 1,









H0574: 1, H0590: 1,









L0738: 1, H0014: 1,









L0747: 1, L0752: 1 and









L0605: 1.


5
HARAA15
460398
15
185–316
155

AR282: 4, AR175: 4,









AR169: 3, AR270: 2,









AR161: 2, AR290: 2,









AR225: 2, AR217: 2,









AR266: 2, AR173: 2,









AR257: 2, AR250: 2,









AR178: 2, AR183: 2,









AR230: 2, AR283: 2,









AR264: 1, AR162: 1,









AR296: 1, AR172: 1,









AR163: 1, AR291: 1,









AR055: 1, AR238: 1,









AR297: 1, AR253: 1,









AR267: 1, AR216: 1,









AR289: 1









T0082: 2


6
HDAAC10
456443
16
 117–1058
156
Arg-15 to Ser-33,
AR173: 10, AR240: 9,








Pro-35 to Cys-41.
AR269: 9, AR183: 9,









AR299: 8, AR253: 6,









AR039: 5, AR282: 4,









AR270: 4, AR313: 4,









AR170: 4, AR246: 4,









AR274: 3, AR096: 3,









AR243: 3, AR161: 3,









AR163: 3, AR225: 2,









AR180: 2, AR053: 2,









AR288: 2, AR207: 2,









AR205: 2, AR255: 2,









AR271: 2, AR275: 2,









AR316: 2, AR245: 2,









AR224: 2, AR162: 2,









AR213: 1, AR175: 1,









AR261: 1, AR199: 1,









AR311: 1, AR235: 1,









AR257: 1, AR211: 1,









AR277: 1, AR060: 1,









AR294: 1









S0358: 12, L0374: 8,









S0470: 4, S0444: 3,









L0748: 3, S0442: 2,









S0354: 2, L0649: 2,









S0374: 2, S0404: 2,









L0749: 2, L0750: 2,









L0002: 1, S0356: 1,









H0489: 1, H0619: 1,









H0497: 1, L0021: 1,









H0085: 1, H0234: 1,









L0738: 1, L0764: 1,









L0771: 1, L0662: 1,









L0804: 1, L0791: 1 and









H0660: 1.


7
HLHFP03
460467
17
224–574
157
Tyr-28 to Phe-34,
AR194: 6, AR186: 6,








Thr-54 to Val-60,
AR169: 6, AR170: 5,








Tyr-73 to Thr-82.
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.


8
HMELR03
456482
18
200–721
158
Asp-17 to Phe-23.
AR292: 21, AR296:









17, AR271: 15, AR298:









14, AR291: 12, AR274:









11, AR294: 11, AR285:









10, AR293: 9, AR286:









9, AR060: 9, AR184:









9, AR182: 8, AR273:









8, AR275: 7, AR192:









7, AR186: 7, AR033:









7, AR104: 7, AR052:









7, AR313: 7, AR290:









7, AR265: 7, AR295:









6, AR202: 6, AR198:









6, AR251: 6, AR089:









6, AR243: 6, AR206:









6, AR270: 6, AR244:









6, AR269: 6, AR309:









5, AR205: 5, AR282:









5, AR300: 5, AR312:









5, AR055: 5, AR316:









5, AR310: 5, AR249:









5, AR268: 4, AR194:









4, AR231: 4, AR061:









4, AR299: 4, AR240:









4, AR053: 4, AR284:









4, AR267: 4, AR185:









4, AR213: 4, AR277:









4, AR248: 4, AR238:









4, AR204: 4, AR253:









3, AR096: 3, AR229:









3, AR039: 3, AR247:









3, AR226: 3, AR246:









3, AR177: 3, AR232:









3, AR289: 3, AR258:









3, AR183: 3, AR237:









3, AR283: 2, AR233:









2, AR175: 2, AR234:









2, AR241: 2, AR256:









2, AR259: 1, AR179:









1, AR266: 1, AR263: 1









H0624: 1, H0341: 1, 









H0333: 1, H0013: 1, 









H0266: 1, H0269: 1 and









S0440: 1.


9
HNGJJ65
456504
19
 84–284
159
Pro-55 to Ser-66.
AR296: 27, AR261:









23, AR287: 23, AR297:









22, AR235: 20, AR260:









18, AR288: 18, AR165:









18, AR164: 17, AR255:









17, AR166: 17, AR291:









15, AR262: 14, AR191:









14, AR236: 14, AR257:









14, AR295: 13, AR258:









12, AR196: 11, AR241:









11, AR285: 10, AR250:









10, AR286: 10, AR190:









10, AR289: 10, AR189:









9, AR162: 9, AR161:









9, AR173: 9, AR163:









9, AR186: 9, AR294:









9, AR188: 9, AR181:









8, AR174: 8, AR246:









8, AR254: 8, AR245:









8, AR256: 8, AR169:









8, AR192: 8, AR178:









8, AR199: 7, AR200:









7, AR176: 7, AR308:









7, AR193: 7, AR210:









7, AR212: 7, AR180:









7, AR172: 7, AR168:









7, AR270: 6, AR204:









6, AR266: 6, AR269:









6, AR293: 6, AR052:









6, AR239: 6, AR211:









6, AR182: 6, AR311:









6, AR229: 6, AR201:









6, AR312: 6, AR197:









6, AR247: 5, AR202:









5, AR271: 5, AR231:









5, AR264: 5, AR221:









5, AR244: 5, AR039:









5, AR215: 5, AR206:









5, AR313: 5, AR183:









5, AR203: 5, AR253:









5, AR179: 5, AR300:









5, AR228: 5, AR171:









5, AR213: 5, AR175:









5, AR251: 5, AR310:









5, AR061: 5, AR290:









5, AR238: 5, AR240:









5, AR053: 5, AR248:









5, AR283: 5, AR089:









5, AR226: 4, AR205:









4, AR195: 4, AR219:









4, AR263: 4, AR268:









4, AR282: 4, AR267:









4, AR198: 4, AR265:









4, AR234: 4, AR298:









4, AR299: 4, AR055:









4, AR233: 4, AR218:









4, AR316: 4, AR273:









4, AR216: 4, AR243:









4, AR309: 4, AR033:









4, AR096: 4, AR185:









4, AR177: 4, AR232:









4, AR060: 3, AR237:









3, AR207: 3, AR284:









3, AR274: 3, AR222:









3, AR230: 3, AR277:









3, AR223: 3, AR252:









3, AR272: 3, AR292:









2, AR104: 2, AR227:









2, AR194: 2, AR217:









2, AR259: 2, AR275:









2, AR170: 2, AR225:









2, AR224: 1, AR214:









1, AR184: 1









S0052: 1


10
HKAFB88
460635
20
238–924
160

AR060: 23, AR316:
22q12.3–q13.2
138981,









20, AR104: 18, AR089:

188826,









18, AR283: 15, AR313:

190040,









12, AR240: 12, AR185:

190040,









11, AR282: 10, AR096:

190040,









9, AR277: 9, AR299:

600850,









8, AR263: 5, AR265:

601669









4, AR249: 3, AR266:









3, AR184: 3, AR310:









2, AR052: 2, AR312:









2, AR271: 2, AR247:









2, AR175: 1









L0755: 22, H0521: 14,









L0766: 9, H0271: 7,









L0659: 7, L0751: 7,









L0757: 7, L0771: 6,









L0806: 5, L0665: 5,









H0580: 4, H0492: 4,









H0646: 4, L0774: 4,









L0740: 4, L0758: 4,









L0601: 4, S0132: 3,









H0551: 3, L0769: 3,









L0794: 3, L0776: 3,









L0789: 3, H0593: 3,









H0435: 3, L0777: 3,









L0731: 3, S0212: 2,









S0360: 2, S0278: 2,









H0641: 2, L0770: 2,









L0764: 2, L0773: 2,









L0648: 2, L0804: 2,









L0775: 2, S0152: 2,









H0522: 2, L0747: 2,









L0750: 2, L0779: 2,









S0436: 2, L0604: 2,









H0653: 2, H0265: 1,









H0556: 1, H0141: 1,









H0585: 1, S0180: 1,









H0661: 1, H0638: 1,









S0418: 1, S0420: 1,









S0442: 1, S0444: 1,









H0728: 1, H0735: 1,









S0045: 1, S0046: 1,









H0550: 1, H0587: 1,









H0497: 1, H0559: 1,









H0069: 1, H0253: 1,









T0048: 1, H0581: 1,









H0744: 1, H0544: 1,









H0046: 1, H0266: 1,









H0179: 1, H0416: 1,









H0286: 1, H0252: 1,









H0039: 1, H0553: 1,









L0055: 1, H0634: 1,









H0477: 1, H0059: 1,









H0494: 1, S0210: 1,









L0640: 1, L0763: 1,









L0637: 1, L5565: 1,









L0761: 1, L0772: 1,









L0800: 1, L0643: 1,









L0553: 1, L0768: 1,









L0803: 1, L0653: 1,









L0382: 1, L0368: 1,









L0793: 1, L0664: 1,









L0565: 1, S0126: 1,









H0683: 1, H0518: 1,









L0716: 1, H0528: 1,









S0406: 1, S3014: 1,









S0028: 1, S0032: 1,









L0746: 1, L0759: 1,









L0361: 1, L0097: 1,









S0192: 1, S0194: 1 and









H0542: 1.


11
HSOAH66
456540
21
196–306
161

AR313: 10, AR277: 9,









AR089: 8, AR299: 7,









AR240: 7, AR060: 7,









AR104: 6, AR316: 6,









AR185: 6, AR283: 5,









AR096: 3, AR282: 2









L0794: 1, L0750: 1,









L0779: 1 and H0343: 1.



HSOAH66
425954
135
136–276
275
Pro-30 to Ser-37.


12
HCRBR74
460694
22
415–711
162
Asp-57 to Gly-64.
AR197: 86, AR195:
4q31–q32
107250,









49, AR198: 30, AR104:

181600,









20, AR033: 17, AR271:

189800,









16, AR201: 13, AR245:

208400,









13, AR192: 12, AR207:

231675,









11, AR309: 11, AR252:

266300









11, AR200: 10, AR039:









10, AR246: 10, AR161:









10, AR162: 10, AR253:









10, AR163: 9, AR205:









9, AR311: 9, AR212:









9, AR089: 8, AR254:









8, AR204: 8, AR243:









8, AR177: 8, AR181:









8, AR224: 8, AR312:









8, AR164: 8, AR053:









8, AR223: 8, AR235:









7, AR308: 7, AR215:









7, AR165: 7, AR274:









7, AR166: 7, AR266:









7, AR176: 7, AR169:









7, AR193: 7, AR211:









7, AR269: 7, AR272:









7, AR264: 7, AR213:









7, AR214: 7, AR263:









7, AR316: 7, AR222:









6, AR171: 6, AR250:









6, AR268: 6, AR228:









6, AR178: 6, AR182:









6, AR229: 6, AR217:









6, AR239: 6, AR242:









6, AR313: 5, AR261:









5, AR168: 5, AR270:









5, AR096: 5, AR183:









5, AR216: 5, AR236:









5, AR247: 5, AR055:









5, AR188: 5, AR267:









5, AR060: 5, AR179:









5, AR233: 5, AR221:









5, AR225: 5, AR210:









5, AR240: 5, AR282:









5, AR290: 5, AR061:









4, AR295: 4, AR196:









4, AR174: 4, AR185:









4, AR291: 4, AR231:









4, AR237: 4, AR299:









4, AR289: 4, AR255:









4, AR285: 4, AR293:









4, AR275: 4, AR226:









4, AR257: 4, AR300:









4, AR175: 4, AR286:









3, AR234: 3, AR297:









3, AR191: 3, AR296:









3, AR283: 3, AR230:









3, AR180: 3, AR189:









3, AR238: 3, AR170:









3, AR173: 3, AR232:









3, AR294: 3, AR262:









3, AR277: 3, AR199:









3, AR288: 3, AR218:









3, AR287: 3, AR227:









3, AR219: 3, AR203:









3, AR190: 2, AR172:









2, AR258: 1









L0758: 15, L0105: 6,









L0439: 5, L0731: 5,









L0438: 4, L0777: 4,









S0388: 3, H0616: 3,









L0769: 3, S0126: 3,









L0763: 2, L0770: 2,









L0784: 2, L0809: 2,









H0144: 2, L0759: 2,









L0411: 1, H0662: 1,









S0300: 1, S0222: 1,









H0455: 1, S0010: 1,









H0327: 1, H0050: 1,









S0316: 1, H0038: 1,









L0794: 1, L0789: 1 and









S0260: 1.


13
HMSJW18
457182
23
 28–297
163
Ser-31 to Ala-37,
AR313: 22, AR089:








Ala-50 to Tyr-55,
21, AR240: 19, AR277:








Phe-63 to Arg-68,
16, AR299: 15, AR316:








His-83 to Pro-89.
14, AR185: 13, AR060:









11, AR096: 11, AR282:









8, AR104: 6, AR283: 5









S0002: 2, S6024: 1,









H0664: 1, H0586: 1,









H0574: 1, H0046: 1,









H0563: 1, H0028: 1,









H0131: 1 and S0428: 1.


14
HHTLF25
461438
24
142–474
164
Ala-28 to Ser-33,
AR251: 168, AR248:
19q13.1
164731,








Ala-76 to Lys-111.
141, AR249: 139,

172400,









AR265: 60, AR253:

172400,









50, AR263: 41, AR096:

180901,









37, AR244: 32, AR268:

180901,









26, AR264: 24, AR290:

221770,









20, AR246: 18, AR240:

248600,









17, AR177: 16, AR267:

600918,









14, AR183: 14, AR270:

602716









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.


15
HMKDD07
460481
25
267–458
165
Glu-45 to Asn-50.
AR263: 43, AR264:









38, AR252: 34, AR311:









34, AR308: 33, AR053:









30, AR312: 29, AR309:









25, AR212: 23, AR218:









21, AR096: 21, AR213:









19, AR219: 19, AR246:









17, AR254: 16, AR253:









14, AR272: 13, AR250:









13, AR240: 12, AR313:









12, AR200: 12, AR189:









9, AR174: 9, AR191:









9, AR224: 9, AR268:









9, AR274: 9, AR165:









9, AR290: 9, AR222:









9, AR195: 9, AR197:









9, AR245: 9, AR164:









9, AR104: 9, AR275:









8, AR173: 8, AR161:









8, AR166: 8, AR162:









8, AR299: 8, AR316:









8, AR185: 8, AR163:









8, AR169: 8, AR223:









8, AR205: 8, AR198:









8, AR183: 8, AR269:









8, AR211: 8, AR225:









7, AR175: 7, AR196:









7, AR039: 7, AR243:









7, AR177: 7, AR171:









7, AR180: 7, AR188:









7, AR190: 7, AR270:









6, AR089: 6, AR221:









6, AR199: 6, AR201:









6, AR033: 6, AR300:









6, AR178: 6, AR172:









6, AR193: 6, AR267:









6, AR170: 6, AR247:









6, AR203: 6, AR229:









6, AR179: 6, AR210:









5, AR271: 5, AR181:









5, AR168: 5, AR204:









5, AR235: 5, AR217:









5, AR216: 5, AR282:









5, AR060: 5, AR277:









4, AR234: 4, AR266:









4, AR261: 4, AR285:









4, AR257: 4, AR226:









4, AR231: 4, AR291:









4, AR288: 4, AR296:









4, AR242: 4, AR297:









4, AR289: 4, AR182:









3, AR230: 3, AR256:









3, AR238: 3, AR207:









3, AR192: 3, AR255:









3, AR295: 3, AR176:









3, AR214: 3, AR293:









3, AR258: 3, AR236:









3, AR262: 3, AR286:









3, AR237: 3, AR055:









3, AR232: 2, AR228:









2, AR227: 2, AR239:









2, AR283: 2, AR061:









2, AR287: 2, AR233:









2, AR294: 2, AR260:









1, AR215: 1









L0502: 2, S0114: 1,









H0402: 1, H0392: 1,









L0586: 1, H0179: 1,









L0804: 1, L0509: 1 and









L0515: 1.


16
HELDY05
456449
26
 79–189
166

AR313: 11, AR089: 5,









AR240: 5, AR060: 5,









AR316: 5, AR277: 5,









AR282: 4, AR104: 4,









AR096: 4, AR299: 4,









AR185: 4, AR283: 4









L0662: 3, S0045: 2,









H0696: 2, H0295: 1,









H0255: 1, S0046: 1,









H0619: 1, H0050: 1,









H0634: 1, H0264: 1,









L0769: 1, L0438: 1,









H0670: 1, H0539: 1,









L0779: 1, L0755: 1 and









S0260: 1.


17
HFIVB57
460449
27
286–402
167

L0757: 2, S0360: 1,









L0040: 1, H0623: 1,









L0761: 1, L0363: 1,









L0804: 1, L0805: 1,









L0776: 1, L0809: 1,









H0547: 1, L0777: 1,









H0653: 1 and S0196: 1.


18
HFPDE69
460450
28
 72–257
168
Ser-4 to Tyr-13.
AR313: 44, AR089:









39, AR277: 35, AR299:









35, AR240: 34, AR316:









25, AR185: 24, AR060:









20, AR096: 18, AR104:









11, AR282: 10, AR283: 7









S0222: 1


19
HMSHM14
461897
29
103–240
169
Met-1 to Ser-6,
AR060: 32, AR104:








Pro-29 to Ser-34.
17, AR089: 16, AR283:









14, AR299: 13, AR316:









12, AR172: 12, AR185:









10, AR277: 9, AR282:









8, AR161: 7, AR162:









7, AR253: 7, AR163:









7, AR171: 7, AR236:









7, AR096: 7, AR250:









6, AR312: 6, AR168:









6, AR235: 6, AR169:









6, AR264: 5, AR274:









5, AR245: 5, AR195:









5, AR240: 5, AR197:









5, AR291: 5, AR313:









5, AR254: 5, AR053:









5, AR246: 4, AR193:









4, AR275: 4, AR295:









4, AR308: 4, AR285:









4, AR272: 4, AR198:









4, AR271: 4, AR212:









4, AR170: 4, AR191:









4, AR311: 4, AR201:









4, AR252: 4, AR269:









4, AR181: 4, AR225:









4, AR309: 4, AR204:









3, AR286: 3, AR033:









3, AR178: 3, AR266:









3, AR222: 3, AR165:









3, AR175: 3, AR257:









3, AR180: 3, AR268:









3, AR221: 3, AR243:









3, AR196: 3, AR176:









3, AR182: 3, AR189:









3, AR190: 3, AR247:









3, AR261: 3, AR293:









3, AR188: 3, AR287:









3, AR173: 3, AR297:









3, AR258: 3, AR300:









3, AR199: 3, AR177:









3, AR183: 3, AR223:









3, AR262: 3, AR289:









3, AR174: 3, AR179:









3, AR232: 3, AR228:









3, AR224: 3, AR288:









2, AR294: 2, AR290:









2, AR233: 2, AR267:









2, AR255: 2, AR210:









2, AR270: 2, AR229:









2, AR296: 2, AR213:









2, AR231: 2, AR238:









2, AR164: 2, AR219:









2, AR200: 2, AR166:









2, AR239: 2, AR226:









2, AR237: 2, AR211:









2, AR217: 2, AR263:









2, AR203: 2, AR256:









2, AR227: 2, AR218:









2, AR061: 2, AR260:









2, AR205: 2, AR055:









1, AR234: 1, AR215:









1, AR216: 1









S0428: 2 and S0002: 1.


20
HSRDE35
460538
30
428–778
170
Asn-43 to Arg-49, 
AR060: 88, AR089:
10








Phe-57 to Cys-65,
68, AR104: 53, AR316:








Pro-93 to Ser-99.
49, AR283: 49, AR299:









34, AR096: 29, AR277:









28, AR185: 25, AR282:









23, AR313: 17, AR240:









17, AR266: 9, AR223:









9, AR222: 9, AR215:









9, AR217: 9, AR214:









8, AR224: 8, AR172:









8, AR229: 7, AR169:









7, AR181: 7, AR216:









7, AR235: 7, AR176:









7, AR168: 7, AR170:









7, AR269: 6, AR267:









6, AR183: 6, AR237:









6, AR177: 6, AR233:









6, AR228: 6, AR221:









6, AR061: 5, AR268:









5, AR182: 5, AR239:









5, AR178: 5, AR171:









5, AR270: 5, AR175:









5, AR231: 5, AR225:









5, AR226: 4, AR261:









4, AR291: 4, AR179:









4, AR243: 4, AR289:









4, AR293: 4, AR232:









4, AR238: 4, AR055:









4, AR236: 4, AR227:









4, AR309: 4, AR053:









4, AR180: 4, AR296:









4, AR274: 4, AR257:









4, AR230: 4, AR234:









3, AR250: 3, AR161:









3, AR162: 3, AR174:









3, AR163: 3, AR290:









3, AR300: 3, AR165:









3, AR247: 3, AR166:









3, AR195: 3, AR173:









3, AR271: 3, AR288:









3, AR286: 3, AR264:









3, AR164: 3, AR287:









3, AR275: 3, AR285:









3, AR255: 3, AR294:









2, AR245: 2, AR033:









2, AR297: 2, AR295:









2, AR191: 2, AR262:









2, AR190: 2, AR201:









2, AR203: 2, AR272:









2, AR308: 2, AR312:









2, AR205: 2, AR311:









1, AR212: 1, AR193:









1, AR254: 1, AR189:









1, AR211: 1, AR188: 1









L0748: 5, H0556: 4,









H0046: 3, L0766: 3,









L0666: 3, L0754: 3,









L0757: 3, H0083: 2,









H0622: 2, H0623: 2,









S0344: 2, L0783: 2,









L0789: 2, L0665: 2,









L0439: 2, S0434: 2,









L0599: 2, L0362: 2,









H0583: 1, H0484: 1,









S0420: 1, S0442: 1,









S0444: 1, S0046: 1,









S0132: 1, S0222: 1,









H0370: 1, H0282: 1,









H0587: 1, S0182: 1,









H0318: 1, H0085: 1,









H0596: 1, H0123: 1,









L0471: 1, H0014: 1,









H0373: 1, H0060: 1,









H0188: 1, S0022: 1,









H0031: 1, H0553: 1,









H0617: 1, H0169: 1,









H0135: 1, H0551: 1,









H0059: 1, T0042: 1,









H0494: 1, H0647: 1,









H0529: 1, L0371: 1,









L0770: 1, L0638: 1,









L0639: 1, L5566: 1,









L0761: 1, L0643: 1,









L0662: 1, L0378: 1,









L0661: 1, L0663: 1,









H0144: 1, L0438: 1,









H0522: 1, S0390: 1,









S3014: 1, L0747: 1,









L0786: 1, L0777: 1,









L0780: 1, L0755: 1,









L0731: 1, L0758: 1,









L0759: 1, S0011: 1,









H0665: 1, S0196: 1,









H0542: 1 and H0543: 1.


21
HNFID82
456494
31
161–286
171
Gln-36 to Lys-41.
H0416: 1 and L0748:









1.


22
HOFNY71
1152170
32
 200–1042
172
Phe-3 to Gly-9,
H0415: 5








Ala-80 to Ala-91,








Asp-119 to Arg-








125,








Ala-167 to Asp-








179,








Trp-195 to Gly-








201.



HOFNY71
456517
136
114–221
276



HOFMS34
704682
137
123–968
277
Phe-3 to Gly-9.


23
HPMBR15
456527
33
262–279
173

H0542: 5, H0423: 5,
7









H0031: 3, L0777: 3,









L0752: 3, H0556: 2,









H0413: 2, H0555: 2,









L0744: 2, L0754: 2,









L0779: 2, H0444: 2,









H0402: 1, S0045: 1,









H0575: 1, H0674: 1,









H0509: 1, L0794: 1,









L0774: 1, L0805: 1,









L0659: 1, L0809: 1,









L0789: 1, H0659: 1,









H0648: 1, L0599: 1,









H0543: 1 and H0422: 1.


24
HSAVD46
456536
34
155–328
174

AR176: 4, AR181: 4,









AR178: 3, AR197: 3,









AR269: 3, AR165: 3,









AR221: 3, AR162: 3,









AR164: 3, AR207: 3,









AR272: 3, AR161: 3,









AR182: 3, AR245: 3,









AR175: 3, AR270: 3,









AR268: 2, AR174: 2,









AR267: 2, AR177: 2,









AR253: 2, AR191: 2,









AR104: 2, AR173: 2,









AR190: 2, AR225: 2,









AR282: 2, AR188: 2,









AR210: 2, AR311: 2,









AR183: 2, AR179: 2,









AR204: 2, AR275: 2,









AR166: 2, AR216: 2,









AR212: 2, AR060: 1,









AR271: 1, AR170: 1,









AR240: 1, AR189: 1,









AR096: 1, AR163: 1,









AR180: 1, AR261: 1,









AR283: 1, AR277: 1,









AR247: 1, AR033: 1,









AR195: 1, AR217: 1,









AR089: 1, AR316: 1,









AR312: 1, AR055: 1,









AR233: 1, AR289: 1,









AR199: 1, AR185: 1,









H0170: 1, S0114: 1,









L0769: 1, L0784: 1,









L0805: 1, L0790: 1,









H0435: 1, H0648: 1,









L0779: 1 and L0777: 1.


25
HCNSB01
460699
35
 552–1091
175
Ser-25 to Gly-31,
AR289: 9, AR039: 8,
15q23–q25
118485,








Gln-149 to Ser-155.
AR248: 7, AR249: 6,

151670,









AR286: 6, AR060: 6,

231680,









AR291: 5, AR053: 5,

272800,









AR274: 5, AR273: 5,

272800,









AR052: 5, AR182: 5,

272800,









AR290: 5, AR270: 4,

276700,









AR104: 4, AR296: 4,

600374,









AR238: 4, AR234: 4,

601780









AR240: 4, AR282: 4,









AR198: 4, AR284: 4,









AR183: 3, AR263: 3,









AR061: 3, AR229: 3,









AR205: 3, AR243: 3,









AR186: 3, AR283: 3,









AR267: 3, AR269: 3,









AR312: 3, AR316: 3,









AR275: 3, AR055: 3,









AR231: 3, AR096: 3,









AR033: 3, AR268: 3,









AR185: 2, AR247: 2,









AR233: 2, AR299: 2,









AR277: 2, AR313: 2,









AR206: 2, AR251: 2,









AR192: 2, AR202: 2,









AR213: 2, AR089: 2,









AR293: 2, AR300: 2,









AR253: 2, AR310: 2,









AR259: 2, AR294: 2,









AR256: 2, AR298: 2,









AR292: 2, AR285: 1,









AR295: 1, AR241: 1,









AR258: 1, AR175: 1,









AR232: 1, AR219: 1,









AR271: 1, AR246: 1,









AR226: 1, AR227: 1,









AR237: 1, AR177: 1









L0777: 9, H0556: 8,









H0620: 7, L0766: 7,









H0521: 7, L0747: 6,









S0360: 5, H0250: 5,









L0751: 5, L0794: 4,









L0754: 4, L0591: 4,









S0354: 3, S0358: 3,









S0278: 3, H0553: 3,









S0144: 3, L0769: 3,









L0773: 3, H0547: 3,









S0404: 3, S3014: 3,









L0743: 3, L0744: 3,









L0439: 3, L0752: 3,









L0731: 3, H0445: 3,









S0218: 2, H0580: 2,









S0132: 2, H0393: 2,









H0550: 2, S0222: 2,









H0486: 2, H0635: 2,









H0575: 2, H0590: 2,









H0251: 2, H0309: 2,









H0457: 2, H0150: 2,









H0050: 2, H0083: 2,









H0266: 2, H0288: 2,









H0135: 2, H0264: 2,









T0041: 2, H0561: 2,









L0761: 2, L0771: 2,









L0768: 2, L0649: 2,









L0804: 2, L0651: 2,









L0518: 2, L0789: 2,









L0666: 2, H0144: 2,









L0565: 2, L0438: 2,









S0330: 2, S0037: 2,









S0027: 2, L0740: 2,









L0745: 2, L0758: 2,









S0434: 2, L0581: 2,









L0593: 2, L0595: 2,









H0542: 2, H0543: 2,









T0002: 1, S0040: 1,









H0717: 1, H0657: 1,









H0656: 1, H0341: 1,









H0483: 1, H0255: 1,









H0305: 1, H0589: 1,









S0420: 1, H0676: 1,









H0208: 1, S0045: 1,









S0046: 1, H0370: 1,









H0392: 1, H0592: 1,









H0587: 1, T0040: 1,









S0280: 1, H0156: 1,









H0706: 1, H0318: 1,









H0310: 1, H0231: 1,









H0545: 1, H0009: 1,









H0565: 1, H0123: 1,









H0012: 1, H0047: 1,









H0197: 1, H0024: 1,









H0057: 1, T0010: 1,









H0267: 1, H0252: 1,









H0688: 1, H0617: 1,









S0364: 1, H0124: 1,









S0036: 1, H0040: 1,









H0488: 1, H0412: 1,









H0059: 1, H0100: 1,









H0494: 1, H0560: 1,









S0448: 1, S0440: 1,









H0131: 1, H0130: 1,









H0633: 1, H0647: 1,









S0344: 1, L0770: 1,









L0643: 1, L0645: 1,









L0764: 1, L0521: 1,









L0364: 1, L0381: 1,









L0774: 1, L0775: 1,









L0806: 1, L0653: 1,









L0776: 1, L0659: 1,









L0809: 1, L0663: 1,









L0664: 1, H0699: 1,









S0374: 1, H0435: 1,









H0648: 1, S0152: 1,









S0044: 1, H0134: 1,









S0406: 1, S0032: 1,









L0749: 1, L0756: 1,









L0779: 1, L0759: 1,









S0260: 1, H0665: 1,









H0136: 1, H0423: 1,









S0424: 1 and H0506: 1.


26
HSDGR57
460599
36
195–329
176
Glu-20 to Glu-27,
AR211: 42, AR210:








Glu-30 to Trp-44.
35, AR296: 27, AR175:









25, AR268: 23, AR174:









22, AR192: 22, AR269:









22, AR191: 20, AR104:









19, AR189: 18, AR177:









18, AR190: 17, AR207:









17, AR181: 17, AR033:









17, AR246: 17, AR270:









16, AR196: 16, AR274:









16, AR201: 16, AR173:









16, AR198: 16, AR180:









15, AR178: 15, AR275:









15, AR224: 14, AR179:









14, AR161: 14, AR163:









14, AR236: 14, AR176:









14, AR235: 13, AR162:









13, AR188: 13, AR197:









13, AR285: 12, AR183:









12, AR225: 12, AR295:









12, AR290: 12, AR195:









12, AR242: 12, AR247:









12, AR245: 12, AR288:









11, AR272: 11, AR243:









11, AR261: 11, AR240:









11, AR222: 11, AR291:









10, AR316: 10, AR255:









10, AR257: 10, AR182:









10, AR233: 10, AR287:









10, AR223: 10, AR193:









10, AR297: 10, AR294:









10, AR262: 10, AR204:









10, AR293: 10, AR267:









10, AR205: 10, AR238:









9, AR089: 9, AR258:









9, AR286: 9, AR231:









9, AR199: 9, AR313:









9, AR200: 9, AR039:









9, AR185: 9, AR234:









9, AR239: 8, AR060:









8, AR289: 8, AR263:









8, AR221: 8, AR228:









8, AR309: 8, AR172:









8, AR096: 8, AR165:









8, AR300: 8, AR260:









8, AR203: 8, AR229:









8, AR164: 8, AR266:









7, AR166: 7, AR227:









7, AR226: 7, AR311:









7, AR250: 7, AR237:









7, AR264: 7, AR169:









7, AR230: 7, AR299:









6, AR171: 6, AR271:









6, AR283: 6, AR256:









6, AR213: 6, AR282:









6, AR168: 6, AR232:









6, AR055: 5, AR312:









5, AR214: 5, AR216:









5, AR212: 5, AR215:









5, AR308: 5, AR061:









4, AR217: 4, AR254:









4, AR277: 4, AR253:









4, AR170: 4, AR218:









3, AR219: 3, AR053: 2









L0744: 8, L0731: 4,









L0439: 3, S0360: 2,









L0766: 2, L0809: 2,









L0438: 2, L0747: 2,









L0756: 2, L0759: 2,









S0358: 1, S0222: 1,









H0455: 1, H0486: 1,









S0010: 1, H0024: 1,









S0388: 1, S0051: 1,









H0644: 1, L0770: 1,









L0769: 1, L0662: 1,









L0649: 1, L0774: 1,









L0651: 1, L0776: 1,









L0635: 1, L0783: 1,









L0663: 1, H0520: 1,









H0547: 1, H0539: 1,









H0436: 1, L0749: 1,









L0757: 1, L0758: 1,









S0031: 1, L0599: 1 and









L0608: 1.


27
HJABB94
456466
37
 74–307
177
Ala-28 to His-41,
AR176: 9, AR225: 9,








Pro-43 to Gln-64.
AR221: 8, AR295: 8,









AR170: 8, AR264: 8,









AR178: 8, AR288: 7,









AR291: 7, AR180: 7,









AR215: 7, AR175: 7,









AR275: 7, AR297: 7,









AR224: 7, AR268: 7,









AR293: 7, AR228: 7,









AR269: 6, AR309: 6,









AR282: 6, AR270: 6,









AR263: 6, AR285: 6,









AR296: 6, AR267: 6,









AR162: 6, AR239: 6,









AR173: 6, AR311: 6,









AR161: 5, AR231: 5,









AR266: 5, AR181: 5,









AR207: 5, AR182: 5,









AR163: 5, AR183: 5,









AR053: 5, AR238: 5,









AR289: 5, AR217: 5,









AR213: 5, AR229: 5,









AR274: 5, AR286: 5,









AR177: 5, AR039: 5,









AR290: 5, AR237: 5,









AR287: 5, AR033: 5,









AR226: 5, AR277: 5,









AR294: 5, AR219: 5,









AR196: 5, AR218: 5,









AR212: 4, AR179: 4,









AR235: 4, AR216: 4,









AR283: 4, AR223: 4,









AR233: 4, AR260: 4,









AR211: 4, AR313: 4,









AR316: 4, AR171: 4,









AR104: 4, AR312: 4,









AR096: 4, AR192: 4,









AR256: 4, AR227: 4,









AR261: 4, AR190: 4,









AR230: 4, AR300: 4,









AR240: 4, AR060: 4,









AR174: 4, AR169: 3,









AR198: 3, AR222: 3,









AR191: 3, AR199: 3,









AR210: 3, AR189: 3,









AR271: 3, AR195: 3,









AR232: 3, AR188: 3,









AR055: 3, AR200: 3,









AR172: 3, AR185: 3,









AR089: 3, AR166: 3,









AR234: 3, AR168: 3,









AR165: 3, AR164: 3,









AR258: 3, AR257: 3,









AR299: 3, AR308: 2,









AR201: 2, AR243: 2,









AR193: 2, AR203: 2,









AR061: 2, AR247: 2,









AR255: 2, AR262: 2,









AR272: 2, AR236: 2,









AR242: 1, AR254: 1









H0624: 1, S0360: 1,









H0586: 1, L0021: 1,









T0041: 1 and L0779: 1.


28
HOFAF39
456516
38
 57–152
178

H0415: 2 and H0414: 1.


29
HTPCS60
456565
39
105–878
179
Pro-35 to Cys-43,
AR060: 5, AR089: 5,








Gln-56 to Lys-67,
AR299: 5, AR282: 5,








Thr-73 to Lys-78,
AR240: 5, AR316: 5,








Tyr-93 to Asp-98,
AR104: 5, AR313: 4,








Ser-116 to Gln-125,
AR277: 3, AR096: 3,








Leu-142 to Phe-
AR185: 2, AR283: 2








151,
L0756: 2, H0657: 1,








Phe-169 to Arg-
H0441: 1, H0270: 1,








174,
L0738: 1, H0083: 1,








Ile-181 to Glu-190,
H0039: 1, H0494: 1,








Thr-243 to Gly-
L0637: 1, L0794: 1,








248.
L0650: 1, L0663: 1,









H0144: 1, H0521: 1,









L0779: 1, L0752: 1,









L0758: 1, S0436: 1,









L0608: 1 and S0412: 1.


30
HKIYO61
456471
40
181–294
180

AR204: 5, AR235: 3,









AR217: 3, AR053: 2,









AR169: 2, AR193: 2,









AR226: 2, AR216: 2,









AR205: 2, AR274: 2,









AR089: 2, AR264: 2,









AR221: 2, AR266: 2,









AR308: 2, AR299: 2,









AR282: 2, AR312: 2,









AR309: 2, AR195: 2,









AR291: 2, AR172: 2,









AR201: 2, AR196: 2,









AR181: 2, AR225: 2,









AR104: 2, AR165: 2,









AR229: 2, AR313: 2,









AR271: 2, AR162: 1,









AR164: 1, AR268: 1,









AR238: 1, AR199: 1,









AR257: 1, AR168: 1,









AR163: 1, AR269: 1,









AR288: 1, AR300: 1,









AR297: 1, AR161: 1,









AR060: 1, AR213: 1,









AR173: 1, AR285: 1,









AR272: 1, AR183: 1,









AR191: 1, AR311: 1,









AR290: 1, AR182: 1,









AR174: 1, AR055: 1,









AR096: 1, AR275: 1,









AR316: 1, AR188: 1,









AR283: 1, AR270: 1,









AR267: 1, AR240: 1,









AR185: 1









H0441: 1


31
HMKCW19
740950
41
 529–1560
181
Leu-8 to Gln-18,
AR283: 19, AR240:








Thr-26 to Lys-33,
18, AR089: 16, AR299:








Met-39 to Cys-46,
9, AR282: 9, AR104:








Ala-62 to Pro-69,
7, AR316: 7, AR313:








Pro-83 to Glu-90.
6, AR060: 4, AR185:









3, AR277: 3, AR096: 1









L0439: 7, L0438: 4,









L0596: 4, L0775: 3,









L0754: 3, L0604: 3,









H0392: 2, L0763: 2,









L0776: 2, L0749: 2,









L0361: 2, S0212: 1,









S0354: 1, S0007: 1,









H0619: 1, H0052: 1,









H0309: 1, H0081: 1,









S6028: 1, H0030: 1,









H0553: 1, H0617: 1,









S0422: 1, L0764: 1,









L0774: 1, L0783: 1,









L0545: 1, S0374: 1,









H0519: 1, H0478: 1,









S3012: 1, L0744: 1,









L0750: 1, L0779: 1,









L0755: 1 and L0603: 1.



HMKCW19
456484
138
188–325
278
Met-1 to Pro-7,








Cys-27 to Val-34.


32
HNFIG36
456495
42
171–311
182

AR277: 9, AR060: 8,









AR104: 7, AR240: 6,









AR096: 6, AR316: 6,









AR283: 6, AR299: 6,









AR089: 5, AR313: 5,









AR185: 5, AR282: 5









L0749: 3, L0761: 2,









H0255: 1, H0271: 1,









H0416: 1, S0426: 1 and









L0780: 1.


33
HBIAS26
460709
43
 57–278
183
Gln-36 to Glu-49, 
AR252: 42, AR250:








Glu-51 to Leu-66,
26, AR254: 20, AR263:








Asp-68 to Ser-73.
19, AR165: 19, AR253:









19, AR164: 17, AR166:









17, AR272: 17, AR161:









16, AR162: 16, AR163:









16, AR264: 15, AR246:









15, AR195: 15, AR197:









15, AR089: 14, AR104:









13, AR245: 12, AR283:









12, AR311: 12, AR313:









12, AR240: 11, AR205:









11, AR309: 11, AR308:









11, AR271: 11, AR199:









11, AR201: 10, AR295:









10, AR096: 10, AR247:









10, AR060: 10, AR203:









9, AR297: 9, AR212:









9, AR282: 9, AR055:









9, AR033: 9, AR198:









9, AR242: 9, AR185:









9, AR053: 8, AR239:









8, AR039: 8, AR312:









8, AR289: 8, AR316:









8, AR291: 8, AR243:









8, AR229: 8, AR193:









8, AR200: 8, AR061:









8, AR275: 8, AR288:









8, AR214: 8, AR285:









8, AR216: 8, AR300:









7, AR204: 7, AR296:









7, AR226: 7, AR235:









7, AR286: 7, AR266:









7, AR174: 7, AR261:









7, AR188: 7, AR255:









7, AR237: 6, AR287:









6, AR257: 6, AR181:









6, AR217: 6, AR299:









6, AR192: 6, AR274:









6, AR238: 6, AR180:









6, AR178: 6, AR182:









6, AR177: 6, AR196:









6, AR189: 6, AR293:









6, AR207: 6, AR210:









5, AR231: 5, AR228:









5, AR234: 5, AR236:









5, AR267: 5, AR211:









5, AR213: 5, AR294:









4, AR258: 4, AR232:









4, AR191: 4, AR183:









4, AR290: 4, AR268:









4, AR179: 4, AR171:









4, AR277: 4, AR223:









4, AR230: 4, AR262:









4, AR190: 4, AR218:









3, AR269: 3, AR222:









3, AR175: 3, AR227:









3, AR225: 3, AR168:









3, AR233: 3, AR221:









3, AR224: 2, AR219:









2, AR256: 2, AR215:









2, AR260: 2, AR270:









2, AR176: 1, AR172:









1, AR173: 1









H0584: 6, H0673: 4,









L0764: 4, L0774: 4,









H0295: 3, H0412: 3,









S0438: 3, L0768: 3,









S0378: 3, S0040: 2,









H0125: 2, H0252: 2,









H0708: 2, L0803: 2,









L0775: 2, L0527: 2,









L0783: 2, H0659: 2,









S0380: 2, L0742: 2,









L0779: 2, L0603: 2,









H0624: 1, T0002: 1,









H0167: 1, H0686: 1,









H0638: 1, S0444: 1,









S0360: 1, S0410: 1,









S0222: 1, H0156: 1,









T0048: 1, S0049: 1,









H0081: 1, L0163: 1,









S0388: 1, H0083: 1,









H0594: 1, H0266: 1,









H0188: 1, H0688: 1,









H0031: 1, H0087: 1,









T0041: 1, S0144: 1,









S0142: 1, S0344: 1,









S0422: 1, S0002: 1,









L0500: 1, L0800: 1,









L0767: 1, L0526: 1,









L0809: 1, S0292: 1,









H0684: 1, S0330: 1,









H0696: 1, S0406: 1,









L0748: 1, L0747: 1,









L0750: 1, L0755: 1,









L0758: 1, S0031: 1,









H0595: 1, L0480: 1,









L0608: 1 and H0352: 1.


34
HE2EC79
460671
44
151–243
184
Thr-14 to Thr-19.
L0759: 3, S0026: 3,









H0624: 1, H0170: 1,









S0040: 1, H0384: 1,









H0013: 1, L0794: 1,









H0144: 1 and L0754: 1.


35
HNDAH54
460486
45
 26–121
185

AR313: 37, AR277:









32, AR089: 28, AR299:









25, AR316: 18, AR185:









18, AR240: 12, AR096:









16, AR060: 12, AR282:









9, AR104: 6, AR283: 4









H0517: 1


36
HNHFU59
460498
46
278–505
186
Pro-54 to Gly-62.
AR203: 6, AR176: 3,









AR282: 3, AR217: 2,









AR213: 2, AR309: 2,









AR270: 2, AR214: 2,









AR225: 2, AR089: 2,









AR283: 2, AR266: 2,









AR216: 2, AR240: 1,









AR193: 1, AR224: 1,









AR277: 1, AR055: 1,









AR182: 1, AR275: 1,









AR201: 1, AR161: 1,









AR313: 1, AR096: 1,









AR286: 1, AR171: 1,









AR294: 1









S0053: 1


37
HE8CV18
456447
47
176–472
187

AR263: 4, AR275: 4,









AR104: 4, AR176: 3,









AR163: 3, AR170: 3,









AR213: 3, AR269: 3,









AR171: 2, AR198: 2,









AR217: 2, AR221: 2,









AR267: 2, AR207: 2,









AR245: 2, AR183: 2,









AR180: 2, AR168: 2,









AR177: 2, AR235: 2,









AR161: 2, AR182: 2,









AR274: 2, AR311: 2,









AR268: 2, AR039: 2,









AR266: 2, AR270: 2,









AR162: 2, AR165: 2,









AR309: 2, AR181: 2,









AR174: 2, AR060: 2,









AR239: 2, AR228: 2,









AR203: 2, AR277: 2,









AR164: 2, AR214: 1,









AR089: 1, AR224: 1,









AR272: 1, AR247: 1,









AR190: 1, AR264: 1,









AR233: 1, AR185: 1,









AR189: 1, AR223: 1,









AR178: 1, AR297: 1,









AR291: 1, AR316: 1,









AR282: 1, AR294: 1









H0619: 3, L0770: 2,









L0806: 2, S0116: 1,









H0587: 1, H0013: 1,









L0769: 1, L0796: 1 and









S0330: 1.


38
HFKEB72
456456
48
 43–240
188
Gln-26 to Gln-34.
AR277: 19, AR283:









18, AR282: 17, AR316:









15, AR313: 13, AR104:









13, AR240: 12, AR299:









12, AR096: 11, AR185:









9, AR089: 9, AR060: 8









H0620: 2, H0012: 1,









H0553: 1, L0803: 1 and









L0779: 1.


39
HTDAD22
456553
49
193–522
189
Ile-2 to Lys-9,
S0414: 3, L0748: 3,








Gln-43 to Phe-49,
L0753: 3, H0486: 2,








Asn-59 to His-69,
L0665: 2, H0445: 2,








Gly-87 to Asp-93.
S0376: 1, H0586: 1,









H0042: 1, H0477: 1,









S0422: 1, L0763: 1,









L0637: 1, L0794: 1,









L0766: 1, L0804: 1,









L0775: 1, L0792: 1,









L0666: 1, H0435: 1,









S0330: 1, L0751: 1,









L0777: 1, L0755: 1 and









S0452: 1.


40
HSKYU29
460531
50
290–445
190
Met-1 to Ala-6.
AR169: 6, AR224: 5,









AR060: 5, AR176: 5,









AR089: 5, AR269: 4,









AR175: 4, AR197: 4,









AR216: 4, AR222: 4,









AR264: 4, AR170: 4,









AR161: 4, AR193: 4,









AR252: 4, AR270: 4,









AR163: 4, AR162: 4,









AR313: 4, AR316: 4,









AR168: 4, AR178: 4,









AR274: 4, AR182: 3,









AR172: 3, AR309: 3,









AR177: 3, AR174: 3,









AR268: 3, AR188: 3,









AR104: 3, AR171: 3,









AR173: 3, AR261: 3,









AR195: 3, AR165: 3,









AR183: 3, AR039: 3,









AR311: 3, AR291: 3,









AR191: 3, AR266: 3,









AR181: 3, AR271: 3,









AR164: 3, AR196: 3,









AR267: 3, AR166: 3,









AR308: 3, AR297: 3,









AR217: 2, AR189: 2,









AR290: 2, AR282: 2,









AR240: 2, AR179: 2,









AR263: 2, AR243: 2,









AR283: 2, AR190: 2,









AR289: 2, AR204: 2,









AR288: 2, AR223: 2,









AR312: 2, AR300: 2,









AR250: 2, AR238: 2,









AR275: 2, AR295: 2,









AR237: 2, AR299: 2,









AR255: 2, AR185: 2,









AR096: 1, AR205: 1,









AR221: 1, AR231: 1,









AR246: 1, AR239: 1,









AR232: 1, AR228: 1,









AR247: 1, AR211: 1,









AR296: 1, AR210: 1,









AR287: 1, AR214: 1,









AR218: 1, AR200: 1









L0748: 21, L0439: 10,









L0777: 10, H0521: 8,









H0144: 7, S3014: 7,









S0474: 6, S0045: 5,









H0266: 5, H0623: 5,









L0758: 5, H0341: 4,









S0212: 4, H0305: 4,









H0581: 4, S0214: 4,









H0616: 4, L0803: 4,









L0663: 4, L0665: 4,









S0206: 4, S0242: 4,









S0418: 3, S0358: 3,









S0010: 3, H0620: 3,









S0003: 3, H0615: 3,









H0038: 3, L0774: 3,









L0666: 3, H0518: 3,









S0027: 3, L0754: 3,









L0749: 3, S0194: 3,









S0040: 2, H0402: 2,









S0420: 2, H0586: 2,









H0486: 2, H0635: 2,









S0280: 2, H0050: 2,









H0024: 2, S0250: 2,









H0169: 2, L0455: 2,









H0090: 2, H0494: 2,









S0422: 2, L0766: 2,









L0659: 2, S0152: 2,









H0436: 2, L0744: 2,









L0740: 2, L0755: 2,









L0588: 2, L0361: 2,









S0192: 2, H0542: 2,









H0624: 1, H0170: 1,









H0171: 1, S0114: 1,









S0218: 1, H0650: 1,









H0656: 1, H0669: 1,









S0278: 1, H0369: 1,









S0222: 1, H0357: 1,









H0574: 1, H0632: 1,









T0040: 1, T0109: 1,









H0013: 1, H0069: 1,









H0075: 1, H0156: 1,









L0021: 1, H0042: 1,









H0575: 1, H0590: 1,









L0105: 1, H0421: 1,









H0052: 1, H0546: 1,









H0046: 1, L0471: 1,









H0012: 1, H0057: 1,









H0014: 1, H0051: 1,









S0051: 1, H0078: 1,









H0060: 1, S6028: 1,









H0271: 1, S0334: 1,









H0622: 1, H0031: 1,









L0142: 1, H0634: 1,









H0551: 1, H0561: 1,









H0641: 1, S0210: 1,









L0640: 1, L0763: 1,









L0770: 1, L0646: 1,









L0662: 1, L0776: 1,









L0527: 1, L0526: 1,









L0382: 1, L0791: 1,









S0428: 1, S0374: 1,









H0435: 1, S0328: 1,









H0539: 1, H0525: 1,









H0522: 1, H0704: 1,









S0390: 1, S0028: 1,









L0747: 1, L0756: 1,









L0731: 1, L0757: 1,









S0031: 1, H0343: 1,









L0592: 1, L0608: 1,









L0594: 1, L0366: 1,









S0011: 1, S0196: 1 and









H0543: 1.


41
HHGDU58
460717
51
235–477
191
Ala-45 to Asp-50,
AR060: 6, AR283: 6,
6q–q15
136550,








Arg-57 to Pro-63.
AR104: 5, AR299: 4,

203310,









AR240: 4, AR185: 4,

269920,









AR089: 3, AR316: 3,

602772









AR277: 2, AR096: 2,









AR282: 2, AR313: 1









H0135: 2, H0333: 1









and S0011: 1.


42
HMDAA66
460472
52
106–201
192

AR060: 7, AR277: 7,









AR240: 6, AR299: 6,









AR089: 6, AR104: 5,









AR283: 5, AR185: 4,









AR316: 4, AR313: 2,









AR096: 2, AR282: 2









H0346: 1


43
HNHFW22
460350
53
228–410
193
Met-25 to Gly-30.
AR252: 5, AR241: 4,









AR161: 3, AR163: 3,









AR235: 3, AR282: 3,









AR251: 3, AR180: 3,









AR162: 3, AR186: 3,









AR202: 3, AR253: 3,









AR263: 3, AR176: 3,









AR207: 3, AR221: 2,









AR265: 2, AR249: 2,









AR230: 2, AR061: 2,









AR272: 2, AR215: 2,









AR312: 2, AR169: 2,









AR184: 2, AR236: 2,









AR266: 2, AR309: 2,









AR206: 2, AR172: 2,









AR289: 2, AR214: 2,









AR292: 2, AR269: 2,









AR313: 2, AR277: 2,









AR053: 2, AR310: 2,









AR165: 2, AR262: 2,









AR166: 2, AR164: 2,









AR096: 1, AR264: 1,









AR195: 1, AR089: 1,









AR217: 1, AR178: 1,









AR247: 1, AR229: 1,









AR270: 1, AR205: 1,









AR283: 1, AR293: 1,









AR228: 1, AR224: 1,









AR191: 1, AR267: 1,









AR055: 1, AR258: 1,









AR284: 1, AR181: 1,









AR240: 1, AR199: 1,









AR299: 1, AR060: 1,









AR257: 1, AR287: 1,









AR300: 1, AR052: 1,









AR233: 1, AR274: 1,









AR225: 1, AR271: 1,









AR227: 1, AR286: 1,









AR294: 1









H0063: 1 and S0053: 1.


44
HSQFP66
460537
54
 96–332
194
Ser-6 to Arg-15.
AR197: 9, AR271: 8,









AR176: 7, AR162: 7,









AR161: 7, AR201: 7,









AR163: 7, AR192: 6,









AR204: 6, AR207: 6,









AR266: 6, AR267: 6,









AR228: 6, AR229: 6,









AR169: 6, AR177: 6,









AR237: 6, AR198: 6,









AR233: 5, AR245: 5,









AR181: 5, AR193: 5,









AR250: 5, AR243: 5,









AR053: 5, AR269: 5,









AR239: 5, AR309: 5,









AR089: 5, AR180: 5,









AR264: 5, AR165: 5,









AR214: 5, AR182: 4,









AR060: 4, AR224: 4,









AR061: 4, AR268: 4,









AR261: 4, AR178: 4,









AR166: 4, AR230: 4,









AR257: 4, AR226: 4,









AR183: 4, AR164: 4,









AR270: 4, AR275: 4,









AR231: 4, AR236: 4,









AR096: 4, AR179: 4,









AR246: 4, AR289: 4,









AR039: 4, AR055: 4,









AR293: 4, AR196: 4,









AR175: 4, AR316: 4,









AR272: 4, AR234: 4,









AR168: 4, AR225: 4,









AR286: 4, AR247: 4,









AR312: 4, AR212: 4,









AR255: 4, AR296: 4,









AR242: 4, AR294: 3,









AR300: 3, AR290: 3,









AR185: 3, AR205: 3,









AR291: 3, AR238: 3,









AR262: 3, AR227: 3,









AR295: 3, AR287: 3,









AR288: 3, AR174: 3,









AR297: 3, AR216: 3,









AR311: 3, AR277: 3,









AR170: 3, AR191: 3,









AR285: 3, AR188: 3,









AR213: 3, AR215: 3,









AR313: 3, AR217: 3,









AR308: 3, AR232: 3,









AR203: 3, AR195: 3,









AR282: 3, AR173: 2,









AR033: 2, AR172: 2,









AR189: 2, AR171: 2,









AR274: 2, AR223: 2,









AR190: 2, AR299: 2,









AR104: 2, AR211: 2,









AR258: 2, AR200: 2,









AR283: 2, AR263: 2,









AR256: 2, AR221: 2,









AR199: 2, AR240: 2,









AR222: 2, AR210: 2,









AR253: 1, AR254: 1,









AR260: 1, AR219: 1,









AR218: 1









S0007: 1, H0555: 1 and









S0026: 1.


45
HNGEV29
442573
55
173–391
195
Lys-9 to Leu-16, 
AR299: 5, AR089: 5, 








Ser-33 to Met-43.
AR313: 5, AR104: 4,









AR283: 4, AR185: 3,









AR060: 3, AR316: 3,









AR282: 2, AR277: 2, 









AR096: 1









S0052: 1



HNGEV29
456498
139
 79–111
279
Met-1 to Asn-6.


46
HUKBH05
457195
56
151–327
196

AR052: 25, AR248:









25, AR253: 20, AR270:









19, AR312: 17, AR309:









17, AR269: 16, AR213:









16, AR265: 16, AR310:









15, AR268: 15, AR053:









15, AR251: 14, AR244:









14, AR291: 13, AR202:









12, AR290: 12, AR274:









12, AR186: 12, AR219:









12, AR218: 11, AR249:









11, AR246: 11, AR247:









11, AR275: 11, AR313:









11, AR241: 11 AR271:









11, AR184: 11, AR033:









11, AR182: 11, AR273:









11, AR204: 10, AR267:









10, AR055: 9, AR192:









9, AR298: 9, AR263:









9, AR266: 9, AR183:









9, AR285: 9, AR198:









9, AR243: 8, AR289:









8, AR185: 8, AR194:









8, AR061: 8, AR286:









8, AR284: 8, AR206:









7, AR177: 7, AR300:









7, AR294: 7, AR295:









7, AR296: 7, AR292:









7, AR234: 7, AR293:









6, AR229: 6, AR238:









6, AR280: 6, AR205:









6, AR315: 6, AR299:









6, AR231: 6, AR096:









6, AR282: 6, AR283:









6, AR314: 6, AR175:









6, AR240: 5, AR316:









5, AR039: 5, AR277:









5, AR104: 5, AR226:









4, AR237: 4, AR258:









4, AR233: 4, AR060:









4, AR256: 4, AR089:









4, AR179: 4, AR259:









3, AR232: 3, AR227:









3, AR281: 2









H0521: 7, H0052: 5,









S0001: 3, H0036: 2,









H0271: 2, H0551: 2,









H0522: 2, H0543: 2,









H0265: 1, H0556: 1,









T0049: 1, S0354: 1,









H0370: 1, H0392: 1,









H0581: 1, H0063: 1,









H0059: 1, H0494: 1,









H0561: 1, H0520: 1,









S0436: 1, L0595: 1,









H0506: 1 and L0600: 1.


47
HATDL27
460380
57
 82–177
197

AR225: 8, AR176: 6,









AR162: 6, AR161: 6,









AR163: 6, AR309: 5,









AR228: 5, AR169: 5,









AR266: 5, AR181: 5,









AR229: 5, AR224: 5,









AR233: 5, AR275: 4,









AR239: 4, AR182: 4,









AR261: 4, AR197: 4,









AR177: 4, AR272: 4,









AR269: 4, AR061: 4,









AR226: 4, AR270: 4,









AR178: 4, AR268: 4,









AR282: 4, AR274: 4,









AR255: 4, AR165: 4,









AR164: 4, AR235: 4,









AR183: 3, AR257: 3,









AR312: 3, AR166: 3,









AR267: 3, AR236: 3,









AR289: 3, AR238: 3,









AR293: 3, AR300: 3,









AR286: 3, AR096: 3,









AR215: 3, AR291: 3,









AR237: 3, AR264: 3,









AR171: 3, AR231: 3,









AR175: 3, AR055: 3,









AR179: 3, AR203: 3,









AR247: 3, AR311: 3,









AR173: 3, AR263: 3,









AR214: 2, AR222: 2,









AR230: 2, AR196: 2,









AR227: 2, AR191: 2,









AR170: 2, AR285: 2,









AR287: 2, AR290: 2,









AR308: 2, AR288: 2,









AR193: 2, AR174: 2,









AR262: 2, AR277: 2,









AR294: 2, AR201: 2,









AR200: 2, AR232: 2,









AR212: 2, AR190: 2,









AR188: 2, AR234: 2,









AR299: 2, AR185: 2,









AR204: 2, AR089: 2,









AR313: 2, AR283: 2,









AR104: 2, AR033: 2,









AR172: 2, AR296: 2,









AR216: 1, AR240: 1,









AR218: 1, AR316: 1,









AR189: 1, AR213: 1,









AR060: 1, AR297: 1,









AR258: 1, AR195: 1,









AR199: 1, AR295: 1









H0156: 1


48
HBJFU48
460392
58
 20–142
198

AR216: 26, AR214:









16, AR217: 14, AR313:









11, AR222: 10, AR245:









10, AR264: 10, AR215:









10, AR224: 10, AR161:









10, AR223: 9, AR163:









9, AR263: 9, AR096:









9, AR225: 9, AR162:









9, AR252: 9, AR089:









8, AR195: 8, AR172:









8, AR197: 8, AR221:









8, AR274: 7, AR201:









7, AR240: 7, AR297:









7, AR247: 7, AR170:









6, AR218: 6, AR282:









6, AR168: 6, AR285:









6, AR196: 6, AR166:









6, AR171: 6, AR272:









6, AR165: 6, AR261:









6, AR246: 6, AR250:









6, AR312: 6, AR183:









6, AR178: 6, AR316:









6, AR164: 6, AR193:









6, AR174: 5, AR169:









5, AR311: 5, AR180:









5, AR299: 5, AR309:









5, AR262: 5, AR200:









5, AR236: 5, AR173:









5, AR176: 5, AR179:









5, AR300: 5, AR295:









5, AR175: 5, AR104:









5, AR210: 5, AR185:









5, AR181: 5, AR291:









5, AR212: 5, AR258:









5, AR188: 5, AR053:









5, AR060: 5, AR213:









5, AR293: 5, AR308:









5, AR191: 4, AR271:









4, AR205: 4, AR257:









4, AR229: 4, AR288:









4, AR199: 4, AR192:









4, AR177: 4, AR269:









4, AR270: 4, AR219:









4, AR287: 4, AR189:









4, AR268: 4, AR182:









4, AR296: 4, AR254:









4, AR266: 4, AR243:









4, AR294: 4, AR275:









4, AR203: 4, AR286:









4, AR253: 3, AR033:









3, AR255: 3, AR290:









3, AR211: 3, AR283:









3, AR260: 3, AR277:









3, AR267: 3, AR233:









3, AR039: 3, AR198:









3, AR231: 3, AR238:









3, AR234: 3, AR190:









3, AR237: 3, AR289:









3, AR226: 3, AR204:









2, AR230: 2, AR256:









2, AR235: 2, AR239:









2, AR055: 2, AR228:









2, AR227: 2, AR232:









1, AR061: 1, AR242: 1









H0255: 2, H0318: 2,









H0341: 1, L0519: 1,









S0053: 1 and L0748: 1.


49
HCUBN59
461443
59
 96–227
199

AR277: 23, AR313:









18, AR089: 15, AR299:









14, AR240: 10, AR316:









10, AR185: 10, AR096:









9, AR060: 6, AR104:









3, AR282: 3









H0306: 1 and L0769: 1.


50
HCUDB38
460783
60
221–346
200

AR245: 5, AR237: 4,









AR227: 3, AR221: 3,









AR205: 3, AR198: 3,









AR171: 3, AR225: 2,









AR232: 2, AR180: 2,









AR207: 2, AR282: 2,









AR311: 2, AR168: 2,









AR269: 2, AR277: 2,









AR182: 2, AR270: 2,









AR287: 1, AR297: 1,









AR226: 1, AR201: 1,









AR061: 1, AR257: 1,









AR294: 1, AR291: 1,









AR193: 1









H0402: 1


51
HDPCO25
460682
61
182–343
201
Pro-22 to His-33,
AR282: 3, AR060: 2,








Ser-42 to Trp-48.
AR316: 1









H0521: 2, H0445: 2,









H0394: 1, H0559: 1,









H0581: 1, L0761: 1,









S0052: 1 and L0750: 1.


52
HDPHI51
460679
62
245–367
202
Gly-2 to Glu-7,
AR195: 9, AR192: 9,








Arg-27 to Gly-34.
AR207: 9, AR215: 8,









AR264: 8, AR225: 7,









AR263: 7, AR311: 7,









AR168: 7, AR309: 7,









AR252: 6, AR172: 6,









AR245: 6, AR161: 6,









AR162: 6, AR163: 6,









AR196: 6, AR223: 6,









AR193: 6, AR177: 6,









AR246: 6, AR224: 6,









AR197: 5, AR308: 5,









AR272: 5, AR214: 5,









AR275: 5, AR222: 5,









AR253: 5, AR176: 5,









AR261: 5, AR295: 5,









AR291: 5, AR171: 5,









AR218: 5, AR221: 5,









AR219: 5, AR188: 5,









AR165: 5, AR096: 5,









AR217: 5, AR238: 5,









AR288: 5, AR164: 5,









AR175: 5, AR166: 5,









AR089: 5, AR271: 5,









AR060: 4, AR240: 4,









AR183: 4, AR201: 4,









AR257: 4, AR169: 4,









AR312: 4, AR316: 4,









AR039: 4, AR274: 4,









AR190: 4, AR191: 4,









AR181: 4, AR178: 4,









AR236: 4, AR216: 4,









AR180: 4, AR205: 4,









AR210: 4, AR270: 4,









AR170: 4, AR277: 4,









AR243: 4, AR235: 4,









AR212: 4, AR104: 4,









AR199: 4, AR189: 4,









AR242: 4, AR213: 4,









AR255: 4, AR289: 4,









AR174: 3, AR285: 3,









AR230: 3, AR286: 3,









AR297: 3, AR299: 3,









AR283: 3, AR313: 3,









AR204: 3, AR287: 3,









AR173: 3, AR247: 3,









AR229: 3, AR269: 3,









AR296: 3, AR182: 3,









AR293: 3, AR266: 3,









AR258: 3, AR198: 3,









AR237: 3, AR262: 3,









AR033: 3, AR239: 3,









AR185: 3, AR231: 3,









AR203: 3, AR200: 3,









AR179: 3, AR211: 3,









AR227: 3, AR268: 3,









AR267: 3, AR294: 3,









AR290: 3, AR234: 3,









AR232: 3, AR226: 3,









AR300: 2, AR250: 2,









AR282: 2, AR256: 2,









AR061: 2, AR053: 2,









AR233: 2, AR260: 2,









AR228: 2, AR055: 2









H0521: 1


53
HSDAG05
460523
63
419–646
203

AR296: 49, AR294:









42, AR252: 36, AR237:









36, AR297: 34, AR287:









33, AR283: 33, AR193:









33, AR033: 32, AR176:









31, AR289: 31, AR293:









31, AR178: 31, AR266:









31, AR233: 30, AR254:









30, AR257: 30, AR185:









30, AR227: 30, AR286:









30, AR230: 29, AR243:









28, AR182: 28, AR229:









28, AR228: 28, AR291:









27, AR313: 27, AR181:









27, AR196: 27, AR174:









26, AR300: 25, AR234:









25, AR261: 25, AR235:









25, AR180: 25, AR231:









25, AR096: 24, AR200:









24, AR203: 24, AR232:









23, AR260: 23, AR061:









23, AR104: 23, AR177:









23, AR255: 23, AR188:









23, AR204: 23, AR191:









22, AR164: 22, AR165:









22, AR179: 22, AR060:









22, AR262: 21, AR239:









21, AR089: 21, AR285:









21, AR312: 21, AR039:









21, AR166: 21, AR238:









21, AR162: 21, AR316:









21, AR161: 21, AR195:









20, AR295: 20, AR197:









20, AR163: 20, AR253:









20, AR258: 20, AR226:









20, AR198: 20, AR236:









19, AR053: 19, AR288:









19, AR246: 19, AR175:









19, AR207: 19, AR308:









19, AR275: 19, AR264:









18, AR256: 18, AR190:









18, AR213: 18, AR267:









18, AR055: 18, AR269:









18, AR183: 18, AR189:









18, AR242: 18, AR250:









17, AR299: 17, AR270:









17, AR282: 17, AR247:









17, AR199: 16, AR272:









16, AR212: 16, AR201:









16, AR240: 15, AR290:









15, AR173: 15, AR309:









15, AR268: 14, AR271:









14, AR311: 14, AR245:









14, AR205: 13, AR263:









13, AR210: 12, AR192:









12, AR277: 12, AR274:









12, AR218: 12, AR219:









11, AR225: 11, AR211:









10, AR170: 10, AR171:









8, AR217: 8, AR169:









7, AR224: 7, AR223:









7, AR172: 7, AR168:









7, AR216: 6, AR222:









6, AR214: 5, AR221:









5, AR215: 4









L0777: 6, L0761: 3,









L0662: 3, L0794: 3,









L0748: 3, H0661: 2,









H0486: 2, L0766: 2,









L0803: 2, L0805: 2,









L0754: 2, L0731: 2,









H0624: 1, S0134: 1,









S0376: 1, S0360: 1,









L0623: 1, S0010: 1,









H0581: 1, H0263: 1,









S0003: 1, L0055: 1,









H0412: 1, H0413: 1,









H0633: 1, S0422: 1,









L0667: 1, L0773: 1,









L0776: 1, L0527: 1,









L0809: 1, L0789: 1,









L0666: 1, L0663: 1,









H0593: 1, H0690: 1,









H0659: 1, H0670: 1,









H0648: 1, H0539: 1,









H0522: 1, H0134: 1,









S0392: 1, H0478: 1,









L0740: 1, L0747: 1,









L0749: 1, L0756: 1,









L0779: 1, L0752: 1,









L0755: 1, L0758: 1,









S0031: 1, S0434: 1 and









S0276: 1.


54
HMWGY01
672222
64
423–734
204
Glu-22 to Ser-33,
H0341: 1








Leu-47 to Ser-55,








Thr-87 to Arg-104.



HMWGY01
456490
140
 17–136
280


55
HSVAQ28
460592
65
149–442
205
Ser-39 to Pro-44.
AR089: 11, AR277:









10, AR240: 10, AR313:









10, AR060: 10, AR299:









9, AR316: 8, AR282:









8, AR185: 7, AR104:









6, AR096: 6, AR283: 6









H0309: 2


56
HGLAM53
615771
66
359–580
206
Ser-32 to Glu-37.
AR163: 8, AR161: 8,









AR162: 4, AR168: 3,









AR214: 3, AR216: 3,









AR311: 3, AR207: 3,









AR192: 3, AR253: 3,









AR171: 3, AR282: 3,









AR195: 2, AR224: 2,









AR180: 2, AR096: 2,









AR274: 2, AR240: 2,









AR225: 2, AR172: 2,









AR089: 2, AR275: 2,









AR205: 2, AR053: 1,









AR283: 1, AR235: 1,









AR197: 1, AR272: 1,









AR261: 1, AR178: 1,









AR199: 1, AR313: 1,









AR296: 1, AR166: 1,









AR312: 1, AR222: 1









L0438: 6, L0439: 6,









L0776: 5, H0428: 4,









L0805: 4, H0687: 3,









L0770: 3, H0651: 3,









L0789: 2, L0756: 2,









L0758: 2, L0361: 2,









S0212: 1, S0001: 1,









H0351: 1, H0052: 1,









H0327: 1, H0178: 1,









L0769: 1, L0767: 1,









L0794: 1, L0807: 1,









L0759: 1 and L0608: 1.



HGLAM53
456461
141
369–572
281
Ser-29 to Glu-34.


57
HLTAI94
456476
67
 47–181
207

AR183: 2, AR242: 2,









AR245: 2, AR252: 2,









AR282: 2, AR222: 2,









AR198: 2, AR309: 2,









AR288: 2, AR096: 1,









AR205: 1, AR168: 1,









AR212: 1, AR195: 1,









AR163: 1, AR295: 1,









AR161: 1, AR299: 1,









AR192: 1









L0766: 3, L0803: 2,









L0666: 2, H0341: 1,









H0090: 1, L0774: 1,









L0805: 1, L0783: 1,









L0528: 1, H0660: 1,









L0740: 1, L0777: 1 and









H0422: 1.


58
HMKAH10
456609
68
 48–212
208

AR263: 10, AR170: 9,









AR235: 9, AR053: 8,









AR224: 8, AR223: 8,









AR264: 8, AR225: 8,









AR169: 8, AR221: 7,









AR168: 7, AR214: 7,









AR217: 7, AR171: 7,









AR215: 6, AR162: 6,









AR161: 6, AR163: 6,









AR165: 6, AR172: 6,









AR164: 6, AR212: 6,









AR309: 6, AR166: 6,









AR222: 6, AR216: 6,









AR311: 6, AR308: 5,









AR261: 5, AR288: 5,









AR295: 5, AR213: 5,









AR312: 5, AR240: 5,









AR196: 4, AR297: 4,









AR275: 4, AR180: 4,









AR291: 4, AR177: 4,









AR313: 4, AR089: 4,









AR277: 4, AR236: 4,









AR181: 4, AR096: 4,









AR285: 4, AR316: 4,









AR286: 3, AR282: 3,









AR174: 3, AR257: 3,









AR258: 3, AR239: 3,









AR033: 3, AR178: 3,









AR060: 3, AR104: 3,









AR176: 3, AR262: 3,









AR299: 3, AR055: 3,









AR191: 3, AR211: 3,









AR185: 3, AR199: 3,









AR283: 3, AR287: 3,









AR175: 3, AR270: 3,









AR188: 3, AR269: 3,









AR234: 3, AR238: 3,









AR200: 3, AR272: 3,









AR173: 3, AR203: 3,









AR255: 3, AR226: 3,









AR219: 3, AR230: 3,









AR210: 2, AR247: 2,









AR293: 2, AR189: 2,









AR061: 2, AR289: 2,









AR232: 2, AR268: 2,









AR300: 2, AR182: 2,









AR229: 2, AR233: 2,









AR190: 2, AR294: 2,









AR231: 2, AR274: 2,









AR237: 2, AR290: 2,









AR256: 2, AR246: 2,









AR227: 2, AR228: 2,









AR296: 2, AR260: 2,









AR267: 2, AR183: 2,









AR179: 2, AR218: 1,









AR193: 1, AR243: 1









H0392: 2, H0052: 1,









H0672: 1, L0779: 1 and









L0758: 1.


59
HSVBH58
757227
69
249–419
209
Pro-10 to Ser-20.
AR282: 5, AR235: 4,









AR221: 3, AR192: 3,









AR195: 3, AR266: 3,









AR242: 3, AR253: 3,









AR191: 2, AR168: 2,









AR217: 2, AR297: 2,









AR264: 2, AR182: 2,









AR246: 2, AR172: 2,









AR283: 2, AR171: 1,









AR193: 1, AR277: 1,









AR289: 1, AR291: 1,









AR257: 1, AR300: 1,









AR055: 1, AR275: 1,









AR286: 1, AR163: 1,









AR216: 1, AR270: 1









H0309: 1



HSVBH58
456549
142
168–236
282


60
HBMWB01
460396
70
 48–236
210
Thr-45 to Tyr-50.
AR277: 144, AR313:









138, AR089: 121,









AR299: 99, AR185:









84, AR240: 79, AR316:









79, AR096: 57, AR060:









53, AR282: 25, AR104:









24, AR283: 12









S0116: 1


61
HBMXP84
460397
71
161–280
211

AR313: 74, AR104:
14q22.1–q24.3
104311,









66, AR185: 56, AR282:

109150,









53, AR299: 52, AR316:

112262,









52, AR096: 51, AR283:

182600,









45, AR277: 36, AR089:

182870,









26, AR060: 22, AR240:

182870,









19

182870,









L0803: 4, L0664: 4,

245200,









L0794: 3, L0804: 3,

600225,









L0665: 3, L0747: 3,

600225,









L0758: 3, L0759: 3,

601208









L0770: 2, L0768: 2,









L0659: 2, L0748: 2,









L0439: 2, L0749: 2,









L0750: 2, L0779: 2,









L0777: 2, H0170: 1,









S0116: 1, H0645: 1,









L0717: 1, H0497: 1,









H0108: 1, L0369: 1,









L0769: 1, L0637: 1,









L0764: 1, L0363: 1,









L0766: 1, L0649: 1,









L0774: 1, L0805: 1,









L0789: 1, L0663: 1,









L0565: 1, H0670: 1,









L0741: 1, L0740: 1,









L0751: 1, L0752: 1,









L0596: 1 and H0543: 1.


62
HLYAF80
460622
72
222–365
212

AR169: 9, AR263: 9,









AR221: 8, AR253: 7,









AR207: 7, AR171: 7,









AR217: 7, AR168: 7,









AR224: 7, AR309: 7,









AR223: 6, AR235: 6,









AR242: 6, AR225: 6,









AR215: 6, AR311: 6,









AR172: 6, AR282: 6,









AR053: 6, AR192: 6,









AR245: 5, AR264: 5,









AR216: 5, AR170: 5,









AR165: 5, AR214: 5,









AR164: 5, AR195: 5,









AR308: 5, AR212: 5,









AR166: 5, AR198: 5,









AR261: 5, AR089: 5,









AR252: 5, AR222: 5,









AR213: 5, AR161: 5,









AR162: 5, AR163: 4,









AR246: 4, AR274: 4,









AR275: 4, AR277: 4,









AR240: 4, AR312: 4,









AR205: 4, AR286: 4,









AR096: 4, AR283: 4,









AR193: 4, AR316: 4,









AR196: 4, AR060: 4,









AR199: 3, AR272: 3,









AR295: 3, AR288: 3,









AR176: 3, AR177: 3,









AR313: 3, AR181: 3,









AR033: 3, AR185: 3,









AR200: 3, AR271: 3,









AR297: 3, AR175: 3,









AR289: 3, AR236: 3,









AR300: 3, AR291: 3,









AR270: 3, AR296: 3,









AR254: 3, AR247: 3,









AR285: 3, AR201: 3,









AR257: 3, AR203: 3,









AR104: 3, AR299: 3,









AR269: 3, AR262: 3,









AR174: 2, AR180: 2,









AR197: 2, AR173: 2,









AR238: 2, AR287: 2,









AR190: 2, AR255: 2,









AR268: 2, AR039: 2,









AR294: 2, AR189: 2,









AR239: 2, AR293: 2,









AR231: 2, AR188: 2,









AR234: 2, AR055: 2,









AR191: 2, AR290: 2,









AR267: 2, AR226: 2,









AR266: 2, AR183: 2,









AR232: 2, AR237: 2,









AR230: 2, AR233: 2,









AR227: 2, AR178: 2,









AR228: 2, AR061: 2,









AR210: 1, AR219: 1,









AR211: 1, AR182: 1,









AR256: 1, AR218: 1,









AR258: 1, AR179: 1,









AR243: 1, AR260: 1,









AR229: 1









H0445: 1


63
HNFDS53
460489
73
200–271
213

AR277: 43, AR316:









36, AR283: 33, AR282:









31, AR104: 30, AR089:









30, AR313: 28, AR096:









25, AR240: 24, AR060:









22, AR299: 21, AR185:









20









L0439: 10, L0758: 4,









L0438: 3, L0751: 3,









L0593: 3, L0748: 2,









S0222: 1, H0271: 1,









S0438: 1, L0769: 1,









L0796: 1, L0809: 1 and









S0052: 1.


64
HNGAX58
460488
74
100–240
214
Lys-33 to Lys-41.
AR254: 7, AR252: 5,









AR215: 5, AR204: 5,









AR176: 4, AR104: 4,









AR282: 4, AR161: 4,









AR313: 4, AR242: 4,









AR162: 4, AR163: 4,









AR096: 4, AR207: 4,









AR274: 4, AR269: 3,









AR165: 3, AR283: 3,









AR164: 3, AR089: 3,









AR296: 3, AR192: 3,









AR166: 3, AR257: 3,









AR300: 3, AR291: 3,









AR289: 3, AR245: 3,









AR264: 3, AR212: 3,









AR271: 3, AR178: 3,









AR183: 3, AR181: 3,









AR293: 3, AR294: 3,









AR308: 3, AR311: 3,









AR299: 3, AR033: 2,









AR263: 2, AR185: 2,









AR243: 2, AR060: 2,









AR213: 2, AR312: 2,









AR039: 2, AR230: 2,









AR295: 2, AR287: 2,









AR286: 2, AR297: 2,









AR173: 2, AR195: 2,









AR224: 2, AR175: 2,









AR316: 2, AR261: 2,









AR174: 2, AR233: 2,









AR188: 2, AR172: 2,









AR237: 2, AR227: 2,









AR201: 2, AR221: 2,









AR229: 2, AR193: 2,









AR203: 2, AR239: 2,









AR223: 2, AR275: 2,









AR232: 2, AR055: 2,









AR234: 2, AR290: 2,









AR179: 2, AR246: 2,









AR228: 2, AR258: 2,









AR247: 2, AR240: 2,









AR225: 2, AR177: 2,









AR210: 2, AR182: 1,









AR205: 1, AR272: 1,









AR061: 1, AR168: 1,









AR189: 1, AR270: 1,









AR288: 1, AR267: 1,









AR190: 1, AR231: 1,









AR277: 1, AR226: 1,









AR285: 1, AR214: 1,









AR256: 1, AR200: 1,









AR191: 1









S0052: 1


65
HOECO90
460505
75
299–415
215
Gly-33 to Lys-38.
AR104: 14, AR299:









12, AR060: 11, AR313:









10, AR089: 9, AR282:









9, AR283: 9, AR096:









9, AR316: 9, AR185:









8, AR240: 6, AR277: 2









L0731: 4, L0771: 3,









L0794: 2, S0126: 2,









L0777: 2, H0341: 1,









S0360: 1, S0300: 1,









H0411: 1, S0222: 1,









H0646: 1, S0422: 1,









L0766: 1, L0803: 1,









L0775: 1, L0526: 1,









L0666: 1, L0663: 1 and









L0362: 1.


66
HSVAY16
460595
76
 52–147
216
Gly-24 to Lys-31.
AR238: 10, AR252: 9,
13









AR191: 8, AR189: 7,









AR176: 7, AR240: 7,









AR190: 7, AR178: 7,









AR173: 7, AR188: 7,









AR245: 6, AR162: 6,









AR161: 6, AR165: 6,









AR174: 6, AR269: 6,









AR163: 6, AR183: 6,









AR181: 6, AR164: 6,









AR236: 6, AR180: 6,









AR290: 6, AR175: 6,









AR166: 5, AR089: 5,









AR286: 5, AR182: 5,









AR258: 5, AR270: 5,









AR179: 5, AR255: 5,









AR033: 5, AR207: 5,









AR246: 5, AR229: 5,









AR201: 5, AR197: 5,









AR177: 5, AR288: 5,









AR291: 5, AR225: 5,









AR257: 5, AR170: 5,









AR231: 4, AR299: 4,









AR268: 4, AR196: 4,









AR266: 4, AR212: 4,









AR172: 4, AR262: 4,









AR233: 4, AR060: 4,









AR247: 4, AR313: 4,









AR300: 4, AR261: 4,









AR053: 4, AR316: 4,









AR282: 4, AR104: 4,









AR312: 4, AR204: 4,









AR295: 4, AR199: 4,









AR237: 4, AR285: 4,









AR294: 4, AR215: 4,









AR228: 4, AR275: 4,









AR234: 4, AR214: 4,









AR223: 3, AR200: 3,









AR309: 3, AR293: 3,









AR198: 3, AR267: 3,









AR296: 3, AR193: 3,









AR185: 3, AR055: 3,









AR297: 3, AR096: 3,









AR210: 3, AR271: 3,









AR203: 3, AR274: 3,









AR235: 3, AR169: 3,









AR260: 3, AR226: 3,









AR205: 3, AR039: 3,









AR289: 3, AR311: 3,









AR287: 3, AR242: 3,









AR195: 3, AR239: 3,









AR277: 3, AR230: 3,









AR283: 3, AR272: 3,









AR216: 3, AR308: 2,









AR250: 2, AR061: 2,









AR213: 2, AR263: 2,









AR243: 2, AR264: 2,









AR224: 2, AR232: 2,









AR218: 2, AR222: 2,









AR227: 2, AR171: 2,









AR211: 2, AR256: 2,









AR168: 1, AR192: 1,









AR219: 1









L0157: 3, L0439: 3,









H0441: 2, H0574: 2,









L0105: 2, T0010: 2,









L0759: 2, S0001: 1,









H0664: 1, H0411: 1,









H0599: 1, S0010: 1,









H0309: 1, L0630: 1,









L0775: 1, L0805: 1,









L0809: 1, L0742: 1 and









H0543: 1.


67
HCFCE10
456437
77
198–335
217

AR161: 6, AR162: 6,









AR163: 6, AR263: 6,









AR269: 5, AR180: 5,









AR264: 5, AR282: 4,









AR175: 4, AR299: 4,









AR311: 4, AR096: 4,









AR226: 4, AR089: 4,









AR170: 4, AR313: 4,









AR178: 4, AR183: 4,









AR238: 4, AR225: 4,









AR274: 4, AR271: 3,









AR291: 3, AR165: 3,









AR211: 3, AR173: 3,









AR164: 3, AR272: 3,









AR240: 3, AR200: 3,









AR213: 3, AR199: 3,









AR235: 3, AR168: 3,









AR189: 3, AR261: 3,









AR308: 3, AR275: 3,









AR188: 3, AR290: 3,









AR169: 3, AR174: 3,









AR297: 3, AR060: 3,









AR182: 3, AR214: 3,









AR191: 3, AR177: 3,









AR181: 3, AR221: 3,









AR287: 3, AR288: 2,









AR257: 2, AR224: 2,









AR179: 2, AR229: 2,









AR300: 2, AR171: 2,









AR196: 2, AR316: 2,









AR277: 2, AR237: 2,









AR262: 2, AR247: 2,









AR166: 2, AR104: 2,









AR268: 2, AR296: 2,









AR176: 2, AR234: 2,









AR267: 2, AR190: 2,









AR295: 2, AR033: 2,









AR232: 2, AR223: 2,









AR270: 2, AR239: 2,









AR172: 2, AR236: 2,









AR233: 2, AR218: 2,









AR293: 2, AR289: 2,









AR231: 2, AR228: 2,









AR285: 2, AR210: 2,









AR309: 2, AR217: 2,









AR294: 2, AR258: 2,









AR185: 2, AR227: 2,









AR312: 2, AR195: 2,









AR222: 1, AR219: 1,









AR230: 1, AR286: 1,









AR198: 1, AR255: 1,









AR203: 1, AR201: 1









H0422: 1


68
HCFNC26
456439
78
 85–159
218
Ile-14 to Thr-24.
H0423: 1


69
HELDZ32
456450
79
148–216
219

AR104: 9, AR299: 7,









AR060: 5, AR313: 4,









AR185: 3, AR283: 3,









AR089: 3, AR316: 3,









AR277: 3, AR282: 2,









AR240: 1









L0750: 5, S0045: 2,









S0212: 1, S0300: 1,









S0010: 1, H0505: 1,









S0049: 1, H0266: 1,









L0598: 1, L0662: 1,









L0809: 1, S0374: 1,









H0696: 1 and L0758: 1.


70
HODAZ26
456514
80
133–225
220

L0747: 4, L0803: 2,









L0608: 2, H0586: 1,









H0563: 1, H0328: 1,









L0483: 1, L0769: 1,









L0637: 1, L0768: 1,









L0791: 1, H0696: 1,









H0436: 1, L0439: 1,









L0755: 1 and H0543: 1.


71
HORBI81
456519
81
 31–135
221

AR282: 2, AR185: 2,









AR060: 1, AR104: 1,









AR316: 1, AR313: 1,









AR089: 1









H0551: 2, L0657: 2,









L0382: 2, H0170: 1,









H0686: 1, S0040: 1,









L0022: 1, T0010: 1,









H0292: 1, L5575: 1,









L0803: 1, L0653: 1,









L0666: 1, L0664: 1 and









L0756: 1.


72
HOSCY73
456520
82
209–295
222

AR313: 18, AR299:









12, AR185: 10, AR104:









7, AR316: 7, AR089:









7, AR096: 6, AR277:









5, AR282: 5, AR060:









4, AR240: 4, AR283: 3









S0003: 3, L0776: 3, 









L0758: 3, H0624: 2,









L0662: 2, L0803: 2,









L0519: 2, L0779: 2,









S0040: 1, H0657: 1,









H0661: 1, S0358: 1, 









H0587: 1, S0010: 1,









H0581: 1, H0014: 1,









H0687: 1, H0328: 1,









H0615: 1, L0055: 1,









H0551: 1, L0564: 1,









T0042: 1, H0494: 1,









L0646: 1, L0767: 1,









L0768: 1, L0794: 1,









L0387: 1, L0766: 1,









L0774: 1, L0651: 1,









L0527: 1, L0657: 1,









L0517: 1, L0791: 1,









H0519: 1, H0659: 1,









H0670: 1, S0328: 1,









H0539: 1, H0521: 1,









H0626: 1, L0741: 1,









L0747: 1, L0749: 1,









L0750: 1, L0756: 1,









L0752: 1, L0608: 1, 









S0194: 1 and S0196: 1.


73
HADFF38
460706
83
 64–165
223

AR282: 4, AR240: 1
17pter-









H0457: 8, H0599: 6,
p13.1









H0424: 6, L0777: 6,









H0556: 4, L0766: 4,









S0046: 3, L0800: 3,









H0436: 3, L0743: 3,









L0748: 3, L0754: 3,









L0758: 3, H0255: 2,









S0358: 2, H0619: 2,









H0393: 2, S0278: 2,









H0253: 2, H0266: 2,









H0412: 2, L0769: 2, 









L0662: 2, L0363: 2,









L0659: 2, L0792: 2,









H0672: 2, H0521: 2,









L0749: 2, L0750: 2,









L0757: 2, T0002: 1,









H0717: 1, H0657: 1,









S0116: 1, H0341: 1,









H0254: 1, H0402: 1,









H0638: 1, S0418: 1,









S0222: 1, H0497: 1,









H0013: 1, H0427: 1,









H0156: 1, H0618: 1,









H0581: 1, H0052: 1,









H0251: 1, H0231: 1,









H0041: 1, H0009: 1,









H0050: 1, H0012: 1,









H0620: 1, H0688: 1,









H0039: 1, H0553: 1,









H0628: 1, H0674: 1,









H0124: 1, H0264: 1,









H0272: 1, H0413: 1,









L0564: 1, S0344: 1,









L0640: 1, L0638: 1,









L0641: 1, L0645: 1,









L0794: 1, L5569: 1,









L0804: 1, L0774: 1,









L0651: 1, L0776: 1,









L0517: 1, L0783: 1,









L0809: 1, L0789: 1,









L0790: 1, L0791: 1,









L0666: 1, L0665: 1,









H0520: 1, S0126: 1,









H0682: 1, H0670: 1,









H0539: 1, S0406: 1,









S0027: 1, L0751: 1,









L0779: 1, L0752: 1,









L0759: 1, S0434: 1,









H0668: 1, H0667: 1,









H0543: 1 and H0506: 1.


74
HFXHN31
427118
84
172–444
224
Glu-31 to Gly-37.
AR096: 12, AR313:









12, AR283: 11, AR282:









10, AR240: 9, AR316:









8, AR060: 7, AR104:









7, AR089: 7, AR185:









7, AR277: 7, AR299: 5









S0001: 1



HFXHN31
456460
143
293–373
283


75
HNGIK21
456500
85
152–238
225

AR060: 6, AR282: 5,









AR104: 4, AR185: 3,









AR316: 3, AR089: 3,









AR283: 3, AR240: 2,









AR299: 1, AR096: 1









S0052: 1


76
HE9FE83
460431
86
351–530
226
Pro-36 to Ala-41.
AR313: 71, AR173:









42, AR165: 36, AR180:









36, AR164: 35, AR258:









34, AR300: 34, AR166:









34, AR218: 33, AR161:









32, AR162: 32, AR229:









32, AR196: 31, AR096:









30, AR089: 30, AR262:









30, AR247: 30, AR240:









29, AR163, 28, AR175:









28, AR178: 28, AR299:









28, AR242: 27, AR179:









26, AR257: 26, AR183:









26, AR199: 26, AR270:









25, AR293: 24, AR219:









24, AR182: 24, AR234:









23, AR181: 23, AR191:









23, AR174: 23, AR269:









22, AR185: 21, AR236:









20, AR226: 20, AR104:









20, AR033: 20, AR238:









20, AR312: 20, AR296:









19, AR233: 19, AR176:









19, AR294: 19, AR260:









19, AR193: 19, AR316:









18, AR060: 18, AR189:









17, AR264: 17, AR203:









17, AR177: 16, AR200:









16, AR297: 16, AR268:









16, AR231: 16, AR188:









16, AR255: 16, AR275:









15, AR237: 15, AR285:









15, AR230: 15, AR287:









15, AR282: 14, AR053:









14, AR291: 13, AR286:









13, AR290: 13, AR267:









13, AR039: 13, AR239:









13, AR192: 12, AR274:









12, AR261: 12, AR277:









12, AR288: 11, AR295:









11, AR227: 11, AR228:









11, AR213: 11, AR211:









11, AR266: 11, AR243:









10, AR204: 10, AR212:









10, AR256: 9, AR190:









9, AR198: 9, AR201:









9, AR254: 8, AR245:









8, AR252: 8, AR250:









8, AR289: 8, AR207:









7, AR308: 7, AR210:









7, AR272: 7, AR195:









7, AR197: 7, AR271:









7, AR170: 7, AR263:









7, AR309: 7, AR283:









6, AR205: 6, AR232:









6, AR055: 6, AR168:









6, AR061: 5, AR253:









5, AR221: 5, AR311:









4, AR235: 4, AR246:









4, AR171: 4, AR216:









3, AR224: 3, AR172:









3, AR217: 3, AR222:









2, AR214: 2, AR215:









2, AR225: 1, AR223: 1









H0265: 1, H0306: 1,









H0252: 1, H0144: 1 and









H0576: 1.


77
HMKDS08
460477
87
230–433
227
Ser-35 to Phe-41.
H0392: 1 and H0555:









1.


78
HNGEM24
460493
88
239–334
228

AR162: 15, AR161:









15, AR163: 14, AR313:









14, AR165: 12, AR164:









11, AR166: 11, AR089:









10, AR229: 10, AR173:









10, AR176: 9, AR178:









9, AR300: 9, AR293:









9, AR060: 8, AR182:









8, AR175: 8, AR247:









8, AR238: 8, AR269:









8, AR257: 8, AR096:









8, AR180: 8, AR242:









7, AR309: 7, AR240:









7, AR266: 7, AR299:









7, AR274: 7, AR264:









7, AR181: 7, AR192:









7, AR275: 7, AR204:









7, AR316: 7, AR233:









7, AR185: 7, AR312:









7, AR226: 7, AR174:









6, AR179: 6, AR277:









6, AR228: 6, AR262:









6, AR267: 6, AR197:









6, AR258: 6, AR270:









6, AR268: 6, AR296:









6, AR282: 6, AR183:









6, AR193: 6, AR291:









6, AR289: 6, AR236:









5, AR177: 5, AR199:









5, AR261: 5, AR272:









5, AR286: 5, AR245:









5, AR255: 5, AR239:









5, AR271: 5, AR201:









5, AR207: 5, AR104:









5, AR234: 5, AR297:









5, AR191: 5, AR053:









5, AR254: 5, AR263:









5, AR237: 5, AR196:









5, AR294: 5, AR283:









5, AR287: 5, AR230:









5, AR227: 4, AR250:









4, AR203: 4, AR231:









4, AR189: 4, AR308:









4, AR290: 4, AR061:









4, AR243: 4, AR033:









4, AR295: 4, AR218:









4, AR285: 4, AR195:









4, AR288: 4, AR222:









4, AR224: 3, AR235:









3, AR188: 3, AR246:









3, AR212: 3, AR205:









3, AR198: 3, AR260:









3, AR213: 3, AR055:









3, AR168: 3, AR215:









3, AR039: 3, AR311:









3, AR200: 3, AR169:









3, AR232: 3, AR219:









3, AR172: 3, AR190:









2, AR214: 2, AR256:









2, AR252: 2, AR171:









2, AR221: 1, AR225:









1, AR216: 1, AR223:









1, AR170: 1, AR253:









1, AR211: 1









S0052: 1


79
HNGFT78
460359
89
 20–127
229

AR060: 6, AR299: 5,









AR283: 4, AR277: 4,









AR282: 4, AR240: 4,









AR316: 3, AR185: 3,









AR104: 3, AR096: 3,









AR313: 3, AR089: 2









S0052: 1


80
HNHDL85
461815
90
194–346
230

AR283: 50, AR096:









35, AR313: 22, AR299:









20, AR282: 17, AR240:









15, AR185: 15, AR089:









14, AR060: 14, AR316:









14, AR104: 13, AR277: 8









S0053: 1


81
HCEIA77
456433
91
785–946
231
Pro-7 to Cys-21,
AR104: 7, AR282: 4,
12








Leu-25 to Ser-30.
AR313: 4, AR185: 2,









AR060: 2, AR299: 2,









AR240: 2, AR316: 2,









AR283: 1, AR096: 1









H0635: 3, L0758: 3,









H0052: 2, L0748: 2,









L0439: 2, H0014: 1,









S6028: 1, L0455: 1,









L0456: 1, H0412: 1,









L0761: 1, L0803: 1,









H0144: 1, L0438: 1,









H0672: 1, L0608: 1 and









H0352: 1.


82
HTNBR95
457175
92
 70–177
232

AR223: 15, AR225:
2









13, AR264: 11, AR309:









10, AR311: 9, AR263:









9, AR235: 9, AR261:









8, AR217: 8, AR169:









8, AR171: 8, AR161:









8, AR162: 8, AR163:









8, AR188: 8, AR221:









8, AR165: 7, AR191:









7, AR196: 7, AR291:









7, AR199: 7, AR164:









7, AR272: 7, AR285:









7, AR166: 7, AR295:









6, AR312: 6, AR224:









5, AR170: 5, AR096:









5, AR274: 5, AR180:









5, AR236: 5, AR308:









5, AR189: 5, AR214:









5, AR168: 5, AR174:









5, AR222: 5, AR190:









5, AR296: 5, AR297:









5, AR275: 5, AR270:









4, AR203: 4, AR288:









4, AR287: 4, AR255:









4, AR089: 4, AR176:









4, AR282: 4, AR246:









4, AR175: 4, AR173:









4, AR316: 4, AR216:









4, AR178: 4, AR262:









4, AR181: 4, AR172:









4, AR257: 4, AR182:









4, AR238: 4, AR177:









4, AR200: 4, AR218:









3, AR258: 3, AR179:









3, AR266: 3, AR240:









3, AR183: 3, AR053:









3, AR293: 3, AR269:









3, AR313: 3, AR229:









3, AR289: 3, AR231:









3, AR286: 3, AR212:









3, AR104: 3, AR290:









3, AR268: 3, AR195:









3, AR299: 3, AR254:









3, AR294: 3, AR260:









3, AR060: 3, AR219:









3, AR237: 3, AR277:









3, AR185: 2, AR234:









2, AR226: 2, AR215:









2, AR300: 2, AR247:









2, AR232: 2, AR233:









2, AR192: 2, AR267:









2, AR239: 2, AR228:









2, AR193: 2, AR243:









2, AR230: 2, AR201:









2, AR250: 2, AR210:









2, AR211: 2, AR256:









1, AR227: 1, AR205:









1, AR213: 1, AR033:









1, AR283: 1, AR061: 1









H0031: 7, L0766: 4,









L0747: 4, S0406: 3,









L0759: 3, H0650: 2,









H0050: 2, L0598: 2,









L0662: 2, L0774: 2,









L0527: 2, L0809: 2,









L0666: 2, L0665: 2,









H0659: 2, L0748: 2,









L0756: 2, L0779: 2,









L0780: 2, L0485: 2,









H0624: 1, H0661: 1,









S0358: 1, S0046: 1,









H0586: 1, H0331: 1,









H0574: 1, H0632: 1,









H0485: 1, H0013: 1,









H0427: 1, L0021: 1,









L0738: 1, S0003: 1,









H0039: 1, H0673: 1,









S0366: 1, H0038: 1,









H0616: 1, T0067: 1,









H0494: 1, H0561: 1,









H0529: 1, L4747: 1,









L0776: 1, L0657: 1,









L0659: 1, L0664: 1,









L0438: 1, H0520: 1,









H0547: 1, H0519: 1,









S0126: 1, H0648: 1,









H0672: 1, S0152: 1,









H0436: 1, L0758: 1,









L0588: 1, L0589: 1,









S0026: 1 and S0276: 1.


83
HUKEX85
457196
93
 35–136
233

AR313: 20, AR277:









20, AR089: 17, AR299:









14, AR240: 12, AR316:









12, AR185: 11, AR060:









10, AR096: 7, AR104:









7, AR282: 6, AR283: 5









H0615: 1, H0059: 1,









S0027: 1, L0759: 1 and









H0665: 1.



HUKEX85
457176
144
 83–448
284
Pro-17 to Thr-26,








Gln-105 to Leu-








111.


84
HWTBM45
456569
94
 69–146
234

AR277: 36, AR313:









31, AR089: 28, AR240:









24, AR299: 21, AR185:









19, AR316: 18, AR060:









14, AR096: 13, AR282:









7, AR104: 7, AR283: 5









H0352: 1


85
HADFK68
683272
95
 91–264
235
Tyr-41 to Phe-47.
AR264: 45, AR311:









37, AR263: 18, AR267:









15, AR309: 15, AR274:









15, AR252: 12, AR312:









11, AR227: 11, AR163:









11, AR161: 11, AR262:









11, AR250: 10, AR308:









10, AR253: 10, AR162:









10, AR175: 9, AR216:









9, AR232: 9, AR272:









9, AR190: 8, AR176:









8, AR231: 8, AR188:









8, AR173: 8, AR269:









8, AR189: 8, AR257:









7, AR255: 7, AR285:









7, AR221: 7, AR239:









7, AR246: 7, AR228:









7, AR254: 7, AR240:









7, AR219: 7, AR191:









6, AR235: 6, AR179:









6, AR234: 6, AR260:









6, AR226: 6, AR237:









6, AR193: 6, AR185:









6, AR218: 5, AR222:









5, AR290: 5, AR247:









5, AR293: 5, AR275:









5, AR223: 5, AR291:









5, AR258: 5, AR053:









5, AR225: 5, AR229:









5, AR165: 4, AR243:









4, AR313: 4, AR261:









4, AR268: 4, AR172:









4, AR200: 4, AR212:









4, AR096: 4, AR288:









4, AR287: 4, AR104:









4, AR217: 4, AR192:









3, AR174: 3, AR238:









3, AR201: 3, AR197:









3, AR300: 3, AR195:









3, AR196: 3, AR230:









3, AR033: 3, AR177:









3, AR316: 3, AR224:









3, AR210: 2, AR198:









2, AR171: 2, AR299:









2, AR169: 2, AR277:









2, AR282: 2, AR061:









2, AR199: 2, AR203:









2, AR297: 2, AR164:









2, AR211: 2, AR256:









2, AR089: 1, AR270:









1, AR233: 1, AR213:









1, AR183: 1, AR271:









1, AR294: 1, AR060:









1, AR266: 1, AR168:









1, AR236: 1, AR180: 1









H0427: 1, L0547: 1









and L0605: 1.



HADFK68
460371
145
 45–125
285


86
HADGG19
467821
96
262–486
236
Leu-37 to Arg-45,
AR104: 7, AR282: 7








Ser-60 to Ser-65.
H0427: 1 and S0196: 1.



HADGG19
460376
146
281–304
286


87
HSDZM95
460524
97
223–351
237

AR060: 6, AR104: 5,









AR282: 5, AR283: 4,









AR089: 3, AR316: 3,









AR240: 3, AR299: 3,









AR277: 2, AR096: 2,









AR185: 2


88
HSNAA55
460535
98
 35–157
238

AR282: 4, AR253: 4,









AR235: 4, AR275: 3,









AR243: 3, AR271: 3,









AR252: 3, AR164: 3,









AR161: 3, AR162: 3,









AR291: 3, AR165: 3,









AR163: 3, AR205: 3,









AR193: 3, AR172: 3,









AR168: 2, AR166: 2,









AR199: 2, AR060: 2,









AR246: 2, AR274: 2,









AR185: 2, AR238: 2,









AR104: 2, AR033: 2,









AR236: 2, AR225: 2,









AR191: 2, AR257: 2,









AR293: 2, AR179: 2,









AR264: 2, AR227: 2,









AR200: 2, AR201: 2,









AR189: 2, AR296: 2,









AR237: 2, AR289: 2,









AR089: 2, AR299: 2,









AR240: 2, AR300: 1,









AR170: 1, AR287: 1,









AR229: 1, AR261: 1,









AR216: 1, AR203: 1,









AR266: 1, AR226: 1,









AR230: 1, AR313: 1,









AR217: 1, AR195: 1,









AR316: 1, AR173: 1,









AR181: 1, AR312: 1,









AR288: 1, AR267: 1,









AR295: 1, AR262: 1,









AR247: 1, AR196: 1,









AR233: 1, AR061: 1,









AR277: 1, AR255: 1









L0752: 2 and H0163: 1.


89
HMDAI51
456480
99
 12–128
239
Phe-22 to Leu-28.
AR060: 7, AR283: 6,









AR240: 6, AR299: 5,









AR104: 4, AR185: 4,









AR316: 4, AR089: 3,









AR096: 2, AR277: 2,









AR282: 2, AR313: 1









H0346: 1


90
HBAFQ54
459927
100
229–372
240

AR282: 141, AR261:









16, AR296: 15, AR295:









13, AR291: 12, AR205:









11, AR264: 11, AR285:









10, AR245: 10, AR286:









10, AR161: 10, AR162:









10, AR165: 10, AR289:









10, AR053: 9, AR163:









9, AR164: 9, AR297:









9, AR309: 9, AR247:









9, AR263: 9, AR287:









9, AR253: 9, AR170:









8, AR266: 8, AR166:









8, AR271: 8, AR312:









8, AR275: 8, AR192:









8, AR252: 8, AR308:









7, AR254: 7, AR257:









7, AR212: 7, AR268:









7, AR243: 7, AR195:









7, AR274: 7, AR235:









7, AR039: 7, AR272:









7, AR246: 7, AR313:









7, AR172: 7, AR197:









7, AR294: 6, AR198:









6, AR210: 6, AR216:









6, AR256: 6, AR258:









6, AR096: 6, AR270:









6, AR250: 6, AR311:









6, AR213: 6, AR168:









6, AR262: 6, AR269:









6, AR240: 6, AR180:









6, AR171: 6, AR217:









6, AR211: 6, AR260:









5, AR183: 5, AR201:









5, AR255: 5, AR175:









5, AR174: 5, AR207:









5, AR300: 5, AR193:









5, AR316: 5, AR188:









5, AR178: 5, AR104:









5, AR173: 5, AR288:









5, AR293: 5, AR204:









5, AR033: 5, AR177:









5, AR238: 5, AR189:









5, AR267: 5, AR196:









4, AR214: 4, AR290:









4, AR199: 4, AR203:









4, AR223: 4, AR242:









4, AR221: 4, AR089:









4, AR191: 4, AR229:









4, AR239: 4, AR200:









4, AR055: 4, AR277:









4, AR283: 4, AR226:









4, AR231: 4, AR182:









4, AR215: 4, AR169:









4, AR190: 3, AR181:









3, AR185: 3, AR299:









3, AR179: 3, AR225:









3, AR234: 3, AR237:









3, AR060: 3, AR219:









3, AR236: 3, AR230:









3, AR061: 3, AR176:









3, AR232: 3, AR233:









2, AR224: 2, AR218:









2, AR228: 2, AR222:









2, AR227: 2









H0411: 1, S0222: 1,









H0432: 1, H0438: 1,









S0038: 1, L0789: 1,









L0664: 1, H0144: 1,









L0438: 1 and H0506: 1.


91
HBGBA14
461874
101
 32–142
241
Pro-20 to Ser-28.
H0617: 6, L0666: 3,









H0188: 2, H0181: 2,









H0196: 1, H0039: 1,









H0673: 1, L0373: 1,









L0775: 1, L0659: 1,









L0809: 1, L0789: 1,









L0663: 1, L0665: 1,









H0658: 1, S0330: 1,









S0378: 1 and S0406: 1.


92
HCFMM26
460729
102
178–342
242

AR235: 6, AR172: 4,









AR263: 4, AR214: 4,









AR252: 4, AR309: 3,









AR183: 3, AR225: 2,









AR168: 2, AR171: 2,









AR282: 2, AR238: 2,









AR246: 2, AR223: 2,









AR277: 2, AR053: 2,









AR264: 2, AR217: 2,









AR200: 2, AR178: 2,









AR245: 2, AR222: 2,









AR257: 2, AR039: 2,









AR191: 2, AR228: 2,









AR205: 1, AR213: 1,









AR237: 1, AR271: 1,









AR311: 1, AR272: 1,









AR226: 1, AR300: 1,









AR164: 1, AR161: 1,









AR162: 1, AR189: 1,









AR210: 1, AR182: 1,









AR297: 1, AR227: 1









H0423: 1


93
HOAAF80
460500
103
303–413
243

AR313: 10, AR060: 7,









AR299: 7, AR282: 5,









AR104: 4, AR240: 4,









AR283: 4, AR089: 4,









AR316: 4, AR185: 4,









AR277: 4, AR096: 2









L0766: 6, S0422: 3,









L0646: 2, L0803: 2,









H0659: 2, H0624: 1,









H0657: 1, H0393: 1,









H0370: 1, H0252: 1,









H0591: 1, L0520: 1 and









H0698: 1.


94
HODCJ90
460501
104
344–472
244

AR060: 10, AR089: 9,









AR313: 9, AR277: 8,









AR316: 7, AR299: 7,









AR240: 7, AR104: 6,









AR096: 5, AR283: 5,









AR185: 5, AR282: 4









L0766: 3, L0439: 2,









L0750: 2, S0376: 1,









S0444: 1, H0427: 1,









L0157: 1, H0050: 1,









H0328: 1, L0055: 1,









H0040: 1, L0803: 1,









H0144: 1, L0748: 1,









L0745: 1, L0779: 1,









S0260: 1, L0604: 1 and









H0542: 1.


95
HNGJU42
456507
105
273–344
245

S0052: 1


96
HAEAV45
554653
106
487–684
246
Met-1 to Trp-12,
L0740: 2, H0550: 1








Arg-33 to Ser-53.
and T0048: 1.



HAEAV45
461863
147
 24–110
287


97
HBJFV28
460387
107
306–467
247
Pro-47 to Asn-53.
AR240: 11, AR060:









10, AR299: 8, AR277:









8, AR313: 7, AR316:









5, AR089: 5, AR104:









5, AR283: 4, AR282:









3, AR185: 3, AR096: 2









L0519: 5, L0747: 2,









L0777: 2, S0001: 1,









H0013: 1, H0318: 1,









S0386: 1, L0803: 1 and









L0749: 1.


98
HDHMB42
460423
108
592–687
248

AR202: 34, AR104: 30,
12









30, AR194: 29, AR244:









21, AR206: 21, AR033:









20, AR246: 20, AR060:









19, AR205: 18, AR241:









18, AR243: 16, AR039:









16, AR263: 16, AR192:









15, AR316: 15, AR204:









14, AR271: 13, AR265:









13, AR281: 13, AR275:









12, AR089: 12, AR186:









12, AR053: 12, AR198:









12, AR310: 12, AR282:









12, AR315: 12, AR299:









12, AR313: 11, AR266:









11, AR251: 10, AR238:









10, AR274: 10, AR240:









10, AR273: 10, AR052:









10, AR283: 10, AR277:









9, AR280: 9, AR312:









9, AR247: 9, AR232:









9, AR284: 9, AR183:









9, AR295: 9, AR309:









9, AR096: 9, AR253:









8, AR213: 8, AR182:









8, AR184: 8, AR270:









8, AR285: 7, AR185:









7, AR289: 7, AR177:









7, AR300: 7, AR227:









7, AR291: 7, AR296:









7, AR175: 7, AR292:









6, AR248: 6, AR269:









6, AR229: 6, AR298:









6, AR286: 6, AR218:









6, AR234: 6, AR268:









6, AR219: 6, AR290:









6, AR294: 6, AR256:









5, AR293: 5, AR258:









5, AR314: 5, AR231:









5, AR233: 4, AR061:









4, AR055: 4, AR267:









4, AR226: 4, AR259:









4, AR249: 3, AR237:









3, AR179: 2









L0438: 6, L0770: 4,









L0766: 4, L0439: 4,









H0013: 3, H0575: 3,









H0050: 3, L0752: 3,









L0757: 3, L0758: 3,









H0556: 2, T0110: 2,









H0572: 2, L0142: 2,









L0803: 2, S0126: 2,









L0748: 2, L0592: 2,









H0265: 1, S0408: 1,









S0132: 1, H0619: 1,









S6016: 1, S0222: 1,









H0083: 1, H0266: 1,









S0250: 1, H0615: 1,









H0428: 1, H0039: 1,









L0455: 1, S0036: 1,









H0591: 1, H0040: 1,









H0616: 1, H0056: 1,









T0041: 1, L0763: 1,









L0769: 1, L0637: 1,









L0767: 1, L0794: 1,









L0804: 1, L0666: 1,









H0648: 1, H0539: 1,









S0152: 1, H0696: 1,









S0406: 1, L0740: 1,









L0756: 1, L0755: 1 and









L0096: 1.


99
HLNAB07
460469
109
187–297
249

AR313: 30, AR173:









20, AR161: 18, AR162:









18, AR163: 18, AR165:









15, AR164: 15, AR180:









14, AR166: 14, AR096:









14, AR196: 14, AR258:









14, AR240: 14, AR182:









13, AR247: 13, AR269:









13, AR175: 13, AR178:









13, AR229: 13, AR179:









13, AR262: 12, AR089:









11, AR174: 11, AR300:









11, AR191: 11, AR257:









11, AR299: 11, AR218:









11, AR219: 10, AR234:









10, AR270: 10, AR181:









10, AR199: 9, AR233:









9, AR185: 9, AR293:









9, AR183: 9, AR238:









9, AR236: 9, AR294:









9, AR200: 9, AR285:









9, AR312: 9, AR296:









8, AR260: 8, AR255:









8, AR264: 8, AR275:









8, AR297: 8, AR316:









8, AR189: 8, AR237:









8, AR226: 8, AR287:









7, AR268: 7, AR060:









7, AR231: 7, AR177:









7, AR033: 7, AR176:









7, AR228: 7, AR267:









7, AR291: 7, AR290:









7, AR225: 6, AR239:









6, AR104: 6, AR188:









6, AR266: 6, AR274:









6, AR286: 6, AR277:









6, AR230: 6, AR288:









6, AR203: 6, AR309:









5, AR261: 5, AR190:









5, AR272: 5, AR053:









5, AR295: 5, AR263:









5, AR282: 5, AR213:









5, AR235: 5, AR308:









4, AR212: 4, AR311:









4, AR232: 4, AR256:









4, AR224: 4, AR242:









4, AR289: 4, AR227:









4, AR172: 3, AR205:









3, AR211: 3, AR217:









3, AR061: 3, AR283:









3, AR169: 3, AR193:









3, AR168: 3, AR215:









3, AR210: 3, AR195:









3, AR207: 2, AR055:









2, AR246: 2, AR216:









2, AR204: 2, AR221:









2, AR243: 2, AR223:









2, AR201: 2, AR214:









2, AR039: 2









H0025: 1


100
HMWDC28
460487
110
124–252
250

AR245: 5, AR176: 5,









AR198: 5, AR161: 5,









AR162: 4, AR204: 4,









AR163: 4, AR207: 4,









AR271: 4, AR309: 4,









AR266: 4, AR164: 4,









AR165: 4, AR166: 4,









AR181: 3, AR221: 3,









AR039: 3, AR252: 3,









AR089: 3, AR254: 3,









AR216: 3, AR182: 3,









AR291: 3, AR177: 3,









AR257: 3, AR224: 3,









AR264: 3, AR312: 3,









AR268: 3, AR238: 3,









AR275: 3, AR296: 3,









AR178: 2, AR179: 2,









AR228: 2, AR215: 2,









AR267: 2, AR196: 2,









AR229: 2, AR295: 2,









AR311: 2, AR055: 2,









AR233: 2, AR282: 2,









AR096: 2, AR270: 2,









AR288: 2, AR269: 2,









AR191: 2, AR246: 2,









AR289: 2, AR053: 2,









AR185: 2, AR300: 2,









AR285: 2, AR286: 2,









AR234: 2, AR236: 2,









AR262: 2, AR316: 2,









AR174: 2, AR255: 2,









AR231: 2, AR313: 2,









AR060: 2, AR201: 2,









AR294: 2, AR287: 2,









AR237: 2, AR243: 2,









AR212: 2, AR240: 2,









AR226: 2, AR232: 2,









AR290: 2, AR283: 2,









AR061: 2, AR261: 2,









AR308: 2, AR168: 2,









AR247: 2, AR203: 2,









AR239: 2, AR253: 2,









AR175: 2, AR277: 2,









AR217: 2, AR293: 1,









AR190: 1, AR272: 1,









AR193: 1, AR227: 1,









AR297: 1, AR213: 1,









AR230: 1, AR258: 1,









AR188: 1, AR180: 1,









AR033: 1, AR195: 1,









AR199: 1, AR183: 1,









AR211: 1, AR235: 1









H0341: 2, L0803: 2,









L0439: 2, L0747: 2,









S0376: 1, S0360: 1,









S0222: 1, H0674: 1,









H0038: 1, L0655: 1,









L0809: 1, L0666: 1,









L0754: 1, L0756: 1,









L0757: 1 and L0591: 1.


101
HSZAF47
456551
111
107–490
251
Gly-16 to Pro-30,
AR253: 151, AR250:
4p16–p15
225500,








Pro-42 to Gly-56,
141, AR254: 138,

600593,








Gly-62 to Gly-77,
AR296: 108, AR260:

602363








Glu-93 to Gly-104,
100, AR252: 98,








Glu-109 to Glu-
AR294: 96, AR180:








114,
90, AR197: 87, AR207:








Pro-121 to Asp-
87, AR256: 86, AR266:








126.
85, AR293: 82, AR297:









81, AR204: 81, AR198:









80, AR176: 78, AR286:









78, AR181: 77, AR242:









76, AR245: 75, AR243:









74, AR235: 73, AR210:









73, AR039: 73, AR218:









72, AR289: 72, AR257:









72, AR192: 71, AR033:









70, AR200: 70, AR219:









69, AR203: 68, AR285:









68, AR246: 68, AR287:









66, AR211: 66, AR178:









66, AR258: 65, AR237:









63, AR201: 62, AR053:









62, AR195: 62, AR295:









61, AR212: 60, AR234:









60, AR175: 60, AR300:









58, AR271: 58, AR230:









58, AR261: 57, AR233:









57, AR229: 57, AR275:









56, AR283: 55, AR309:









55, AR177: 55, AR183:









55, AR213: 55, AR199:









54, AR179: 53, AR272:









53, AR269: 52, AR060:









52, AR191: 51, AR205:









50, AR262: 50, AR228:









50, AR291: 50, AR182:









50, AR240: 49, AR173:









48, AR288: 47, AR190:









47, AR236: 47, AR193:









47, AR239: 47, AR268:









46, AR174: 46, AR188:









46, AR227: 46, AR255:









45, AR185: 45, AR196:









45, AR290: 44, AR055:









43, AR231: 43, AR282:









42, AR232: 42, AR264:









42, AR270: 41, AR189:









41, AR274: 40, AR267:









40, AR061: 39, AR170:









38, AR096: 37, AR316:









37, AR238: 37, AR299:









36, AR312: 36, AR104:









34, AR226: 34, AR172:









34, AR169: 31, AR171:









31, AR313: 30, AR247:









29, AR161: 29, AR311:









29, AR162: 28, AR089:









28, AR225: 27, AR163:









27, AR168: 26, AR165:









25, AR263: 24, AR164:









24, AR308: 23, AR166:









23, AR223: 22, AR217:









21, AR277: 20, AR221:









20, AR224: 20, AR216:









19, AR222: 18, AR215:









15, AR214: 14









H0013: 1, H0321: 1









L0792: 1 and L0779: 1.


102
HTADX17
457172
112
 84–512
252
Glu-15 to Arg-23,
AR227: 8, AR293: 7,








Asn-79 to Gly-84.
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.


103
HFTCU19
735139
113
137–802
253
His-2 to Lys-7,
AR282: 7, AR235: 6,








Ser-28 to Glu-35.
AR196: 4, AR161: 4,









AR162: 4, AR163: 4,









AR195: 4, AR285: 4,









AR295: 4, AR178: 4,









AR261: 4, AR089: 4,









AR274: 4, AR291: 4,









AR297: 4, AR173: 4,









AR269: 4, AR240: 4,









AR185: 4, AR270: 4,









AR170: 3, AR287: 3,









AR177: 3, AR255: 3,









AR165: 3, AR288: 3,









AR189: 3, AR172: 3,









AR191: 3, AR164: 3,









AR264: 3, AR166: 3,









AR183: 3, AR300: 3,









AR247: 3, AR188: 3,









AR236: 3, AR175: 3,









AR262: 3, AR308: 3,









AR180: 3, AR296: 3,









AR293: 3, AR277: 3,









AR254: 3, AR053: 3,









AR033: 3, AR174: 3,









AR201: 3, AR104: 2,









AR289: 2, AR039: 2,









AR096: 2, AR179: 2,









AR182: 2, AR176: 2,









AR311: 2, AR316: 2,









AR286: 2, AR181: 2,









AR271: 2, AR237: 2,









AR200: 2, AR290: 2,









AR198: 2, AR257: 2,









AR204: 2, AR168: 2,









AR246: 2, AR294: 2,









AR229: 2, AR266: 2,









AR190: 2, AR275: 2,









AR217: 2, AR268: 2,









AR238: 2, AR258: 2,









AR060: 2, AR210: 2,









AR230: 2, AR231: 2,









AR239: 2, AR234: 2,









AR312: 2, AR309: 2,









AR205: 2, AR233: 2,









AR228: 2, AR225: 2,









AR213: 2, AR260: 2,









AR242: 2, AR212: 2,









AR272: 2, AR193: 2,









AR216: 2, AR199: 1,









AR211: 1, AR055: 1,









AR299: 1, AR226: 1,









AR203: 1, AR283: 1,









AR218: 1, AR061: 1,









AR313: 1, AR219: 1,









AR223: 1, AR192: 1









L0439: 8, H0620: 7,









L0779: 7, H0265: 4,









L0764: 4, L0743: 4,









H0012: 3, L0766: 3,









L0775: 3, L0776: 3,









L0655: 3, L0809: 3,









L0747: 3, L0750: 3,









L0777: 3, L0755: 3,









L0591: 3, H0556: 2,









T0002: 2, H0717: 2,









H0663: 2, S0358: 2,









S0132: 2, H0393: 2,









H0013: 2, H0428: 2,









H0622: 2, T0041: 2,









S0150: 2, H0529: 2,









L0770: 2, L0372: 2,









L0771: 2, L0794: 2,









L0805: 2, L0659: 2,









H0658: 2, S0152: 2,









S0406: 2, L0754: 2,









L0758: 2, S0434: 2,









L0588: 2, L0592: 2,









L0601: 2, H0685: 1,









H0295: 1, T0049: 1,









S0134: 1, H0657: 1,









H0656: 1, S0282: 1,









H0662: 1, S0420: 1,









S0354: 1, S0408: 1,









H0728: 1, H0733: 1,









S0468: 1, H0208: 1,









S0476: 1, H0645: 1,









H0369: 1, H0550: 1,









H0461: 1, H0438: 1,









H0586: 1, H0497: 1,









T0109: 1, H0156: 1,









T0048: 1, H0505: 1,









S0049: 1, H0007: 1,









H0052: 1, H0309: 1,









H0263: 1, H0596: 1,









H0231: 1, H0327: 1,









H0046: 1, H0178: 1,









H0172: 1, H0123: 1,









H0081: 1, L0471: 1,









H0051: 1, S6028: 1,









H0266: 1, H0267: 1,









H0292: 1, H0688: 1,









H0039: 1, H0604: 1,









H0553: 1, H0644: 1,









H0606: 1, S0366: 1,









H0063: 1, H0551: 1,









H0264: 1, H0268: 1,









H0102: 1, H0561: 1,









H0207: 1, S0344: 1,









S0422: 1, L0520: 1,









L0763: 1, L0371: 1,









L0769: 1, L0761: 1,









L0521: 1, L0662: 1,









L0767: 1, L0768: 1,









L0364: 1, L0649: 1,









L0651: 1, L0806: 1,









L0783: 1, L0382: 1,









L0787: 1, L0664: 1,









L0665: 1, S0053: 1,









H0144: 1, S0374: 1,









S0148: 1, L0352: 1,









H0547: 1, S0126: 1,









H0690: 1, H0682: 1,









H0435: 1, H0659: 1,









H0670: 1, S0330: 1,









S0332: 1, S0027: 1,









S0028: 1, L0749: 1,









L0752: 1, L0753: 1,









L0759: 1, S0031: 1,









S0436: 1, L0608: 1,









S0011: 1, H0668: 1,









H0667: 1 and H0352: 1.



HFTCU19
456457
148
157–327
288


104
HEBFL88
420660
114
 76–192
254

AR277: 11, AR089: 8,









AR313: 8, AR240: 7,









AR316: 6, AR060: 5,









AR299: 5, AR185: 4,









AR104: 3, AR096: 3,









AR283: 1, AR282: 1









S0007: 3


105
HJMAV91
461886
115
251–349
255

AR252: 180, AR246:









93, AR264: 87, AR263:









83, AR053: 82, AR311:









80, AR254: 70, AR308:









66, AR312: 58, AR245:









58, AR309: 58, AR250:









57, AR253: 49, AR096:









49, AR213: 47, AR272:









46, AR218: 43, AR212:









42, AR243: 42, AR219:









38, AR205: 36, AR198:









32, AR039: 30, AR197:









30, AR313: 29, AR240:









28, AR201: 27, AR195:









26, AR200: 24, AR192:









23, AR164: 22, AR274:









21, AR224: 21, AR271:









21, AR242: 20, AR222:









20, AR316: 20, AR223:









20, AR089: 19, AR185:









19, AR193: 19, AR033:









18, AR189: 18, AR204:









18, AR161: 18, AR269:









18, AR104: 18, AR191:









18, AR165: 17, AR174:









17, AR275: 17, AR162:









17, AR163: 16, AR210:









16, AR166: 16, AR173:









16, AR290: 16, AR299:









15, AR207: 15, AR247:









15, AR188: 15, AR270:









15, AR190: 15, AR199:









14, AR211: 14, AR300:









14, AR221: 14, AR183:









14, AR175: 14, AR178:









13, AR225: 13, AR196:









13, AR177: 13, AR203:









12, AR268: 12, AR282:









11, AR060: 11, AR267:









11, AR180: 11, AR179:









10, AR172: 10, AR288:









9, AR296: 9, AR181:









9, AR266: 9, AR255:









9, AR291: 9, AR176:









9, AR295: 9, AR234:









8, AR238: 8, AR277:









8, AR285: 8, AR170:









8, AR229: 8, AR169:









8, AR261: 8, AR235:









8, AR289: 7, AR256:









7, AR262: 7, AR297:









7, AR293: 7, AR168:









7, AR286: 7, AR257:









7, AR258: 7, AR214:









7, AR215: 7, AR237:









7, AR216: 7, AR283:









7, AR231: 6, AR182:









6, AR236: 6, AR287:









6, AR226: 6, AR239:









6, AR230: 5, AR260:









5, AR294: 5, AR171:









5, AR232: 5, AR228:









5, AR217: 4, AR233:









4, AR055: 4, AR227:









4, AR061: 4









H0169: 2, H0545: 1









and L0526: 1.


106
HSDJJ82
460602
116
 79–237
256
Pro-45 to Gln-52.
AR264: 10, AR250: 7,









AR253: 7, AR263: 7,









AR254: 7, AR309: 6,









AR197: 6, AR053: 5,









AR198: 5, AR308: 4,









AR312: 4, AR212: 4,









AR313: 4, AR213: 4,









AR311: 4, AR161: 4,









AR252: 4, AR162: 4,









AR163: 4, AR271: 4,









AR096: 4, AR246: 3,









AR195: 3, AR168: 3,









AR266: 3, AR224: 3,









AR291: 3, AR089: 3,









AR183: 3, AR165: 3,









AR164: 3, AR216: 3,









AR166: 3, AR178: 3,









AR175: 3, AR268: 3,









AR215: 3, AR269: 3,









AR225: 3, AR201: 3,









AR270: 3, AR176: 3,









AR282: 2, AR182: 2,









AR235: 2, AR228: 2,









AR242: 2, AR221: 2,









AR272: 2, AR316: 2,









AR191: 2, AR240: 2,









AR199: 2, AR257: 2,









AR181: 2, AR297: 2,









AR190: 2, AR193: 2,









AR299: 2, AR174: 2,









AR060: 2, AR229: 2,









AR189: 2, AR275: 2,









AR200: 2, AR290: 2,









AR295: 2, AR236: 2,









AR177: 2, AR196: 2,









AR261: 2, AR277: 2,









AR243: 2, AR104: 2,









AR293: 2, AR171: 2,









AR300: 2, AR185: 2,









AR239: 2, AR227: 2,









AR237: 2, AR033: 2,









AR289: 2, AR226: 2,









AR233: 2, AR296: 1,









AR287: 1, AR179: 1,









AR188: 1, AR205: 1,









AR231: 1, AR217: 1,









AR173: 1, AR219: 1,









AR247: 1, AR232: 1,









AR230: 1, AR267: 1,









AR262: 1, AR288: 1,









AR283: 1, AR218: 1,









AR061: 1, AR203: 1,









AR258: 1









S0260: 1


107
HTLDP69
456560
117
330–776
257
Gln-21 to Gly-33,
H0593: 3, L0592: 3,
20pter-








Gln-55 to Glu-60.
S0420: 1, H0549: 1,
q11.23









H0441: 1, H0438: 1,









H0253: 1, L0794: 1,









L0774: 1, L0658: 1,









L0666: 1 and L0663: 1.


108
HPEBT80
460513
118
 21–173
258

AR060: 6, AR240: 5,









AR282: 4, AR283: 4,









AR277: 4, AR299: 4,









AR104: 4, AR316: 3,









AR089: 3, AR185: 3,









AR313: 2, AR096: 2









H0166: 1


109
HCNAV36
460695
119
505–645
259
Leu-40 to His-46.
AR200: 94, AR309:









94, AR272: 88, AR198:









85, AR308: 85, AR312:









76, AR274: 71, AR263:









70, AR195: 69, AR264:









68, AR311: 64, AR212:









57, AR245: 54, AR197:









52, AR053: 51, AR205:









50, AR252: 49, AR096:









49, AR271: 47, AR213:









44, AR177: 41, AR275:









40, AR207: 40, AR174:









35, AR164: 34, AR313:









33, AR196: 33, AR261:









33, AR162: 32, AR163:









32, AR161: 32, AR165:









32, AR247: 31, AR170:









30, AR166: 29, AR295:









29, AR181: 29, AR246:









28, AR216: 27, AR214:









27, AR240: 25, AR168:









24, AR089: 24, AR222:









24, AR316: 24, AR253:









24, AR243: 23, AR297:









23, AR224: 23, AR285:









22, AR199: 22, AR193:









21, AR104: 21, AR282:









21, AR293: 21, AR254:









20, AR296: 19, AR185:









19, AR201: 19, AR172:









19, AR192: 18, AR225:









18, AR039: 18, AR188:









18, AR189: 17, AR060:









17, AR283: 17, AR217:









16, AR218: 16, AR223:









16, AR250: 16, AR215:









16, AR169: 15, AR262:









15, AR299: 14, AR221:









14, AR231: 14, AR294:









14, AR242: 13, AR229:









13, AR190: 13, AR211:









13, AR287: 13, AR238:









12, AR210: 12, AR235:









12, AR171: 12, AR061:









12, AR300: 12, AR179:









12, AR219: 12, AR288:









11, AR204: 11, AR277:









11, AR255: 11, AR237:









11, AR236: 10, AR191:









10, AR203: 10, AR176:









10, AR230: 10, AR290:









10, AR232: 9, AR234:









9, AR226: 9, AR291:









9, AR286: 9, AR289:









8, AR033: 8, AR180:









8, AR239: 8, AR258:









7, AR257: 7, AR055:









7, AR266: 6, AR270:









6, AR178: 6, AR268:









5, AR233: 5, AR183:









5, AR267: 5, AR175:









5, AR228: 5, AR173:









4, AR182: 4, AR269:









4, AR227: 3, AR256:









3, AR260: 2









L0771: 8, L0438: 8,









L0439: 7, L0759: 7,









L0744: 5, L0659: 4,









L0731: 4, L0794: 3,









H0624: 2, H0716: 2,









S0442: 2, H0545: 2,









H0068: 2, H0412: 2,









L0520: 2, L0766: 2,









L0775: 2, L0651: 2,









L0740: 2, L0754: 2,









L0747: 2, L0756: 2,









L0779: 2, L0588: 2,









H0542: 2, H0171: 1,









S0282: 1, S0420: 1,









S0354: 1, S0358: 1,









S0444: 1, S0360: 1,









S0410: 1, S0468: 1,









S0007: 1, S0045: 1,









S0046: 1, S0132: 1,









S0476: 1, H0645: 1,









H0393: 1, L0717: 1,









H0351: 1, H0369: 1,









H0486: 1, H0013: 1,









H0575: 1, H0251: 1,









H0085: 1, H0544: 1,









H0546: 1, S0051: 1,









T0010: 1, S0003: 1,









H0030: 1, H0644: 1,









H0628: 1, H0032: 1,









H0212: 1, H0591: 1,









H0038: 1, H0040: 1,









H0268: 1, H0413: 1,









S0438: 1, S0472: 1,









S0210: 1, H0529: 1,









L0763: 1, L0770: 1,









L0667: 1, L0641: 1,









L0768: 1, L0389: 1,









L0803: 1, L0378: 1,









L0655: 1, L0807: 1,









L0809: 1, L0793: 1,









L0666: 1, L0665: 1,









H0144: 1, S0374: 1,









L0352: 1, H0520: 1,









H0519: 1, S0126: 1,









H0435: 1, H0658: 1,









H0670: 1, H0648: 1,









H0672: 1, S0044: 1,









S0027: 1, L0780: 1,









L0755: 1, S0031: 1,









H0444: 1, S0434: 1,









L0592: 1, L0599: 1,









S0026: 1 and H0665: 1.


110
HNFIU96
460611
120
170–409
260

AR313: 16, AR242:









13, AR089: 13, AR161:









12, AR162: 12, AR165:









11, AR053: 11, AR163:









11, AR164: 11, AR166:









11, AR204: 10, AR060:









9, AR192: 9, AR312:









8, AR299: 8, AR197:









8, AR096: 8, AR316:









7, AR198: 7, AR193:









7, AR212: 7, AR213:









6, AR309: 6, AR282:









6, AR277: 6, AR201:









6, AR264: 6, AR039:









6, AR300: 5, AR275:









5, AR254: 5, AR185:









5, AR195: 5, AR104:









5, AR308: 5, AR274:









5, AR271: 5, AR263:









5, AR180: 5, AR243:









5, AR253: 5, AR246:









5, AR240: 5, AR283:









4, AR196: 4, AR205:









4, AR236: 4, AR247:









4, AR229: 4, AR272:









4, AR169: 4, AR235:









4, AR311: 4, AR183:









4, AR174: 4, AR177:









4, AR257: 4, AR178:









3, AR181: 3, AR207:









3, AR179: 3, AR262:









3, AR296: 3, AR170:









3, AR250: 3, AR191:









3, AR266: 3, AR217:









3, AR267: 3, AR168:









3, AR182: 3, AR261:









3, AR189: 3, AR224:









3, AR238: 3, AR228:









3, AR270: 3, AR286:









2, AR234: 2, AR033:









2, AR199: 2, AR294:









2, AR226: 2, AR297:









2, AR200: 2, AR230:









2, AR237: 2, AR293:









2, AR223: 2, AR233:









2, AR188: 2, AR239:









2, AR287: 2, AR061:









2, AR203: 2, AR269:









2, AR055: 2, AR173:









2, AR268: 2, AR176:









2, AR289: 2, AR291:









2, AR285: 2, AR172:









2, AR190: 2, AR225:









2, AR290: 2, AR171:









2, AR245: 2, AR258:









2, AR231: 2, AR175:









1, AR227: 1, AR216:









1, AR288: 1, AR214:









1, AR295: 1, AR210: 1









H0416: 1


111
HGLDE38
460647
121
 31–216
261

AR313: 26, AR089:









25, AR299: 22, AR277:









22, AR316: 16, AR240:









16, AR185: 16, AR060:









15, AR104: 11, AR096:









11, AR282: 8, AR283: 7









H0351: 1 and L0748:









1.


112
HODDB05
456515
122
244–276
262

AR060: 3, AR282: 3,









AR240: 2, AR104: 2,









AR283: 2, AR316: 1,









AR089: 1, AR299: 1









S0360: 2, L0779: 2,









L0759: 2, S0418: 1,









S0420: 1, H0052: 1,









H0328: 1, L0804: 1,









L0777: 1 and L0589: 1.


113
HNGAC63
460491
123
214–261
263

AR313: 23, AR089:









20, AR277: 17, AR240:









17, AR299: 15, AR316:









13, AR060: 13, AR185:









12, AR104: 9, AR096:









9, AR282: 6, AR283: 6









S0052: 1


114
HTADV27
456552
124
 69–131
264

AR250: 5, AR266: 5,









AR271: 5, AR180: 5,









AR176: 5, AR228: 4,









AR162: 4, AR163: 4,









AR161: 4, AR267: 4,









AR291: 4, AR183: 3,









AR269: 3, AR229: 3,









AR264: 3, AR182: 3,









AR201: 3, AR165: 3,









AR243: 3, AR060: 3,









AR171: 3, AR204: 3,









AR164: 3, AR293: 3,









AR270: 3, AR178: 3,









AR268: 3, AR230: 3,









AR235: 3, AR205: 3,









AR181: 3, AR175: 3,









AR197: 3, AR294: 3,









AR173: 3, AR272: 3,









AR239: 3, AR289: 3,









AR295: 2, AR296: 2,









AR168: 2, AR237: 2,









AR309: 2, AR283: 2,









AR285: 2, AR238: 2,









AR297: 2, AR287: 2,









AR096: 2, AR053: 2,









AR193: 2, AR290: 2,









AR288: 2, AR061: 2,









AR224: 2, AR225: 2,









AR231: 2, AR172: 2,









AR185: 2, AR179: 2,









AR227: 2, AR233: 2,









AR226: 2, AR311: 2,









AR232: 2, AR282: 2,









AR166: 2, AR240: 2,









AR199: 2, AR260: 2,









AR256: 2, AR213: 2,









AR236: 2, AR316: 2,









AR055: 2, AR089: 2,









AR300: 2, AR286: 2,









AR234: 2, AR313: 1,









AR210: 1, AR257: 1,









AR215: 1, AR211: 1,









AR207: 1, AR217: 1,









AR212: 1, AR216: 1,









AR169: 1









H0069: 1


115
HCUFZ62
461448
125
233–388
265
Pro-9 to Trp-18,
H0402: 1, S0418: 1,
17qter








Thr-20 to Ala-27.
H0521: 1 and L0752: 1.


116
HSSJN64
460596
126
 70–255
266

AR060: 8, AR299: 7,









AR277: 7, AR313: 6,









AR089: 6, AR240: 6,









AR316: 6, AR185: 6,









AR096: 5, AR283: 5,









AR104: 5, AR282: 3









H0135: 1


117
HTEHH53
456555
127
 22–651
267
Pro-20 to Trp-25,
AR060: 4, AR283: 3,








Arg-33 to Thr-38,
AR240: 2, AR316: 2,








Asn-51 to Ile-56,
AR299: 2, AR282: 1,








Gly-82 to Ser-91,
AR089: 1, AR185: 1








Lys-151 to Arg-
H0038: 4, H0253: 2,








156.
L0779: 2, H0618: 1,









H0616: 1, L0794: 1 and









L0758: 1.


118
HSIDI15
460528
128
273–494
268

AR060: 8, AR104: 7,









AR283: 6, AR240: 5,









AR089: 5, AR299: 4,









AR316: 4, AR277: 4,









AR185: 4, AR282: 3,









AR313: 3, AR096: 2









H0036: 1 and H0444:









1.


119
HE9HW52
460432
129
122–226
269

AR299: 34, AR283:
17p13.2









15, AR240: 14, AR277:









14, AR104: 13, AR316:









12, AR282: 12, AR060:









11, AR089: 11, AR313:









8, AR096: 8, AR185: 8









L0747: 4, S0358: 3,









S0408: 3, S0440: 3,









L0758: 3, H0716: 2,









S0442: 2, L0157: 2,









H0529: 2, L0764: 2,









L0774: 2, L0776: 2,









H0521: 2, H0522: 2,









L0779: 2, H0662: 1,









H0638: 1, S0444: 1,









S0222: 1, H0625: 1,









S0448: 1, L0371: 1,









L0770: 1, L0796: 1,









L0775: 1, L0809: 1,









L0791: 1, L0666: 1,









H0144: 1, H0520: 1,









H0436: 1, L0748: 1,









L0755: 1, L0731: 1 and









L0603: 1.


120
HLWCF05
460619
130
155–328
270

AR196: 15, AR235: 9,









AR271: 8, AR261: 8,









AR309: 8, AR214: 7,









AR188: 7, AR199: 7,









AR191: 7, AR223: 6,









AR263: 6, AR218: 6,









AR189: 6, AR222: 6,









AR198: 5, AR165: 5,









AR312: 5, AR164: 5,









AR275: 5, AR295: 5,









AR166: 5, AR240: 5,









AR308: 5, AR190: 5,









AR311: 5, AR282: 4,









AR264: 4, AR224: 4,









AR161: 4, AR162: 4,









AR096: 4, AR216: 4,









AR163: 4, AR217: 4,









AR039: 4, AR195: 4,









AR089: 4, AR296: 4,









AR177: 4, AR246: 4,









AR285: 4, AR288: 4,









AR200: 4, AR210: 4,









AR219: 4, AR175: 4,









AR183: 4, AR168: 4,









AR236: 4, AR207: 4,









AR253: 4, AR174: 4,









AR299: 4, AR178: 4,









AR192: 3, AR060: 3,









AR203: 3, AR181: 3,









AR238: 3, AR316: 3,









AR213: 3, AR257: 3,









AR212: 3, AR237: 3,









AR245: 3, AR173: 3,









AR268: 3, AR242: 3,









AR250: 3, AR104: 3,









AR274: 3, AR182: 3,









AR272: 3, AR270: 3,









AR269: 3, AR291: 3,









AR221: 3, AR053: 3,









AR262: 3, AR225: 3,









AR258: 3, AR226: 3,









AR289: 3, AR176: 3,









AR232: 2, AR234: 2,









AR193: 2, AR277: 2,









AR211: 2, AR239: 2,









AR267: 2, AR300: 2,









AR287: 2, AR172: 2,









AR205: 2, AR297: 2,









AR294: 2, AR180: 2,









AR231: 2, AR313: 2,









AR185: 2, AR229: 2,









AR171: 2, AR033: 2,









AR286: 2, AR290: 2,









AR293: 2, AR197: 2,









AR233: 2, AR215: 2,









AR243: 2, AR201: 2,









AR061: 2, AR227: 2,









AR179: 2, AR228: 2,









AR283: 1, AR255: 1,









AR247: 1, AR260: 1,









AR230: 1, AR266: 1









L0439: 5, L0740: 5,









S0010: 3, H0521: 3,









L0749: 3, H0341: 2,









H0040: 2, S0422: 2,









L0662: 2, L0519: 2,









L0754: 2, S0434: 2,









S0412: 2, S0114: 1,









H0657: 1, H0638: 1,









S0358: 1, S0444: 1,









L0022: 1, H0553: 1,









H0598: 1, H0163: 1,









H0623: 1, H0100: 1,









L0598: 1, H0529: 1,









L0768: 1, L0775: 1,









L0527: 1, L0659: 1,









L0663: 1, H0648: 1,









H0672: 1, H0696: 1,









S0406: 1, H0436: 1,









H0727: 1, L0755: 1 and









L0758: 1.


121
HFGAL10
714960
131
179–466
271

L0439: 7, L0438: 4,









L0748: 3, S0346: 2,









L0758: 2, L0592: 2,









H0009: 1, H0178: 1,









H0569: 1, H0040: 1,









L0518: 1, L0783: 1,









L0809: 1, H0144: 1,









L0753: 1, L0581: 1 and









S0412: 1.



HFGAL10
456455
149
179–289
289


122
HSLBF69
456539
132
 107–1324
272
Ala-21 to Arg-27,
AR277: 17, AR313:








Asp-40 to Arg-45,
13, AR089: 12, AR299:








Glu-97 to Thr-110,
12, AR240: 12, AR282:








Glu-117 to Lys-
10, AR316: 9, AR185:








128,
7, AR060: 7, AR096:








Arg-175 to Lys-
7, AR283: 4, AR104: 4








182,








Pro-207 to Gly-220,
L0745: 2, H0341: 1,








Val-253 to Ile-272.
S0152: 1, S0028: 1,









L0779: 1 and H0543: 1.


123
HTEDS39
745350
133
205–444
273
Asn-20 to Pro-25,
AR052: 20, AR309:








Ser-48 to Asp-65.
15, AR310: 15, AR053:









13, AR312: 12, AR265:









11, AR313: 11, AR213:









10, AR186: 10, AR241:









10, AR204: 9, AR263:









8, AR192: 8, AR251:









7, AR055: 7, AR244:









7, AR275: 7, AR271:









7, AR246: 6, AR061:









6, AR316: 6, AR060:









6, AR198: 6, AR273:









6, AR270: 6, AR185:









6, AR274: 6, AR205:









6, AR243: 6, AR202:









6, AR249: 5, AR206:









5, AR089: 5, AR253:









5, AR033: 5, AR104:









5, AR299: 5, AR300:









4, AR240: 4, AR266:









4, AR269: 4, AR233:









4, AR229: 3, AR277:









3, AR096: 3, AR282:









3, AR283: 3, AR286:









3, AR295: 3, AR218:









3, AR293: 3, AR183:









3, AR177: 3, AR219:









3, AR247: 3, AR039:









3, AR268: 3, AR175:









3, AR238: 3, AR237:









3, AR292: 3, AR184:









3, AR248: 3, AR226:









2, AR231: 2, AR182:









2, AR256: 2, AR294:









2, AR284: 2, AR290:









2, AR267: 2, AR285:









1, AR179: 1, AR258:









1, AR232: 1, AR227: 1









H0656: 3, L0766: 3,









H0518: 3, H0542: 3,









H0341: 2, H0038: 2,









H0616: 2, L0794: 2,









H0525: 2, L0748: 2,









L0731: 2, H0543: 2,









H0423: 2, H0221: 1,









H0158: 1, H0159: 1,









H0036: 1, H0571: 1,









H0109: 1, H0090: 1,









H0591: 1, H0560: 1,









H0561: 1, L0763: 1,









L0662: 1, L0775: 1,









L0655: 1, L0663: 1,









H0648: 1, H0436: 1 and









H0422: 1.



HTEDS39
456554
150
227–289
290


















TABLE 1C





Gene No. X:
Clone ID:
Preferred Indication Identifier

















1
HCHAA63
Cancer


2
HCNSP40
Cancer


3
HBMXN79
Cancer


4
HGBGV89
Digestive


5
HARAA15
Neural/Sensory


6
HDAAC10
Cardiovascular,




Digestive,




Reproductive


7
HLHFP03
Respiratory


8
HMELR03
Cancer


9
HNGJJ65
Immune/Hematopoetic


10
HKAFB88
Cancer


11
HSOAH66
Digestive


12
HCRBR74
Cancer


13
HMSJW18
Cancer


14
HHTLF25
Cancer


15
HMKDD07
Immune/Hematopoetic,




Neural/Sensory


16
HELDY05
Cancer


17
HFIVB57
Digestive,




Musculoskeletal,




Reproductive


18
HFPDE69
Neural/Sensory


19
HMSHM14
Immune/Hematopoetic


20
HSRDE35
Cancer


21
HNFID82
Immune/Hematopoetic


22
HOFNY71
Reproductive


23
HPMBR15
Cancer


24
HSAVD46
Immune/Hematopoetic,




Mixed Fetal,




Reproductive


25
HCNSB01
Cancer


26
HSDGR57
Cancer


27
HJABB94
Cancer


28
HOFAF39
Reproductive


29
HTPCS60
Cancer


30
HKIYO61
Excretory


31
HMKCW19
Cancer


32
HNFIG36
Immune/Hematopoetic


33
HBIAS26
Cancer


34
HE2EC79
Connective/Epithelial,




Mixed Fetal,




Neural/Sensory


35
HNDAH54
Cancer


36
HNHFU59
Immune/Hematopoetic


37
HE8CV18
Cancer


38
HFKEB72
Excretory,




Reproductive


39
HTDAD22
Cancer


40
HSKYU29
Cancer


41
HHGDU58
Musculoskeletal


42
HMDAA66
Neural/Sensory


43
HNHFW22
Immune/Hematopoetic


44
HSQFP66
Excretory,




Neural/Sensory


45
HNGEV29
Immune/Hematopoetic


46
HUKBH05
Cancer


47
HATDL27
Endocrine


48
HBJFU48
Immune/Hematopoetic


49
HCUBN59
Immune/Hematopoetic


50
HCUDB38
Immune/Hematopoetic


51
HDPCO25
Immune/Hematopoetic


52
HDPHI51
Immune/Hematopoetic


53
HSDAG05
Cancer


54
HMWGY01
Immune/Hematopoetic


55
HSVAQ28
Cancer


56
HGLAM53
Immune/Hematopoetic,




Neural/Sensory,




Reproductive


57
HLTAI94
Immune/Hematopoetic,




Reproductive


58
HMKAH10
Neural/Sensory,




Reproductive


59
HSVBH58
Cancer


60
HBMWB01
Immune/Hematopoetic


61
HBMXP84
Cancer


62
HLYAF80
Immune/Hematopoetic


63
HNFDS53
Digestive,




Immune/Hematopoetic,




Neural/Sensory


64
HNGAX58
Immune/Hematopoetic


65
HOECO90
Cancer


66
HSVAY16
Cancer


67
HCFCE10
Immune/Hematopoetic


68
HCFNC26
Immune/Hematopoetic


69
HELDZ32
Cancer


70
HODAZ26
Cancer


71
HORBI81
Cancer


72
HOSCY73
Cancer


73
HADFF38
Cancer


74
HFXHN31
Neural/Sensory


75
HNGIK21
Immune/Hematopoetic


76
HE9FE83
Immune/Hematopoetic,




Mixed Fetal,




Musculoskeletal


77
HMKDS08
Excretory,




Neural/Sensory


78
HNGEM24
Immune/Hematopoetic


79
HNGFT78
Immune/Hematopoetic


80
HNHDL85
Immune/Hematopoetic


81
HCEIA77
Cancer


82
HTNBR95
Cancer


83
HUKEX85
Musculoskeletal,




Reproductive


84
HWTBM45
Cancer


85
HADFK68
Connective/Epithelial


86
HADGG19
Connective/Epithelial,




Musculoskeletal


87
HSDZM95
Cancer


88
HSNAA55
Cancer


89
HMDAI51
Neural/Sensory


90
HBAFQ54
Cancer


91
HBGBA14
Cardiovascular,




Digestive,




Reproductive


92
HCFMM26
Immune/Hematopoetic


93
HOAAF80
Cancer


94
HODCJ90
Cancer


95
HNGJU42
Immune/Hematopoetic


96
HAEAV45
Cardiovascular,




Reproductive


97
HBJFV28
Immune/Hematopoetic,




Mixed Fetal,




Neural/Sensory


98
HDHMB42
Cancer


99
HLNAB07
Immune/Hematopoetic


100
HMWDC28
Cancer


101
HSZAF47
Mixed Fetal


102
HTADX17
Immune/Hematopoetic,




Reproductive


103
HFTCU19
Cancer


104
HEBFL88
Neural/Sensory


105
HJMAV91
Reproductive


106
HSDJJ82
Neural/Sensory


107
HTLDP69
Cancer


108
HPEBT80
Reproductive


109
HCNAV36
Cancer


110
HNFIU96
Immune/Hematopoetic


111
HGLDE38
Cancer


112
HODDB05
Digestive,




Neural/Sensory,




Reproductive


113
HNGAC63
Immune/Hematopoetic


114
HTADV27
Immune/Hematopoetic


115
HCUFZ62
Immune/Hematopoetic


116
HSSJN64
Musculoskeletal


117
HTEHH53
Reproductive


118
HSIDI15
Digestive,




Immune/Hematopoetic


119
HE9HW52
Cancer


120
HLWCF05
Cancer


121
HFGAL10
Mixed Fetal,




Neural/Sensory,




Reproductive


122
HSLBF69
Immune/Hematopoetic,




Musculoskeletal,




Reproductive


123
HTEDS39
Cancer





















TABLE 1D






SEQ ID
CONTIG
BAC ID:
SEQ ID
EXON


Clone ID:
NO: X
ID:
A
NO: B
From–To




















HGBGV89
14
460353
AC018512
673
1–776


HGBGV89
14
460353
AC022305
674
1–878


HGBGV89
14
460353
AC002518
675
1–150


HGBGV89
14
460353
AC016135
676
1–845


HDAAC10
16
456443
AL354714
677
1–108







284–347







1641–1741







2964–3212







3853–4020







4458–4591







5755–5859







8350–8630


HLHFP03
17
460467
AC011976
678
1–467







1023–1116







2101–2317







2529–2630







2860–3097


HLHFP03
17
460467
AC011976
679
1–391


HNGJJ65
19
456504
AL096710
680
1–1118


HNGJJ65
19
456504
AL096710
681
1–200


HSOAH66
21
456540
AC018994
682
1–1160


HMSJW18
23
457182
AC027821
683
1–512







566–939







1198–4045







4663–6657







7057–7231







8567–8907







9310–10137


HMSJW18
23
457182
AC021317
684
1–702







756–1129







1388–4230







4848–5994







6014–6828







8738–9078







9481–9868


HMSJW18
23
457182
AC027821
685
1–152


HMSJW18
23
457182
AC021317
686
1–530


HMSJW18
23
457182
AC021317
687
1–171


HMKDD07
25
460481
AC011040
688
1–84







168–725


HMKDD07
25
460481
AC024107
689
1–84







168–725


HMKDD07
25
460481
AC011040
690
1–326


HMKDD07
25
460481
AC011040
691
1–283


HMKDD07
25
460481
AC024107
692
1–326


HMKDD07
25
460481
AC024107
693
1–283


HFIVB57
27
460449
AC002394
694
1–1987


HFIVB57
27
460449
AC022278
695
1–1037


HFIVB57
27
460449
AC002394
696
1–2600


HFIVB57
27
460449
AC002394
697
1–625


HFIVB57
27
460449
AC022278
698
1–406


HFIVB57
27
460449
AC022278
699
1–416


HFPDE69
28
460450
AL121656
700
1–336







465–1681







1822–4167


HFPDE69
28
460450
AP001584
701
1–533


HFPDE69
28
460450
AC011127
702
1–534


HFPDE69
28
460450
AC022046
703
1–534


HFPDE69
28
460450
AP001584
704
1–538


HFPDE69
28
460450
AC011127
705
1–538


HFPDE69
28
460450
AC022046
706
1–538


HSRDE35
30
460538
AL359388
707
1–181







393–787







1320–1765







2360–2464







2693–3483







3675–4014







4182–4364







4775–5145







5496–5761







5903–6113







6239–6336







6640–6750







6878–7490







7623–8237







8539–8633







9183–9620







9801–10270







10409–10542







10602–11201


HSRDE35
30
460538
AL359388
708
1–1183







1213–1844







1932–2343







2451–2492


HSRDE35
30
460538
AL359388
709
1–346


HPMBR15
33
456527
AC069304
710
1–63







671–992







2513–2588







4756–6584


HPMBR15
33
456527
AC069304
711
1–2735


HSAVD46
34
456536
AC012518
712
1–741


HSAVD46
34
456536
AC012385
713
1–741


HSAVD46
34
456536
AC026491
714
1–741


HSAVD46
34
456536
AC012518
715
1–271


HSAVD46
34
456536
AC012518
716
1–254


HSAVD46
34
456536
AC012385
717
1–271


HSAVD46
34
456536
AC012385
718
1–254


HSAVD46
34
456536
AC026491
719
1–254


HSAVD46
34
456536
AC026491
720
1–271


HSDGR57
36
460599
AL133477
721
1–159







558–841







1831–2076







3589–3776







3906–4593







4749–5424







5483–6838


HSDGR57
36
460599
AL133477
722
1–396


HSDGR57
36
460599
AL133477
723
1–116


HNFIG36
42
456495
AC068936
724
1–1055


HNFIG36
42
456495
AC006405
725
1–1055


HNFIG36
42
456495
AC068936
726
1–456


HNFIG36
42
456495
AC068936
727
1–42







513–758







912–1566


HNFIG36
42
456495
AC006405
728
1–246







400–1055


HNFIG36
42
456495
AC006405
729
1–456


HE2EC79
44
460671
AC007554
730
1–266







2842–4768


HE2EC79
44
460671
AC020899
731
1–266







2842–4767


HE2EC79
44
460671
AC007554
732
1–605


HE2EC79
44
460671
AC007554
733
1–424


HE2EC79
44
460671
AC020899
734
1–393


HE2EC79
44
460671
AC020899
735
1–606


HNDAH54
45
460486
AC073127
736
1–380







2590–2966


HE8CV18
47
456447
AL137061
737
1–1428


HE8CV18
47
456447
AL137061
738
1–490


HFKEB72
48
456456
AC044801
739
1–1383


HFKEB72
48
456456
AC008515
740
1–1383


HFKEB72
48
456456
AC020960
741
1–1384


HFKEB72
48
456456
AC044801
742
1–402


HFKEB72
48
456456
AC044801
743
1–305


HFKEB72
48
456456
AC008515
744
1–402


HFKEB72
48
456456
AC008515
745
1–305


HFKEB72
48
456456
AC020960
746
1–305


HFKEB72
48
456456
AC020960
747
1–402


HTDAD22
49
456553
AL035086
748
1–64







221–3787







5411–5599







7385–7410


HTDAD22
49
456553
AL359638
749
1–64







221–3787







5418–5606







7394–7421


HTDAD22
49
456553
AL035086
750
1–510


HTDAD22
49
456553
AL359638
751
1–510


HHGDU58
51
460717
AL035694
752
1–131







3726–4036







9191–9552







10640–12003


HHGDU58
51
460717
AL035694
753
1–3503


HHGDU58
51
460717
AL035694
754
1–293


HMDAA66
52
460472
AL022160
755
1–571


HNHFW22
53
460350
AC021895
756
1–737


HNHFW22
53
460350
AC025839
757
1–737


HNHFW22
53
460350
AC021605
758
1–737


HNHFW22
53
460350
AC021895
759
1–256


HNHFW22
53
460350
AC025839
760
1–290







443–782


HNHFW22
53
460350
AC021605
761
1–290







443–782


HNGEV29
55
442573
AC068978
762
1–1819


HNGEV29
55
442573
AC055739
763
1–1551


HNGEV29
55
442573
AC068978
764
1–488


HDPCO25
61
460682
AC048331
765
1–1608


HDPCO25
61
460682
AC023797
766
1–1608


HDPCO25
61
460682
AC023415
767
1–1608


HDPCO25
61
460682
AC069244
768
1–1603


HDPCO25
61
460682
AC055725
769
1–1607


HDPCO25
61
460682
AC048331
770
1–485


HDPCO25
61
460682
AC048331
771
1–2166


HDPCO25
61
460682
AC023797
772
1–2165


HDPCO25
61
460682
AC023797
773
1–485


HDPCO25
61
460682
AC023415
774
1–2165


HDPCO25
61
460682
AC023415
775
1–486


HDPCO25
61
460682
AC069244
776
1–485


HDPCO25
61
460682
AC055725
777
1–485


HDPCO25
61
460682
AC055725
778
1–404


HMWGY01
64
672222
AC011276
779
1–723


HSVAQ28
65
460592
AL117344
780
1–874







1393–1503


HSVAQ28
65
460592
AL117344
781
1–323


HLTAI94
67
456476
AL357565
782
1–2550







2558–4290







4478–4536







4607–4782







5153–5858







6249–7013


HLTAI94
67
456476
AL357565
783
1–555


HMKAH10
68
456609
AC018638
784
1–868


HMKAH10
68
456609
AC018638
785
1–613


HMKAH10
68
456609
AC018638
786
1–402


HSVBH58
69
757227
AC005071
787
1–513







2138–2341







2878–3828







4039–4407







4438–5711







6609–6900







6969–7501







7547–7645







7882–8496







8569–9146







9288–11595







11642–11927







12146–12299







12591–12757







13029–13369







13719–14460







14563–14623







14709–15271







15352–16582







16831–17249







17276–17914







17947–18016







18327–19493







19719–20635







21586–21816







24795–25126







26501–26923







27475–28264







28742–29110







29565–29655







29657–29867







31272–31718


HSVBH58
69
757227
AC005073
788
1–513







2138–2341







2878–3828







4039–4407







4438–5711







6598–6908







6969–7501







7547–7645







7882–8496







8569–9146







9288–11595







11642–11927







12146–12299







12591–12757







13029–13369







13719–14460







14563–14623







14696–15271







15352–16582







16831–17249







17276–17914







17947–18016







18327–19493







19719–20635







21271–22015







22432–22985







23034–23359







24795–25234







26499–26923







27475–28264







28742–29110







29565–29655







29657–29867







30539–30779







31272–31718


HSVBH58
69
757227
AC005071
789
1–1715







2229–2480







2882–3799







5384–5472







5637–5962







6637–7520







7764–8274







8396–12010







13347–14042







14076–14293







14296–17861







17931–18121







18344–21358


HSVBH58
69
757227
AC005071
790
1–1300


HSVBH58
69
757227
AC005073
791
1–853


HSVBH58
69
757227
AC005073
792
1–1715







2222–2480







2882–3799







5384–5472







5637–5940







6637–7520







7849–8274







8396–11766







11769–12006







12486–12589







12647–12760







12798–13105







13347–14042







14076–14293







14296–17861







17923–18121







18344–21676


HBMWB01
70
460396
AC069362
793
1–131


HBMWB01
70
460396
AL162727
794
1–135


HBMWB01
70
460396
AC031987
795
1–125


HBMWB01
70
460396
AC009899
796
1–175


HBMWB01
70
460396
AC009097
797
1–101


HBMWB01
70
460396
AC027584
798
1–162


HBMWB01
70
460396
AC008470
799
1–129


HBMWB01
70
460396
AC023309
800
1–193


HBMWB01
70
460396
AC073446
801
1–140


HBMWB01
70
460396
AC024475
802
1–187


HBMWB01
70
460396
AC068682
803
1–153


HBMWB01
70
460396
AC022696
804
1–356


HBMWB01
70
460396
AC068755
805
1–190


HBMWB01
70
460396
AC027544
806
1–158


HBMWB01
70
460396
AC011036
807
1–193


HBMWB01
70
460396
AC025975
808
1–136


HBMWB01
70
460396
AL353659
809
1–202


HBMWB01
70
460396
AC016142
810
1–150


HBMWB01
70
460396
AL356976
811
1–354


HBMWB01
70
460396
AC023864
812
1–142


HBMWB01
70
460396
AC073219
813
1–123


HBMWB01
70
460396
AC027584
814
1–368


HBMWB01
70
460396
AC073446
815
1–52







2626–2925


HBMWB01
70
460396
AC023864
816
1–1485







1590–4704


HLYAF80
72
460622
AC022867
817
1–774


HNFDS53
73
460489
AC067779
818
1–2044


HNFDS53
73
460489
AC069260
819
1–41







2947–3765







5305–7348


HNFDS53
73
460489
AC011871
820
1–41







2949–3767







5307–7349


HNFDS53
73
460489
AC067779
821
1–819


HNGAX58
74
460488
AC016922
822
1–1078







4685–5073


HNGAX58
74
460488
AC016922
823
1–457


HOECO90
75
460505
AC069028
824
1–74







1997–2055







2714–2795







3576–3824







5130–7046


HOECO90
75
460505
AC069028
825
1–586


HOECO90
75
460505
AC069028
826
1–387


HCFCE10
77
456437
AL137013
827
1–2702







2814–4633


HCFCE10
77
456437
AL137013
828
1–120







175–422


HCFNC26
78
456439
AL078605
829
1–2416







2632–3173


HCFNC26
78
456439
AL078605
830
1–552


HELDZ32
79
456450
AC025019
831
1–2121


HELDZ32
79
456450
AC025019
832
1–573


HELDZ32
79
456450
AC025019
833
1–40







1158–2410


HODAZ26
80
456514
AC002081
834
1–46







722–823







4405–4543







6062–6326







7019–7192







7651–7793







8742–9001







9263–9455







10724–10821







11497–11557







26499–26989







27624–27924







35425–35895







37785–38077







38301–39344


HODAZ26
80
456514
AC026985
835
1–85


HODAZ26
80
456514
AC025036
836
1–148


HODAZ26
80
456514
AC011953
837
1–126


HODAZ26
80
456514
AC002081
838
1–585


HADFF38
83
460706
AC015651
839
1–87







236–382







1043–1235







1763–1913







2600–2734







3232–3368







3741–3920







4272–4829







5048–9161


HADFF38
83
460706
AC046185
840
1–87







236–382







1043–1235







1763–1913







2600–2734







3232–3368







3741–3920







4272–4829







5048–8404


HADFF38
83
460706
AC018571
841
1–87







236–382







1044–1236







1764–1914







2601–2735







3233–3369







3742–3921







4273–4830







5049–9162


HADFF38
83
460706
AC015651
842
1–1052







1137–1554







1594–1822







2098–2459


HADFF38
83
460706
AC015651
843
1–146


HADFF38
83
460706
AC046185
844
1–146


HADFF38
83
460706
AC018571
845
1–1052







1137–1554







1594–1822







2098–2460


HADFF38
83
460706
AC018571
846
1–146


HNGIK21
85
456500
AC053510
847
1–972


HNGIK21
85
456500
AC019248
848
1–976


HNGIK21
85
456500
AL365215
849
1–976


HNGIK21
85
456500
AC053510
850
1–695


HNGIK21
85
456500
AC019248
851
1–695


HNGIK21
85
456500
AL365215
852
1–695


HNGEM24
88
460493
AC064796
853
1–918


HNGFT78
89
460359
AC004518
854
1–575


HNHDL85
90
461815
AL139082
855
1–809


HNHDL85
90
461815
AL139082
856
1–161


HWTBM45
94
456569
AC012089
857
1–985


HWTBM45
94
456569
AC026674
858
1–985


HWTBM45
94
456569
AC031988
859
1–985


HWTBM45
94
456569
AC012089
860
1–87


HWTBM45
94
456569
AC026674
861
1–87


HADFK68
95
683272
AC068301
862
1–808


HADFK68
95
683272
AC017097
863
1–782


HADFK68
95
683272
AC068301
864
1–315


HADFK68
95
683272
AC017097
865
1–315


HADGG19
96
467821
AL157706
866
1–796


HADGG19
96
467821
AL157706
867
1–159


HSNAA55
98
460535
AC069481
868
1–666


HSNAA55
98
460535
AC021148
869
1–33







116–172







1121–1561







1926–2479







2949–3195







3910–4441







4480–5181







6069–6137







6349–6453







7931–8051


HSNAA55
98
460535
AC021148
870
1–288


HSNAA55
98
460535
AC021148
871
1–619


HBAFQ54
100
459927
AL139255
872
1–463







491–2034


HBAFQ54
100
459927
AC020781
873
1–2787


HBAFQ54
100
459927
AL139255
874
1–302


HOAAF80
103
460500
AC022405
875
1–962


HOAAF80
103
460500
AC022405
876
1–232


HOAAF80
103
460500
AC022405
877
1–91


HODCJ90
104
460501
AC022202
878
1–1993


HODCJ90
104
460501
AC022202
879
1–165


HODCJ90
104
460501
AC022202
880
1–319


HNGJU42
105
456507
AC022517
881
1–585


HNGJU42
105
456507
AC021618
882
1–585


HNGJU42
105
456507
AC022517
883
1–402







740–863







2738–2918







4192–4419







6120–6195







6478–6530







6816–7232







7994–8528







8602–8957







9113–9750







10204–10452







11284–11730







11784–12169







12502–13010







13106–13268







13638–13821







15845–17074







17617–17868







18764–19306







20191–22459


HNGJU42
105
456507
AC021618
884
1–1960


HAEAV45
106
554653
AP001605
885
1–781


HAEAV45
106
554653
AP001604
886
1–781


HAEAV45
106
554653
AC008590
887
1–921


HAEAV45
106
554653
AP001604
888
1–106


HDHMB42
108
460423
AC023790
889
1–69







685–1098







1401–2011







2026–3517


HDHMB42
108
460423
AC023790
890
1–527


HDHMB42
108
460423
AC023790
891
1–2146


HLNAB07
109
460469
AC018462
892
1–669


HLNAB07
109
460469
AC018462
893
1–156


HMWDC28
110
460487
AL133323
894
1–1807







1942–3408


HMWDC28
110
460487
AL158212
895
1–1807







1942–3408


HMWDC28
110
460487
AL133323
896
1–559


HMWDC28
110
460487
AL158212
897
1–559


HTADX17
112
457172
AL357565
898
1–937







945–1293







2068–2336







2732–3109


HTADX17
112
457172
AL357565
899
1–104


HFTCU19
113
735139
AC003665
900
1–72







154–584







631–1003







2259–2372







2850–3031







4325–4493







4746–4901







6234–6374







6636–6747







7066–8259


HFTCU19
113
735139
AC003665
901
1–580







851–995







1224–1296







1314–1663







1930–1975







2724–2905







2968–3098







3283–3328







5121–5230







5331–5689


HFTCU19
113
735139
AC003665
902
1–450


HEBFL88
114
420660
AP001113
903
1–699


HEBFL88
114
420660
AP001028
904
1–699


HEBFL88
114
420660
AC010966
905
1–699


HEBFL88
114
420660
AP001113
906
1–268


HEBFL88
114
420660
AP001028
907
1–268


HEBFL88
114
420660
AC010966
908
1–268


HJMAV91
115
461886
AC011331
909
1–860


HJMAV91
115
461886
AP001991
910
1–860


HJMAV91
115
461886
AC019039
911
1–860


HJMAV91
115
461886
AC011331
912
1–477


HJMAV91
115
461886
AC019039
913
1–477


HSDJJ82
116
460602
AL050309
914
1–507


HSDJJ82
116
460602
AL353698
915
1–507


HSDJJ82
116
460602
AL050309
916
1–305


HSDJJ82
116
460602
AL353698
917
1–306


HTLDP69
117
456560
AL109804
918
1–241







1302–1559







2542–2945







2963–3071







3383–3546







3736–3786







3931–3992







4194–4319







4417–5235


HTLDP69
117
456560
AC017113
919
1–241







1299–1556







2539–2942







2960–3068







3380–3543







3733–3783







3928–3989







4191–4317







4413–5227


HTLDP69
117
456560
AL109804
920
1–2633


HTLDP69
117
456560
AC017113
921
1–189







522–1840


HCNAV36
119
460695
AC004837
922
1–139







909–1208







4162–4709







4838–7963


HCNAV36
119
460695
AC002486
923
1–553


HCNAV36
119
460695
AC004837
924
1–435


HCNAV36
119
460695
AC002486
925
1–119







127–334


HNFIU96
120
460611
AC006238
926
1–691







2472–2631


HNFIU96
120
460611
AC006238
927
1–280


HNFIU96
120
460611
AC006238
928
1–302


HGLDE38
121
460647
AL109865
929
1–785







1835–1975







2149–2485







2492–2956







3022–3565


HGLDE38
121
460647
AL136133
930
1–464







4670–5003







5007–5118







6448–6567







9700–9758







11372–11503







12913–13037







13505–13584







16314–16701







16719–16960







17351–17823







19146–19309







19358–19755







19781–20565







21615–21755







21929–22265







22272–22736







22802–23345







25663–25902







26797–27290







28801–28967







31831–31998







33023–33083







33972–34255







35078–35661







35818–36281







36431–36561







36671–38855


HGLDE38
121
460647
AL109865
931
1–398


HODDB05
122
456515
AL109932
932
1–509







710–1055







1360–1542







1556–2698







2760–4553







4641–4979







5138–5439







5483–5934







6062–7306


HODDB05
122
456515
AL109932
933
1–217







5647–5868







6872–7181







7867–8132







8544–8868







9514–9842







11024–11474







11478–12017


HODDB05
122
456515
AL109932
934
1–1358


HNGAC63
123
460491
AC004099
935
1–607


HNGAC63
123
460491
AC004099
936
1–184


HNGAC63
123
460491
AC004099
937
1–381


HSSJN64
126
460596
AC021396
938
1–725


HSSJN64
126
460596
AC021396
939
1–102


HLWCF05
130
460619
AC026451
940
1–958


HLWCF05
130
460619
AC009097
941
1–4163


HLWCF05
130
460619
AC026451
942
1–394


HLWCF05
130
460619
AC009097
943
1–103


HLWCF05
130
460619
AC009097
944
1–394


HFGAL10
131
714960
AC005805
945
1–2401


HFGAL10
131
714960
AC005805
946
1–190


HFGAL10
131
714960
AC005805
947
1–270





















TABLE 1E







AA SEQ ID





Gene No.
Clone ID:
NO: Y
Biological Activity
Exemplary Activity Assay
Preferred Indication







7
HLHFP03
157
Activation of T-
Kinase assay. JNK and p38
Preferred indications include





Cell p38 or JNK
kinase assays for signal
neoplastic diseases (e.g., as





Signaling Pathway.
transduction that regulate cell
described below under






proliferation, activation, or
“Hyperproliferative






apoptosis are well known in
Disorders”), blood disorders






the art and may be used or
(e.g., as described below under






routinely modified to assess
“Immune Activity”,






the ability of polypeptides of
“Cardiovascular Disorders”,






the invention (including
and/or “Blood-Related






antibodies and agonists or
Disorders”), and infection






antagonists of the invention) to
(e.g., an infectious disease as






promote or inhibit immune cell
described below under






(e.g. T-cell) proliferation,
“Infectious Disease”). Highly






activation, and apoptosis.
preferred indications include






Exemplary assays for JNK and
autoimmune diseases (e.g.,






p38 kinase activity that may be
rheumatoid arthritis, systemic






used or routinely modified to
lupus erythematosis, multiple






test JNK and p38 kinase-
sclerosis and/or as described






induced activity of
below) and






polypeptides of the invention
immunodeficiencies (e.g., as






(including antibodies and
described below). Additional






agonists or antagonists of the
highly preferred indications






invention) include the assays
include inflammation and






disclosed in Forrer et al., Biol
inflammatory disorders.






Chem 379(8–9): 1101–1110
Highly preferred indications






(1998); Gupta et al., Exp Cell
also include neoplastic






Res 247(2): 495–504 (1999);
diseases (e.g., leukemia,






Kyriakis JM, Biochem Soc
lymphoma, and/or as described






Symp 64: 29–48 (1999); Chang
below under






and Karin, Nature
“Hyperproliferative






410(6824): 37–40 (2001); and
Disorders”). Highly preferred






Cobb MH, Prog Biophys Mol
indications include neoplasms






Biol 71(3–4): 479–500 (1999);
and cancers, such as, leukemia,






the contents of each of which
lymphoma, prostate, breast,






are herein incorporated by
lung, colon, pancreatic,






reference in its entirety. T
esophageal, stomach, brain,






cells that may be used
liver, and urinary cancer. Other






according to these assays are
preferred indications include






publicly available (e.g.,
benign dysproliferative






through the ATCC).
disorders and pre-neoplastic






Exemplary mouse T cells that
conditions, such as, for






may be used according to these
example, hyperplasia,






assays include the CTLL cell
metaplasia, and/or dysplasia.






line, which is an IL-2
Preferred indications include






dependent suspension-culture
arthritis, asthma, AIDS,






cell line with cytotoxic
allergy, anemia, pancytopenia,






activity.
leukopenia, thrombocytopenia,







Hodgkin's disease, acute







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.


14
HHTLF25
164
Production of IL-10
IL-10 FMAT. Assays for
A highly preferred





and downregulation
immunomodulatory proteins
embodiment of the invention





of immune
produced by activated T cells,
includes a method for





responses
B cells, and monocytes that
stimulating the production of






exhibit anti-inflammatory
IL-10. An alternative






activity and downregulate
preferred embodiment of the






monocyte/macrophage
invention includes a method






function and expression of
for inhibiting the production of






cytokines are well known in
IL-10.






the art and may be used or
Highly preferred indications






routinely modified to assess
include inflammation and






the ability of the polypeptides
inflammatory disorders (e.g.






of the invention (including
inflammatory bowel disease).






antibodies and agonists or
An additional highly preferred






antagonists of the invention) to
indication includes






mediate immunomodulation,
inflammatory bowel disease.






regulate inflammatory
Additional highly preferred






activities, and modulate
indications include blood






immune cell function and
disorders (e.g., as described






cytokine production.
below under “Immune






Exemplary assays that test for
Activity” (e.g. autoimmune






immunomodulatory proteins
disorders), “Blood-Related






evaluate the production of
Disorders”, and/or






cytokines, such as IL-10, and
““Cardiovascular Disorders””).






the downmodulation of
Highly preferred indications






immune responses. Such
include autoimmune diseases






assays that may be used or
(e.g., rheumatoid arthritis,






routinely modified to test
systemic lupus erythematosis,






immunomodulatory activity of
multiple sclerosis and/or as






polypeptides of the invention
described below) and






(including antibodies and
immunodeficiencies (e.g., as






agonists or antagonists of the
described below).






invention) include the assays
Preferred indications include






disclosed in Miraglia et al. J
neoplastic diseases (e.g.,






Biomolecular Screening 4: 193–204
leukemia, lymphoma,






(1999); Rowland et al.,
melanoma, and/or as described






““Lymphocytes: a practical
below under






approach”” Chapter 6: 138–160
“Hyperproliferative






(2000); and Koning et al.,
Disorders”). Preferred






Cytokine 9(6): 427–436 (1997),
indications include neoplasms






the contents of each of which
and cancers, such as, for






are herein incorporated by
example, leukemia, lymphoma,






reference in its entirety.
melanoma, and prostate,






Human T cells that may be
breast, lung, colon, pancreatic,






used according to these assays
esophageal, stomach, brain,






may be isolated using
liver and urinary cancer. Other






techniques disclosed herein or
preferred indications include






otherwise known in the art.
benign dysproliferative






Human T cells are primary
disorders and pre-neoplastic






human lymphocytes that
conditions, such as, for






mature in the thymus and
example, hyperplasia,






express a T cell receptor and
metaplasia, and/or dysplasia.






CD3, CD4, or CD8. These
Preferred indications include






cells mediate humoral or cell-
anemia, pancytopenia,






mediated immunity and may
leukopenia, thrombocytopenia,






be preactivated to enhance
Hodgkin's disease, acute






responsiveness to
lymphocytic anemia (ALL),






immunomodulatory factors.
plasmacytomas, multiple







myeloma, Burkitt's lymphoma,







Crohn's disease, arthritis,







AIDS, granulomatous 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.







An additional preferred







indication is infection (e.g., as







described below under







““Infectious Disease””).


19
HMSHM14
169
Activation of
Assays for the activation of
Preferred indications include





transcription
transcription through the
blood disorders (e.g., as





through cAMP
cAMP response element are
described below under





response element in
well-known in the art and may
“Immune Activity”, “Blood-





immune cells (such
be used or routinely modified
Related Disorders”, and/or





as T-cells).
to assess the ability of
““Cardiovascular Disorders””),






polypeptides of the invention
and infection (e.g., an






(including antibodies and
infectious disease as described






agonists or antagonists of the
below under “Infectious






invention) to increase cAMP
Disease”). Preferred






and regulate CREB
indications include






transcription factors, and
autoimmune diseases (e.g.,






modulate expression of genes
rheumatoid arthritis, systemic






involved in a wide variety of
lupus erythematosis, multiple






cell functions. Exemplary
sclerosis and/or as described






assays for transcription
below), immunodeficiencies






through the cAMP response
(e.g., as described below),






element that may be used or
boosting a T cell-mediated






routinely modified to test
immune response, and






cAMP-response element
suppressing a T cell-mediated






activity of polypeptides of the
immune response. Additional






invention (including antibodies
preferred indications include






and agonists or antagonists of
inflammation and






the invention) include assays
inflammatory disorders.






disclosed in Berger et al., Gene
Highly preferred indications






66: 1–10 (1998); Cullen and
include neoplastic diseases






Malm, Methods in Enzymol
(e.g., leukemia, lymphoma,






216: 362–368 (1992); Henthorn
and/or as described below






et al., Proc Natl Acad Sci USA
under “Hyperproliferative






85: 6342–6346 (1988); Black et
Disorders”). Highly preferred






al., Virus Genes 15(2): 105–117
indications include neoplasms






(1997); and Belkowski et al., J
and cancers, such as, for






Immunol 161(2): 659–665
example, leukemia, lymphoma






(1998), the contents of each of
(e.g., T cell lymphoma,






which are herein incorporated
Burkitt's lymphoma, non-






by reference in its entirety. T
Hodgkins lymphoma,






cells that may be used
Hodgkin's disease), melanoma,






according to these assays are
and prostate, breast, lung,






publicly available (e.g.,
colon, pancreatic, esophageal,






through the ATCC).
stomach, brain, liver and






Exemplary mouse T cells that
urinary cancer. Other preferred






may be used according to these
indications include benign






assays include the CTLL cell
dysproliferative disorders and






line, which is a suspension
pre-neoplastic conditions, such






culture of IL-2 dependent
as, for example, hyperplasia,






cytotoxic T cells.
metaplasia, and/or dysplasia.







Preferred indications include







anemia, pancytopenia,







leukopenia, thrombocytopenia,







acute lymphocytic anemia







(ALL), 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.


19
HMSHM14
169
Activation of
Assays for the activation of
A preferred embodiment of





transcription
transcription through the
the invention includes a





through serum
Serum Response Element
method for inhibiting (e.g.,





response element in
(SRE) are well-known in the
reducing) TNF alpha





immune cells (such
art and may be used or
production. An alternative





as T-cells).
routinely modified to assess
preferred embodiment of the






the ability of polypeptides of
invention includes a method






the invention (including
for stimulating (e.g.,






antibodies and agonists or
increasing) TNF alpha






antagonists of the invention) to
production. Preferred






regulate the serum response
indications include blood






factors and modulate the
disorders (e.g., as described






expression of genes involved
below under “Immune






in growth. Exemplary assays
Activity”, “Blood-Related






for transcription through the
Disorders”, and/or






SRE that may be used or
““Cardiovascular Disorders””),






routinely modified to test SRE
Highly preferred indications






activity of the polypeptides of
include autoimmune diseases






the invention (including
(e.g., rheumatoid arthritis,






antibodies and agonists or
systemic lupus erythematosis,






antagonists of the invention)
Crohn's disease, multiple






include assays disclosed in
sclerosis and/or as described






Berger et al., Gene 66: 1–10
below), immunodeficiencies






(1998); Cullen and Malm,
(e.g., as described below),






Methods in Enzymol 216: 362–368
boosting a T cell-mediated






(1992); Henthorn et al.,
immune response, and






Proc Natl Acad Sci USA
suppressing a T cell-mediated






85: 6342–6346 (1988); and
immune response. Additional






Black et al., Virus Genes
highly preferred indications






12(2): 105–117 (1997), the
include inflammation and






content of each of which are
inflammatory disorders, and






herein incorporated by
treating joint damage in






reference in its entirety. T
patients with rheumatoid






cells that may be used
arthritis. An additional highly






according to these assays are
preferred indication is sepsis.






publicly available (e.g.,
Highly preferred indications






through the ATCC).
include neoplastic diseases






Exemplary mouse T cells that
(e.g., leukemia, lymphoma,






may be used according to these
and/or as described below






assays include the CTLL cell
under “Hyperproliferative






line, which is an IL-2
Disorders”). Additionally,






dependent suspension culture
highly preferred indications






of T cells with cytotoxic
include neoplasms and






activity.
cancers, such as, for example,







leukemia, lymphoma,







melanoma, glioma (e.g.,







malignant glioma), solid







tumors, 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.







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”).


19
HMSHM14
169
Production of
MCP-1 FMAT. Assays for
A highly preferred





MCP-1
immunomodulatory proteins
embodiment of the invention






that are produced by a large
includes a method for






variety of cells and act to
stimulating (e.g., increasing)






induce chemotaxis and
MCP-1 production. An






activation of monocytes and T
alternative highly preferred






cells are well known in the art
embodiment of the invention






and may be used or routinely
includes a method for






modified to assess the ability
inhibiting (e.g., reducing)






of polypeptides of the
MCP-1 production. A highly






invention (including antibodies
preferred indication is






and agonists or antagonists of
infection (e.g., an infectious






the invention) to mediate
disease as described below






immunomodulation, induce
under “Infectious Disease”).






chemotaxis, and modulate
Additional highly preferred






immune cell activation.
indications include






Exemplary assays that test for
inflammation and






immunomodulatory proteins
inflammatory disorders.






evaluate the production of cell
Preferred indications include






surface markers, such as
blood disorders (e.g., as






monocyte chemoattractant
described below under






protein (MCP), and the
“Immune Activity”, “Blood-






activation of monocytes and T
Related Disorders”, and/or






cells. Such assays that may be
““Cardiovascular Disorders””).






used or routinely modified to
Highly preferred indications






test immunomodulatory and
include autoimmune diseases






diffferentiation activity of
(e.g., rheumatoid arthritis,






polypeptides of the invention
systemic lupus erythematosis,






(including antibodies and
multiple sclerosis and/or as






agonists or antagonists of the
described below) and






invention) include assays
immunodeficiencies (e.g., as






disclosed in Miraglia et al., J
described below). Preferred






Biomolecular Screening 4: 193–204
indications also include






(1999); Rowland et al.,
anemia, pancytopenia,






““Lymphocytes: a practical
leukopenia, thrombocytopenia,






approach”” Chapter 6: 138–160
Hodgkin's disease, acute






(2000); Satthaporn and
lymphocytic anemia (ALL),






Eremin, J R Coll Surg Ednb
plasmacytomas, multiple






45(1): 9–19 (2001); and
myeloma, Burkitt's lymphoma,






Verhasselt et al., J Immunol
arthritis, AIDS, granulomatous






158: 2919–2925 (1997), the
disease, inflammatory bowel






contents of each of which are
disease, sepsis, neutropenia,






herein incorporated by
neutrophilia, psoriasis,






reference in its entirety.
suppression of immune






Human dendritic cells that may
reactions to transplanted






be used according to these
organs and tissues,






assays may be isolated using
hemophilia, hypercoagulation,






techniques disclosed herein or
diabetes mellitus, endocarditis,






otherwise known in the art.
meningitis (bacterial and






Human dendritic cells are
viral), Lyme Disease, asthma,






antigen presenting cells in
and allergy Preferred






suspension culture, which,
indications also include






when activated by antigen
neoplastic diseases (e.g.,






and/or cytokines, initiate and
leukemia, lymphoma, and/or as






upregulate T cell proliferation
described below under






and functional activities.
“Hyperproliferative







Disorders”). Highly preferred







indications include neoplasms







and cancers, such as, leukemia,







lymphoma, 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.


27
HJABB94
177
Insulin Secretion
Assays for measuring secretion
A highly preferred






of insulin are well-known in
indication is diabetes mellitus.






the art and may be used or
An additional highly preferred






routinely modified to assess
indication is a complication






the ability of polypeptides of
associated with diabetes (e.g.,






the invention (including
diabetic retinopathy, diabetic






antibodies and agonists or
nephropathy, kidney disease






antagonists of the invention) to
(e.g., renal failure,






stimulate insulin secretion.
nephropathy and/or other






For example, insulin secretion
diseases and disorders as






is measured by FMAT using
described in the ““Renal






anti-rat insulin antibodies.
Disorders”” section below),






Insulin secretion from
diabetic neuropathy, nerve






pancreatic beta cells is
disease and nerve damage






upregulated by glucose and
(e.g., due to diabetic






also by certain
neuropathy), blood vessel






proteins/peptides, and
blockage, heart disease, stroke,






disregulation is a key
impotence (e.g., due to diabetic






component in diabetes.
neuropathy or blood vessel






Exemplary assays that may be
blockage), seizures, mental






used or routinely modified to
confusion, drowsiness,






test for stimulation of insulin
nonketotic hyperglycemic-






secretion (from pancreatic
hyperosmolar coma,






cells) by polypeptides of the
cardiovascular disease (e.g.,






invention (including antibodies
heart disease, atherosclerosis,






and agonists or antagonists of
microvascular disease,






the invention) include assays
hypertension, stroke, and other






disclosed in: Shimizu, H., et
diseases and disorders as






al., Endocr J, 47(3): 261–9
described in the






(2000); Salapatek, A.M., et al.,
““Cardiovascular Disorders””






Mol Endocrinol, 13(8): 1305–17
section below), dyslipidemia,






(1999); Filipsson, K., et al.,
endocrine disorders (as






Ann N Y Acad Sci, 865: 441–4
described in the ““Endocrine






(1998); Olson, L.K., et al., J
Disorders”” section below),






Biol Chem, 271(28): 16544–52
neuropathy, vision impairment






(1996); and, Miraglia S et. al.,
(e.g., diabetic retinopathy and






Journal of Biomolecular
blindness), ulcers and impaired






Screening, 4: 193–204 (1999),
wound healing, and infection






the contents of each of which
(e.g., infectious diseases and






is herein incorporated by
disorders as described in the






reference in its entirety.
““Infectious Diseases”” section






Pancreatic cells that may be
below, especially of the






used according to these assays
urinary tract and skin), carpal






are publicly available (e.g.,
tunnel syndrome and






through the ATCC) and/or
Dupuytren's contracture).






may be routinely generated.
An additional highly preferred






Exemplary pancreatic cells that
indication is obesity and/or






may be used according to these
complications associated with






assays include HITT15 Cells.
obesity. Additional highly






HITT15 are an adherent
preferred indications include






epithelial cell line established
weight loss or alternatively,






from Syrian hamster islet cells
weight gain. Aditional






transformed with SV40. These
highly preferred indications are






cells express glucagon,
complications associated with






somatostatin, and
insulin resistance.






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.


44
HSQFP66
194
Stimulation of
Assays for measuring secretion
A highly preferred





insulin secretion
of insulin are well-known in
indication is diabetes mellitus.





from pancreatic
the art and may be used or
An additional highly preferred





beta cells.
routinely modified to assess
indication is a complication






the ability of polypeptides of
associated with diabetes (e.g.,






the invention (including
diabetic retinopathy, diabetic






antibodies and agonists or
nephropathy, kidney disease






antagonists of the invention) to
(e.g., renal failure,






stimulate insulin secretion.
nephropathy and/or other






For example, insulin secretion
diseases and disorders as






is measured by FMAT using
described in the ““Renal






anti-rat insulin antibodies.
Disorders”” section below),






Insulin secretion from
diabetic neuropathy, nerve






pancreatic beta cells is
disease and nerve damage






upregulated by glucose and
(e.g., due to diabetic






also by certain
neuropathy), blood vessel






proteins/peptides, and
blockage, heart disease, stroke,






disregulation is a key
impotence (e.g., due to diabetic






component in diabetes.
neuropathy or blood vessel






Exemplary assays that may be
blockage), seizures, mental






used or routinely modified to
confusion, drowsiness,






test for stimulation of insulin
nonketotic hyperglycemic-






secretion (from pancreatic
hyperosmolar coma,






cells) by polypeptides of the
cardiovascular disease (e.g.,






invention (including antibodies
heart disease, atherosclerosis,






and agonists or antagonists of
microvascular disease,






the invention) include assays
hypertension, stroke, and other






disclosed in: Ahren, B., et al.,
diseases and disorders as






Am J Physiol, 277(4 Pt
described in the






2): R959–66 (1999); Li, M., et
““Cardiovascular Disorders””






al., Endocrinology,
section below), dyslipidemia,






138(9): 3735–40 (1997); Kim,
endocrine disorders (as






K.H., et al., FEBS Lett,
described in the ““Endocrine






377(2): 237–9 (1995); and,
Disorders”” section below),






Miraglia S et. al., Journal of
neuropathy, vision impairment






Biomolecular Screening,
(e.g., diabetic retinopathy and






4: 193–204 (1999), the contents
blindness), ulcers and impaired






of each of which is herein
wound healing, and infection






incorporated by reference in its
(e.g., infectious diseases and






entirety. Pancreatic cells that
disorders as described in the






may be used according to these
““Infectious Diseases”” section






assays are publicly available
below, especially of the






(e.g., through the ATCC)
urinary tract and skin), carpal






and/or may be routinely
tunnel syndrome and






generated. Exemplary
Dupuytren's contracture).






pancreatic cells that may be
An additional highly preferred






used according to these assays
indication is obesity and/or






include rat INS-1 cells. INS-1
complications associated with






cells are a semi-adherent cell
obesity. Additional highly






line established from cells
preferred indications include






isolated from an X-ray induced
weight loss or alternatively,






rat transplantable insulinoma.
weight gain.






These cells retain
Aditional highly preferred






characteristics typical of native
indications are complications






pancreatic beta cells includind
associated with insulin






glucose inducible insulin
resistance.






secretion. References: Asfari






et al. Endocrinology 1992






130: 167.


48
HBJFU48
198
Activation of
Kinase assay. Kinase assays,
A highly preferred





Adipocyte ERK
for example an Elk-1 kinase
embodiment of the invention





Signaling Pathway
assay, for ERK signal
includes a method for






transduction that regulate cell
stimulating adipocyte






proliferation or differentiation
proliferation. An alternative






are well known in the art and
highly preferred embodiment






may be used or routinely
of the invention includes a






modified to assess the ability
method for inhibiting






of polypeptides of the
adipocyte proliferation.






invention (including antibodies
A highly preferred






and agonists or antagonists of
embodiment of the invention






the invention) to promote or
includes a method for






inhibit cell proliferation,
stimulating adipocyte






activation, and differentiation.
differentiation. An alternative






Exemplary assays for ERK
highly preferred embodiment






kinase activity that may be
of the invention includes a






used or routinely modified to
method for inhibiting






test ERK kinase-induced
adipocyte differentiation.






activity of polypeptides of the
A highly preferred






invention (including antibodies
embodiment of the invention






and agonists or antagonists of
includes a method for






the invention) include the
stimulating (e.g., increasing)






assays disclosed in Forrer et
adipocyte activation. An






al., Biol Chem 379(8–9): 1101–1110
alternative highly preferred






(1998); Le Marchand-
embodiment of the invention






Brustel Y, Exp Clin
includes a method for






Endocrinol Diabetes
inhibiting the activation of






107(2): 126–132 (1999);
(e.g., decreasing) and/or






Kyriakis JM, Biochem Soc
inactivating adipocytes.






Symp 64: 29–48 (1999); Chang
Highly preferred indications






and Karin, Nature
include endocrine disorders






410(6824): 37–40 (2001); and
(e.g., as described below under






Cobb MH, Prog Biophys Mol
““Endocrine Disorders””).






Biol 71(3–4): 479–500 (1999);
Highly preferred indications






the contents of each of which
also include neoplastic






are herein incorporated by
diseases (e.g., lipomas,






reference in its entirety.
liposarcomas, and/or as






Mouse adipocyte cells that
described below under






may be used according to these
“Hyperproliferative






assays are publicly available
Disorders”). Preferred






(e.g., through the ATCC).
indications include blood






Exemplary mouse adipocyte
disorders (e.g., hypertension,






cells that may be used
congestive heart failure, blood






according to these assays
vessel blockage, heart disease,






include 3T3-L1 cells. 3T3-L1
stroke, impotence and/or as






is an adherent mouse
described below under






preadipocyte cell line that is a
“Immune Activity”,






continuous substrain of 3T3
“Cardiovascular Disorders”,






fibroblast cells developed
and/or “Blood-Related






through clonal isolation and
Disorders”), immune disorders






undergo a pre-adipocyte to
(e.g., as described below under






adipose-like conversion under
““Immune Activity””), neural






appropriate differentiation
disorders (e.g., as described






conditions known in the art.
below under ““Neural Activity







and Neurological Diseases””),







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, 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.


48
HBJFU48
198
Production of IL-6
IL-6 FMAT. IL-6 is produced
A highly preferred






by T cells and has strong
embodiment of the invention






effects on B cells. IL-6
includes a method for






participates in IL-4 induced
stimulating (e.g., increasing)






IgE production and increases
IL-6 production. An alternative






IgA production (IgA plays a
highly preferred embodiment






role in mucosal immunity).
of the invention includes a






IL-6 induces cytotoxic T cells.
method for inhibiting (e.g.,






Deregulated expression of IL-6
reducing) IL-6 production.






has been linked to autoimmune
A highly preferrred indication






disease, plasmacytomas,
is the stimulation or






myelomas, and chronic
enhancement of mucosal






hyperproliferative diseases.
immunity. Highly preferred






Assays for immunomodulatory
indications include blood






and differentiation factor
disorders (e.g., as described






proteins produced by a large
below under “Immune






variety of cells where the
Activity”, “Blood-Related






expression level is strongly
Disorders”, and/or






regulated by cytokines, growth
““Cardiovascular Disorders””),






factors, and hormones are well
and infection (e.g., as






known in the art and may be
described below under






used or routinely modified to
“Infectious Disease”). Highly






assess the ability of
preferred indications include






polypeptides of the invention
autoimmune diseases (e.g.,






(including antibodies and
rheumatoid arthritis, systemic






agonists or antagonists of the
lupus erythematosis, multiple






invention) to mediate
sclerosis and/or as described






immunomodulation and
below) and






differentiation and modulate T
immunodeficiencies (e.g., as






cell proliferation and function.
described below). Highly






Exemplary assays that test for
preferred indications also






immunomodulatory proteins
include boosting a B cell-






evaluate the production of
mediated immune response






cytokines, such as IL-6, and
and alternatively suppressing a






the stimulation and
B cell-mediated immune






upregulation of T cell
response. Highly preferred






proliferation and functional
indications include






activities. Such assays that
inflammation and






may be used or routinely
inflammatory disorders.






modified to test
Additional highly preferred






immunomodulatory and
indications include asthma and






diffferentiation activity of
allergy. Highly preferred






polypeptides of the invention
indications include neoplastic






(including antibodies and
diseases (e.g., myeloma,






agonists or antagonists of the
plasmacytoma, leukemia,






invention) include assays
lymphoma, melanoma, and/or






disclosed in Miraglia et al., J
as described below under






Biomolecular Screening 4:
“Hyperproliferative






193–204(1999); Rowland et al.,
Disorders”). Highly preferred






““Lymphocytes: a practical
indications include neoplasms






approach”” Chapter 6: 138–160
and cancers, such as, myeloma,






(2000); and Verhasselt et al., J
plasmacytoma, leukemia,






Immunol 158: 2919–2925
lymphoma, melanoma, and






(1997), the contents of each of
prostate, breast, lung, colon,






which are herein incorporated
pancreatic, esophageal,






by reference in its entirety.
stomach, brain, liver and






Human dendritic cells that may
urinary cancer. Other preferred






be used according to these
indications include benign






assays may be isolated using
dysproliferative disorders and






techniques disclosed herein or
pre-neoplastic conditions, such






otherwise known in the art.
as, for example, hyperplasia,






Human dendritic cells are
metaplasia, and/or dysplasia.






antigen presenting cells in
Preferred indications include






suspension culture, which,
anemia, pancytopenia,






when activated by antigen
leukopenia, thrombocytopenia,






and/or cytokines, initiate and
Hodgkin's disease, acute






upregulate T cell proliferation
lymphocytic anemia (ALL),






and functional activities.
multiple myeloma, Burkitt's







lymphoma, 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, and Lyme Disease.







An additonal preferred







indication is infection (e.g., an







infectious disease as described







below under ““Infectious







Disease””).


51
HDPCO25
201
Regulation of
Assays for the regulation of
A highly preferred





viability and
viability and proliferation of
indication is diabetes mellitus.





proliferation of
cells in vitro are well-known in
An additional highly preferred





pancreatic beta
the art and may be used or
indication is a complication





cells.
routinely modified to assess
associated with diabetes (e.g.,






the ability of polypeptides of
diabetic retinopathy, diabetic






the invention (including
nephropathy, kidney disease






antibodies and agonists or
(e.g., renal failure,






antagonists of the invention) to
nephropathy and/or other






regulate viability and
diseases and disorders as






proliferation of pancreatic beta
described in the ““Renal






cells. For example, the Cell
Disorders”” section below),






Titer-Glo luminescent cell
diabetic neuropathy, nerve






viability assay measures the
disease and nerve damage






number of viable cells in
(e.g., due to diabetic






culture based on quantitation
neuropathy), blood vessel






of the ATP present which
blockage, heart disease, stroke,






signals the presence of
impotence (e.g., due to diabetic






metabolically active cells.
neuropathy or blood vessel






Exemplary assays that may be
blockage), seizures, mental






used or routinely modified to
confusion, drowsiness,






test regulation of viability and
nonketotic hyperglycemic-






proliferation of pancreatic beta
hyperosmolar coma,






cells by polypeptides of the
cardiovascular disease (e.g.,






invention (including antibodies
heart disease, atherosclerosis,






and agonists or antagonists of
microvascular disease,






the invention) include assays
hypertension, stroke, and other






disclosed in: Ohtani KI, et al.,
diseases and disorders as






Endocrinology, 139(1): 172–8
described in the






(1998); Krautheim A, et al,
““Cardiovascular Disorders””






Exp Clin Endocrinol Diabetes,
section below), dyslipidemia,






107 (1): 29–34 (1999), the
endocrine disorders (as






contents of each of which is
described in the ““Endocrine






herein incorporated by
Disorders”” section below),






reference in its entirety.
neuropathy, vision impairment






Pancreatic cells that may be
(e.g., diabetic retinopathy and






used according to these assays
blindness), ulcers and impaired






are publicly available (e.g.,
wound healing, and infection






through the ATCC) and/or
(e.g., infectious diseases and






may be routinely generated.
disorders as described in the






Exemplary pancreatic cells that
““Infectious Diseases”” section






may be used according to these
below, especially of the






assays include HITT15 Cells.
urinary tract and skin), carpal






HITT15 are an adherent
tunnel syndrome and






epithelial cell line established
Dupuytren's contracture).






from Syrian hamster islet cells
An additional highly preferred






transformed with SV40. These
indication is obesity and/or






cells express glucagon,
complications associated with






somatostatin, and
obesity. Additional highly






glucocorticoid receptors. The
preferred indications include






cells secrete insulin, which is
weight loss or alternatively,






stimulated by glucose and
weight gain.






glucagon and suppressed by
Aditional highly preferred






somatostatin or
indications are complications






glucocorticoids. ATTC# CRL-
associated with insulin






1777 Refs: Lord and
resistance.






Ashcroft. Biochem. J. 219:






547–551; Santerre et al. Proc.






Natl. Acad. Sci. USA 78:






4339–4343, 1981.


51
HDPCO25
201
Activation of
Assays for the activation of
Highly preferred indications





transcription
transcription through the
include inflammation and





through NFKB
NFKB response element are
inflammatory disorders.





response element in
well-known in the art and may
Highly preferred indications





immune cells (such
be used or routinely modified
include blood disorders (e.g.,





as T-cells).
to assess the ability of
as described below under






polypeptides of the invention
“Immune Activity”, “Blood-






(including antibodies and
Related Disorders”, and/or






agonists or antagonists of the
““Cardiovascular Disorders””).






invention) to regulate NFKB
Highly preferred indications






transcription factors and
include autoimmune diseases






modulate expression of
(e.g., rheumatoid arthritis,






immunomodulatory genes.
systemic lupus erythematosis,






Exemplary assays for
multiple sclerosis and/or as






transcription through the
described below), and






NFKB response element that
immunodeficiencies (e.g., as






may be used or rountinely
described below). An






modified to test NFKB-
additional highly preferred






response element activity of
indication is infection (e.g.,






polypeptides of the invention
AIDS, and/or an infectious






(including antibodies and
disease as described below






agonists or antagonists of the
under “Infectious Disease”).






invention) include assays
Highly preferred indications






disclosed in Berger et al., Gene
include neoplastic diseases






66: 1–10 (1998); Cullen and
(e.g., melanoma, leukemia,






Malm, Methods in Enzymol
lymphoma, and/or as described






216: 362–368 (1992); Henthorn
below under






et al., Proc Natl Acad Sci U.S.A.
“Hyperproliferative






85: 6342–6346 (1988); Black et al.,
Disorders”). Highly preferred






Virus Gnes 15(2): 105–117
indications include neoplasms






(1997); and Fraser et al.,
and cancers, such






29(3): 838–844 (1999), the
as, melanoma, renal cell






contents of each of which are
carcinoma, leukemia,






herein incorporated by
lymphoma, and prostate,






reference in its entirety. T
breast, lung, colon, pancreatic,






cells that may be used
esophageal, stomach, brain,






according to these assays are
liver and urinary cencer. Other






publicly available (e.g.,
preferred indications include






through the ATCC).
benign dysproliferative






Exemplary human T cells that
disorders and pre-neoplastic






may be used according to these
conditions, such as, for






assays include the SUPT cell
example, hyperplasia,






line, which is a suspension
metaplasia, and/or dysplasia.






culture of IL-2 and IL-4
Preferred indications also






responsive T cells.
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,







sepsis, neutropenia,







neutrophilia, psoriasis,







hemophilia, hypercoagulation,







diabetes mellitus, endocarditis,







meningitis, Lyme Disease,







suppression of immune







reactions to transplanted







organs, asthma and allergy.


52
HDPHI51
202
Regulation of
Assays for the regulation of
A highly preferred





transcription
transcription through the FAS
indication is diabetes mellitus.





through the FAS
promoter element are well-
An additional highly preferred





promoter element
known in the art and may be
indication is a complication





in hepatocytes
used or routinely modified to
associated with diabetes (e.g.,






assess the ability of
diabetic retinopathy, diabetic






polypeptides of the invention
nephropathy, kidney disease






(including antibodies and
(e.g., renal failure,






agonists or antagonists of the
nephropathy and/or other






invention) to activate the FAS
diseases and disorders as






promoter element in a reporter
described in the ““Renal






construct and to regulate
Disorders”” section below),






transcription of FAS, a key
diabetic neuropathy, nerve






enzyme for lipogenesis. FAS
disease and nerve damage






promoter is regulated by many
(e.g., due to diabetic






transcription factors including
neuropathy), blood vessel






SREBP. Insulin increases FAS
blockage, heart disease, stroke,






gene transcription in livers of
impotence (e.g., due to diabetic






diabetic mice. This
neuropathy or blood vessel






stimulation of transcription is
blockage), seizures, mental






also somewhat glucose
confusion, drowsiness,






dependent. Exemplary assays
nonketotic hyperglycemic-






that may be used or routinely
hyperosmolar coma,






modified to test for FAS
cardiovascular disease (e.g.,






promoter element activity (in
heart disease, atherosclerosis,






hepatocytes) by polypeptides
microvascular disease,






of the invention (including
hypertension, stroke, and other






antibodies and agonists or
diseases and disorders as






antagonists of the invention)
described in the






include assays disclosed in
““Cardiovascular Disorders””






Xiong, S., et al., Proc Natl
section below), dyslipidemia,






Acad Sci U.S.A., 97(8): 3948–53
endocrine disorders (as






(2000); Roder, K., et al.,
described in the ““Endocrine






Eur J Biochem, 260(3): 743–51
Disorders”” section below),






(1999); Oskouian B, et al.,
neuropathy, vision impairment






Biochem J, 317 (Pt 1): 257–65
(e.g., diabetic retinopathy and






(1996); Berger, et al., Gene
blindness), ulcers and impaired






66: 1–10 (1988); and, Cullen,
wound healing, and infection






B., et al., Methods in Enzymol.
(e.g., infectious diseases and






216: 362–368 (1992), the
disorders as described in the






contents of each of which is
““Infectious Diseases”” section






herein incorporated by
below, especially of the






reference in its entirety.
urinary tract and skin), carpal






Hepatocytes that may be used
tunnel syndrome and






according to these assays, such
Dupuytren's contracture).






as H4IIE cells, are publicly
An additional highly preferred






available (e.g., through the
indication is obesity and/or






ATCC) and/or may be
complications associated with






routinely generated.
obesity. Additional highly






Exemplary hepatocytes that
preferred indications include






may be used according to these
weight loss or alternatively,






assays include rat liver
weight gain. Aditional






hepatoma cell line(s) inducible
highly preferred indications are






with glucocorticoids, insulin,
complications associated with






or cAMP derivatives.
insulin resistance.


52
HDPHI51
202
Activation of
Assays for the activation of
A highly preferred





transcription
transcription through the
indication is allergy.





through STAT6
Signal Transducers and
Another highly preferred





response element in
Activators of Transcription
indication is asthma.





immune cells (such
(STAT6) response element are
Additional highly preferred





as T-cells).
well-known in the art and may
indications include






be used or routinely modified
inflammation and






to assess the ability of
inflammatory disorders.






polypeptides of the invention
Preferred indications include






(including antibodies and
blood disorders (e.g., as






agonists or antagonists of the
described below under






invention) to regulate STAT6
“Immune Activity”, “Blood-






transcription factors and
Related Disorders”, and/or






modulate the expression of
““Cardiovascular Disorders””).






multiple genes. Exemplary
Preferred indications include






assays for transcription
autoimmune diseases (e.g.,






through the STAT6 response
rheumatoid arthritis, systemic






element that may be used or
lupus erythematosis, multiple






routinely modified to test
sclerosis and/or as described






STAT6 response element
below) and






activity of the polypeptides of
immunodeficiencies (e.g., as






the invention (including
described below).






antibodies and agonists or
Preferred indications include






antagonists of the invention)
neoplastic diseases (e.g.,






include assays disclosed in
leukemia, lymphoma,






Berger et al., Gene 66: 1–10
melanoma, and/or as described






(1998); Cullen and Malm,
below under






Methods in Enzymol 216: 362–368
“Hyperproliferative






(1992); Henthorn et al.,
Disorders”). Preferred






Proc Natl Acad Sci U.S.A.
indications include neoplasms






85: 6342–6346 (1988); Georas
and cancers, such as, leukemia,






et al., Blood 92(12): 4529–4538
lymphoma, melanoma, and






(1998); Moffatt et al.,
prostate, breast, lung, colon,






Transplantation 69(7): 1521–1523
pancreatic, esophageal,






(2000); Curiel et al., Eur
stomach, brain, liver and






J Immunol 27(8): 1982–1987
urinary cancer. Other preferred






(1997); and Masuda et al., J
indications include benign






Biol Chem 275(38): 29331–29337
dysproliferative disorders and






(2000), the contents of
pre-neoplastic conditions, such






each of which are herein
as, for example, hyperplasia,






incorporated by reference in its
metaplasia, and/or dysplasia.






entirety. T cells that may be
Preferred indications include






used according to these assays
anemia, pancytopenia,






are publicly available (e.g.,
leukopenia, thrombocytopenia,






through the ATCC).
Hodgkin's disease, acute






Exemplary T cells that may be
lymphocytic anemia (ALL),






used according to these assays
plasmacytomas, multiple






include the SUPT cell line,
myeloma, Burkitt's lymphoma,






which is a suspension culture
arthritis, AIDS, granulomatous






of IL-2 and IL-4 responsive T
disease, inflammatory bowel






cells.
disease, sepsis, neutropenia,







neutrophilia, psoriasis,







suppression of immune







reactions to transplanted







organs and 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””).


62
HLYAF80
212
Activation of
Assays for the activation of
A highly preferred





transcription
transcription through the
indication is allergy.





through STAT6
Signal Transducers and
Another highly preferred





response element in
Activators of Transcription
indication is asthma.





immune cells (such
(STAT6) response element are
Additional highly preferred





as natural killer
well-known in the art and may
indications include





cells).
be used or routinely modified
inflammation and






to assess the ability of
inflammatory disorders.






polypeptides of the invention
Preferred indications include






(including antibodies and
blood disorders (e.g., as






agonists or antagonists of the
described below under






invention) to regulate STAT6
“Immune Activity”, “Blood-






transcription factors and
Related Disorders”, and/or






modulate the expression of
““Cardiovascular Disorders””).






multiple genes. Exemplary
Preferred indications include






assays for transcription
autoimmune diseases (e.g.,






through the STAT6 response
rheumatoid arthritis, systemic






element that may be used or
lupus erythematosis, multiple






routinely modified to test
sclerosis and/or as described






STAT6 response element
below) and






activity of the polypeptides of
immunodeficiencies (e.g., as






the invention (including
described below).






antibodies and agonists or
Preferred indications include






antagonists of the invention)
neoplastic diseases (e.g.,






include assays disclosed in
leukemia, lymphoma,






Berger et al., Gene 66: 1–10
melanoma, and/or as described






(1998); Cullen and Malm,
below under






Methods in Enzymol 216: 362–368
“Hyperproliferative






(1992); Henthorn et al.,
Disorders”). Preferred






Proc Natl Acad Sci USA
indications include neoplasms,






85: 6342–6346 (1988); Georas
such as, for example,






et al., Blood 92(12): 4529–4538
leukemia, lymphoma,






(1998); Moffatt et al.,
melanoma, and prostate,






Transplantation 69(7): 1521–1523
breast, lung, colon, pancreatic,






(2000); Curiel et al., Eur
esophageal, stomach, brain,






J Immunol 27(8): 1982–1987
liver and urinary cancer. Other






(1997); and Masuda et al., J
preferred indications include






Biol Chem 275(38): 29331–29337
benign dysproliferative






(2000), the contents of
disorders and pre-neoplastic






each of which are herein
conditions, such as, for






incorporated by reference in its
example, hyperplasia,






entirety. T cells that may be
metaplasia, and/or dysplasia.






used according to these assays
Preferred indications include






are publicly available (e.g.,
anemia, pancytopenia,






through the ATCC).
leukopenia, thrombocytopenia,






Exemplary rat natural killer
Hodgkin's disease, acute






cells that may be used
lymphocytic anemia (ALL),






according to these assays are
plasmacytomas, multiple






publicly available (e.g.,
myeloma, Burkitt's lymphoma,






through the ATCC).
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, and Lyme Disease.







Additional preferred







indications include infection







(e.g., an infectious disease as







described below under







““Infectious Disease””).


100
HMWDC28
250
Stimulation of
Assays for measuring secretion
A highly preferred





insulin secretion
of insulin are well-known in
indication is diabetes mellitus.





from pancreatic
the art and may be used or
An additional highly preferred





beta cells.
routinely modified to assess
indication is a complication






the ability of polypeptides of
associated with diabetes (e.g.,






the invention (including
diabetic retinopathy, diabetic






antibodies and agonists or
nephropathy, kidney disease






antagonists of the invention) to
(e.g., renal failure,






stimulate insulin secretion.
nephropathy and/or other






For example, insulin secretion
diseases and disorders as






is measured by FMAT using
described in the ““Renal






anti-rat insulin antibodies.
Disorders”” section below),






Insulin secretion from
diabetic neuropathy, nerve






pancreatic beta cells is
disease and nerve damage






upregulated by glucose and
(e.g., due to diabetic






also by certain
neuropathy), blood vessel






proteins/peptides, and
blockage, heart disease, stroke,






disregulation is a key
impotence (e.g., due to diabetic






component in diabetes.
neuropathy or blood vessel






Exemplary assays that may be
blockage), seizures, mental






used or routinely modified to
confusion, drowsiness,






test for stimulation of insulin
nonketotic hyperglycemic-






secretion (from pancreatic
hyperosmolar coma,






cells) by polypeptides of the
cardiovascular disease (e.g.,






invention (including antibodies
heart disease, atherosclerosis,






and agonists or antagonists of
microvascular disease,






the invention) include assays
hypertension, stroke, and other






disclosed in: Ahren, B., et al.,
diseases and disorders as






Am J Physiol, 277(4 Pt
described in the






2): R959–66 (1999); Li, M., et
““Cardiovascular Disorders””






al., Endocrinology,
section below), dyslipidemia,






138(9): 3735–40 (1997); Kim,
endocrine disorders (as






K. H., et al., FEBS Lett,
described in the ““Endocrine






377(2): 237–9 (1995); and,
Disorders”” section below),






Miraglia S et. al., Journal of
neuropathy, vision impairment






Biomolecular Screening,
(e.g., diabetic retinopathy and






4: 193–204 (1999), the contents
blindness), ulcers and impaired






of each of which is herein
wound healing, and infection






incorporated by reference in its
(e.g., infectious diseases and






entirety. Pancreatic cells that
disorders as described in the






may be used according to these
““Infectious Diseases”” section






assays are publicly available
below, especially of the






(e.g., through the ATCC)
urinary tract and skin), carpal






and/or may be routinely
tunnel syndrome and






generated. Exemplary
Dupuytren's contracture).






pancreatic cells that may be
An additional highly preferred






used according to these assays
indication is obesity and/or






include rat INS-1 cells. INS-1
complications associated with






cells are a semi-adherent cell
obesity. Additional highly






line established from cells
preferred indications include






isolated from an X-ray induced
weight loss or alternatively,






rat transplantable insulinoma.
weight gain.






These cells retain
Aditional highly preferred






characteristics typical of native
indications are complications






pancreatic beta cells including
associated with insulin






glucose inducible insulin
resistance.






secretion. References: Asfari






et al. Endocrinology 1992






130: 167.


102
HTADX17
252
Activation of
Assays for the activation of
Highly preferred indications





transcription
transcription through the
include blood disorders (e.g.,





through NFAT
Nuclear Factor of Activated T
as described below under





response in immune
cells (NFAT) response element
“Immune Activity”, “Blood-





cells (such as T-
are well-known in the art and
Related Disorders”, and/or





cells).
may be used or routinely
““Cardiovascular Disorders””).






modified to assess the ability
Highly preferred indications






of polypeptides of the
include autoimmune diseases






invention (including antibodies
(e.g., rheumatoid arthritis,






and agonists or antagonists of
systemic lupus erythematosis,






the invention) to regulate
multiple sclerosis and/or as






NFAT transcription factors and
described below),






modulate expression of genes
immunodeficiencies (e.g., as






involved in
described below), boosting a T






immunomodulatory functions.
cell-mediated immune






Exemplary assays for
response, and suppressing a T






transcription through the
cell-mediated immune






NFAT response element that
response. Additional highly






may be used or routinely
preferred indications include






modified to test NFAT-
inflammation and






response element activity of
inflammatory disorders. An






polypeptides of the invention
additional highly preferred






(including antibodies and
indication is infection (e.g., an






agonists or antagonists of the
infectious disease as described






invention) include assays
below under “Infectious






disclosed in Berger et al., Gene
Disease”). Preferred






66: 1–10 (1998); Cullen and
indications include neoplastic






Malm, Methods in Enzymol
diseases (e.g., leukemia,






216: 362–368 (1992); Henthorn
lymphoma, and/or as described






et al., Proc Natl Acad Sci USA
below under






85: 6342–6346 (1988); Serfling
“Hyperproliferative






et al., Biochim Biophys Acta
Disorders”). Preferred






1498(1): 1–18 (2000); De Boer
indications include neoplasms






et al., Int J Biochem Cell Biol
and cancers, such as, for






31(10): 1221–1236 (1999);
example, leukemia, lymphoma,






Fraser et al., Eur J Immunol
and prostate, breast, lung,






29(3): 838–844 (1999); and
colon, pancreatic, esophageal,






Yeseen et al., J Biol Chem
stomach, brain, liver and






268(19): 14285–14293 (1993),
urinary cancer. Other preferred






the contents of each of which
indications include benign






are herein incorporated by
dysproliferative disorders and






reference in its entirety. T
pre-neoplastic conditions, such






cells that may be used
as, for example, hyperplasia,






according to these assays are
metaplasia, and/or dysplasia.






publicly available (e.g.,
Preferred indications also






through the ATCC).
include anemia, pancytopenia,






Exemplary human T cells that
leukopenia, thrombocytopenia,






may be used according to these
Hodgkin's disease, acute






assays include the JURKAT
lymphocytic anemia (ALL),






cell line, which is a suspension
plasmacytomas, multiple






culture of leukemia cells that
myeloma, Burkitt's lymphoma,






produce IL-2 when stimulated.
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,







asthma and allergy.


102
HTADX17
252
Activation of
Assays for the activation of
Highly preferred indications





transcription
transcription through the
include neoplastic diseases





through GAS
Gamma Interferon Activation
(e.g., leukemia, lymphoma,





response element in
Site (GAS) response element
and/or as described below





immune cells (such
are well-known in the art and
under “Hyperproliferative





as T-cells).
may be used or routinely
Disorders”). Highly preferred






modified to assess the ability
indications include neoplasms






of polypeptides of the
and cancers, such as, for






invention (including antibodies
example, leukemia, lymphoma






and agonists or antagonists of
(e.g., T cell lymphoma,






the invention) to regulate
Burkitt's lymphoma, non-






STAT transcription factors and
Hodgkins lymphoma,






modulate gene expression
Hodgkin's disease), melanoma,






involved in a wide variety of
and prostate, breast, lung,






cell functions. Exemplary
colon, pancreatic, esophageal,






assays for transcription
stomach, brain, liver and






through the GAS response
urinary cancer. Other preferred






element that may be used or
indications include benign






routinely modified to test
dysproliferative disorders and






GAS-response element activity
pre-neoplastic conditions, such






of polypeptides of the
as, for example, hyperplasia,






invention (including antibodies
metaplasia, and/or dysplasia.






and agonists or antagonists of
Preferred indications include






the invention) include assays
autoimmune diseases (e.g.,






disclosed in Berger et al., Gene
rheumatoid arthritis, systemic






66: 1–10 (1998); Cullen and
lupus erythematosis, multiple






Malm, Methods in Enzymol
sclerosis and/or as described






216: 362–368 (1992); Henthorn
below), immunodeficiencies






et al., Proc Natl Acad Sci USA
(e.g., as described below),






85: 6342–6346 (1988);
boosting a T cell-mediated






Matikainen et al., Blood
immune response, and






93(6): 1980–1991 (1999); and
suppressing a T cell-mediated






Henttinen et al., J Immunol
immune response. Additional






155(10): 4582–4587 (1995), the
preferred indications include






contents of each of which are
inflammation and






herein incorporated by
inflammatory disorders.






reference in its entirety.
Highly preferred indications






Exemplary human T cells,
include blood disorders (e.g.,






such as the MOLT4 cell line,
as described below under






that may be used according to
“Immune Activity”, “Blood-






these assays are publicly
Related Disorders”, and/or






available (e.g., through the
““Cardiovascular Disorders””),






ATCC).
and infection (e.g., viral







infections, tuberculosis,







infections associated with







chronic granulomatosus







disease and malignant







osteoporosis, and/or an







infectious disease as described







below under “Infectious







Disease”). An additional







preferred indication is







idiopathic pulmonary fibrosis.







Preferred indications include







anemia, pancytopenia,







leukopenia, thrombocytopenia,







acute lymphocytic anemia







(ALL), 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.


102
HTADX17
252
Activation of
Assays for the activation of
Highly preferred indications





transcription
transcription through the
include inflammation and





through NFKB
NFKB response element are
inflammatory disorders.





response element in
well-known in the art and may
Highly preferred indications





immune cells (such
be used or routinely modified
include blood disorders (e.g.,





as T-cells).
to assess the ability of
as described below under






polypeptides of the invention
“Immune Activity”, “Blood-






(including antibodies and
Related Disorders”, and/or






agonists or antagonists of the
““Cardiovascular Disorders””).






invention) to regulate NFKB
Highly preferred indications






transcription factors and
include autoimmune diseases






modulate expression of
(e.g., rheumatoid arthritis,






immunomodulatory genes.
systemic lupus erythematosis,






Exemplary assays for
multiple sclerosis and/or as






transcription through the
described below), and






NFKB response element that
immunodeficiencies (e.g., as






may be used or rountinely
described below). An






modified to test NFKB-
additional highly preferred






response element activity of
indication is infection (e.g.,






polypeptides of the invention
AIDS, and/or an infectious






(including antibodies and
disease as described below






agonists or antagonists of the
under “Infectious Disease”).






invention) include assays
Highly preferred indications






disclosed in Berger et al., Gene
include neoplastic diseases






66: 1–10 (1998); Cullen and
(e.g., melanoma, leukemia,






Malm, Methods in Enzymol
lymphoma, and/or as described






216: 362–368 (1992); Henthorn
below under






et al., Proc Natl Acad Sci USA
“Hyperproliferative






85: 6342–6346 (1988); Black et
Disorders”). Highly preferred






al., Virus Gnes 15(2): 105–117
indications include neoplasms






(1997); and Fraser et al.,
and cancers, such as, for






29(3): 838–844 (1999), the
example, melanoma, renal cell






contents of each of which are
carcinoma, leukemia,






herein incorporated by
lymphoma, and prostate,






reference in its entirety.
breast, lung, colon, pancreatic,






Exemplary human T cells,
esophageal, stomach, brain,






such as the MOLT4, that may
liver and urinary cancer. Other






be used according to these
preferred indications include






assays are publicly available
benign dysproliferative






(e.g., through the ATCC).
disorders and pre-neoplastic







conditions, such as, for







example, hyperplasia,







metaplasia, and/or dysplasia.







Preferred indications also







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, sepsis, neutropenia,







neutrophilia, psoriasis,







hemophilia, hypercoagulation,







diabetes mellitus, endocarditis,







meningitis, Lyme Disease,







suppression of immune







reactions to transplanted







organs, asthma and allergy.


103
HFTCU19
253
Activation of
Assays for the activation of
Preferred embodiments of the





transcription
transcription through the
invention include using





through NFKB
NFKB response element are
polypeptides of the invention





response element in
well-known in the art and may
(or antibodies, agonists, or





neuronal cells (such
be used or routinely modified
antagonists thereof) in





as SKNMC cells).
to assess the ability of
detection, diagnosis,






polypeptides of the invention
prevention, and/or treatment of






(including antibodies and
Neurological Diseases and






agonists or antagonists of the
Disorders (e.g. Alzheimer's






invention) to regulate NFKB
Disease, Parkinson's Disease,






transcription factors and
Brain Cancer, Seizures).






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 JS, 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).






Exemplary neuronal cells that






may be used according to these






assays include the SKNMC






neuronal cell line.


106
HSDJJ82
256
Protection from
Caspase Apoptosis Rescue.
A highly preferred





Endothelial Cell
Assays for caspase apoptosis
embodiment of the invention





Apoptosis.
rescue are well known in the
includes a method for






art and may be used or
stimulating endothelial cell






routinely modified to assess
growth. An alternative highly






the ability of the polypeptides
preferred embodiment of the






of the invention (including
invention includes a method






antibodies and agonists or
for inhibiting endothelial cell






antagonists of the invention) to
growth. A highly preferred






inhibit caspase protease-
embodiment of the invention






mediated apoptosis.
includes a method for






Exemplary assays for caspase
stimulating endothelial cell






apoptosis that may be used or
proliferation. An alternative






routinely modified to test
highly preferred embodiment






caspase apoptosis rescue of
of the invention includes a






polypeptides of the invention
method for inhibiting






(including antibodies and
endothelial cell proliferation.






agonists or antagonists of the
A highly preferred






invention) include the assays
embodiment of the invention






disclosed in Romeo et al.,
includes a method for






Cardiovasc Res 45(3): 788–794
stimulating endothelial cell






(2000); Messmer et al., Br J
growth. An alternative highly






Pharmacol 127(7): 1633–1640
preferred embodiment of the






(1999); and J Atheroscler
invention includes a method






Thromb 3(2): 75–80 (1996);
for inhibiting endothelial cell






the contents of each of which
growth. A highly preferred






are herein incorporated by
embodiment of the invention






reference in its entirety.
includes a method for






Endothelial cells that may be
stimulating apoptosis of






used according to these assays
endothelial cells. An






are publicly available (e.g.,
alternative highly preferred






through commercial sources).
embodiment of the invention






Exemplary endothelial cells
includes a method for






that may be used according to
inhibiting (e.g., decreasing)






these assays include bovine
apoptosis of endothelial cells.






aortic endothelial cells
A highly preferred






(bAEC), which are an example
embodiment of the invention






of endothelial cells which line
includes a method for






blood vessels and are involved
stimulating angiogenisis. An






in functions that include, but
alternative highly preferred






are not limited to,
embodiment of the invention






angiogenesis, vascular
includes a method for






permeability, vascular tone,
inhibiting angiogenesis. A






and immune cell extravasation.
highly preferred embodiment







of the invention includes a







method for reducing cardiac







hypertrophy. An 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.


120
HLWCF05
270
Activation of
Kinase assay. Kinase assays,
A highly preferred





Adipocyte PI3
for example an GSK-3 assays,
embodiment of the invention





Kinase Signalling
for PI3 kinase signal
includes a method for





Pathway
transduction that regulate
increasing adipocyte survival






glucose metabolism and cell
An alternative highly preferred






survival are well-known in the
embodiment of the invention






art and may be used or
includes a method for






routinely modified to assess
decreasing adipocyte survival.






the ability of polypeptides of
A preferred embodiment of the






the invention (including
invention includes a method






antibodies and agonists or
for stimulating adipocyte






antagonists of the invention) to
proliferation. An alternative






promote or inhibit glucose
highly preferred embodiment






metabolism and cell survival.
of the invention includes a






Exemplary assays for PI3
method for inhibiting






kinase activity that may be
adipocyte proliferation. A






used or routinely modified to
preferred embodiment of the






test PI3 kinase-induced activity
invention includes a method






of polypeptides of the
for stimulating adipocyte






invention (including antibodies
differentiation. An alternative






and agonists or antagonists of
highly preferred embodiment






the invention) include assays
of the invention includes a






disclosed in Forrer et al., Biol
method for inhibiting






Chem 379(8–9): 1101–1110
adipocyte differentiation.






(1998); Nikoulina et al.,
Highly preferred indications






Diabetes 49(2): 263–271
include endocrine disorders






(2000); and Schreyer et al.,
(e.g., as described below under






Diabetes 48(8): 1662–1666
““Endocrine Disorders””).






(1999), the contents of each of
Preferred indications include






which are herein incorporated
neoplastic diseases (e.g.,






by reference in its entirety.
lipomas, liposarcomas, and/or






Mouse adipocyte cells that
as described below under






may be used according to these
“Hyperproliferative






assays are publicly available
Disorders”), blood disorders






(e.g., through the ATCC).
(e.g., hypertension, congestive






Exemplary mouse adipocyte
heart failure, blood vessel






cells that may be used
blockage, heart disease, stroke,






according to these assays
impotence and/or as described






include 3T3-L1 cells. 3T3-L1
below under “Immune






is an adherent mouse
Activity”, “Cardiovascular






preadipocyte cell line that is a
Disorders”, and/or “Blood-






continous substrain of 3T3
Related Disorders”), immune






fibroblast cells developed
disorders (e.g., as described






through clonal isolation and
below under ““Immune






undergo a pre-adipocyte to
Activity””), neural disorders






adipose-like conversion under
(e.g., as described below under






appropriate differentiation
““Neural Activity and






conditions known in the art.
Neurological Diseases””), 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, 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.







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. Highly preferred







indications include neoplasms







and cancer, such as, lipoma,







liposarcoma, lymphoma,







leukemia and breast, colon,







and kidney cancer. Additional







highly preferred indications







include melanoma, prostate,







lung, 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.


120
HLWCF05
270
Activation of
Assays for the activation of
Preferred indications





transcription
transcription through the AP1
include neoplastic diseases





through AP1
response element are well-
(e.g., as described below under





response element in
known in the art and may be
“Hyperproliferative





immune cells (such
used or routinely modified to
Disorders”), blood disorders





as T-cells).
assess the ability of
(e.g., as described below under






polypeptides of the invention
“Immune Activity”,






(including antibodies and
“Cardiovascular Disorders”,






agonists or antagonists of the
and/or “Blood-Related






invention) to modulate growth
Disorders”), and infection






and other cell functions.
(e.g., an infectious disease as






Exemplary assays for
described below under






transcription through the AP1
“Infectious Disease”). Highly






response element that may be
preferred indications include






used or routinely modified to
autoimmune diseases (e.g.,






test AP1-response element
rheumatoid arthritis, systemic






activity of polypeptides of the
lupus erythematosis, multiple






invention (including antibodies
sclerosis and/or as described






and agonists or antagonists of
below) and






the invention) include assays
immunodeficiencies (e.g., as






disclosed in Berger et al., Gene
described below). Additional






66: 1–10 (1988); Cullen and
highly preferred indications






Malm, Methods in Enzymol
include inflammation and






216: 362–368 (1992); Henthorn
inflammatory disorders.






et al., Proc Natl Acad Sci USA
Highly preferred indications






85: 6342–6346 (1988);
also include neoplastic






Rellahan et al., J Biol Chem
diseases (e.g., leukemia,






272(49): 30806–30811 (1997);
lymphoma, and/or as described






Chang et al., Mol Cell Biol
below under






18(9): 4986–4993 (1998); and
“Hyperproliferative






Fraser et al., Eur J Immunol
Disorders”). Highly preferred






29(3): 838–844 (1999), the
indications include neoplasms






contents of each of which are
and cancers, such as, leukemia,






herein incorporated by
lymphoma, prostate, breast,






reference in its entirety.
lung, colon, pancreatic,






Human T cells that may be
esophageal, stomach, brain,






used according to these assays
liver, and urinary cancer. Other






are publicly available (e.g.,
preferred indications include






through the ATCC).
benign dysproliferative






Exemplary human T cells that
disorders and pre-neoplastic






may be used according to these
conditions, such as, for






assays include the SUPT cell
example, hyperplasia,






line, which is an IL-2 and IL-4
metaplasia, and/or dysplasia.






responsive suspension-culture
Preferred indications include






cell line.
arthritis, asthma, AIDS,







allergy, anemia, pancytopenia,







leukopenia, thrombocytopenia,







Hodgkin's disease, acute







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.


120
HLWCF05
270
Activation of
Assays for the activation of
A highly preferred





transcription
transcription through the CD28
embodiment of the invention





through CD28
response element are well-
includes a method for





response element in
known in the art and may be
stimulating T cell proliferation.





immune cells (such
used or routinely modified to
An alternative highly preferred





as T-cells).
assess the ability of
embodiment of the invention






polypeptides of the invention
includes a method for






(including antibodies and
inhibiting T cell proliferation.






agonists or antagonists of the
A highly preferred






invention) to stimulate IL-2
embodiment of the invention






expression in T cells.
includes a method for






Exemplary assays for
activating T cells. An






transcription through the CD28
alternative highly preferred






response element that may be
embodiment of the invention






used or routinely modified to
includes a method for






test CD28-response element
inhibiting the activation of






activity of polypeptides of the
and/or inactivating T cells.






invention (including antibodies
A highly preferred






and agonists or antagonists of
embodiment of the invention






the invention) include assays
includes a method for






disclosed in Berger et al., Gene
stimulating (e.g., increasing)






66: 1–10 (1998); Cullen and
IL-2 production. An alternative






Malm, Methods in Enzymol
highly preferred embodiment






216: 362–368 (1992); Henthorn
of the invention includes a






et al., Proc Natl Acad Sci USA
method for inhibiting (e.g.,






85: 6342–6346 (1988);
reducing) IL-2 production.






McGuire and Iacobelli, J
Additional highly preferred






Immunol 159(3): 1319–1327
indications include






(1997); Parra et al., J Immunol
inflammation and






166(4): 2437–2443 (2001); and
inflammatory disorders.






Butscher et al., J Biol Chem
Highly preferred indications






3(1): 552–560 (1998), the
include autoimmune diseases






contents of each of which are
(e.g., rheumatoid arthritis,






herein incorporated by
systemic lupus erythematosis,






reference in its entirety. T
multiple sclerosis and/or as






cells that may be used
described below),






according to these assays are
immunodeficiencies (e.g., as






publicly available (e.g.,
described below), boosting a T






through the ATCC).
cell-mediated immune






Exemplary human T cells that
response, and suppressing a T






may be used according to these
cell-mediated immune






assays include the SUPT cell
response. Highly preferred






line, which is a suspension
indications include neoplastic






culture of IL-2 and IL-4
diseases (e.g., melanoma, renal






responsive T cells.
cell carcinoma, leukemia,







lymphoma, and/or as described







below under







“Hyperproliferative







Disorders”). Highly preferred







indications include neoplasms







and cancers, such as, for







example, melanoma (e.g.,







metastatic melanoma), renal







cell carcinoma (e.g., metastatic







renal cell carcinoma),







leukemia, lymphoma (e.g,. T







cell 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.







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.


120
HLWCF05
270
Activation of
Assays for the activation of
Highly preferred indications





transcription
transcription through the
include blood disorders (e.g.,





through NFAT
Nuclear Factor of Activated T
as described below under





response element in
cells (NFAT) response element
“Immune Activity”, “Blood-





immune cells (such
are well-known in the art and
Related Disorders”, and/or





as T-cells).
may be used or routinely
““Cardiovascular Disorders””).






modified to assess the ability
Highly preferred indications






of polypeptides of the
include autoimmune diseases






invention (including antibodies
(e.g., rheumatoid arthritis,






and agonists or antagonists of
systemic lupus erythematosis,






the invention) to regulate
multiple sclerosis and/or as






NFAT transcription factors and
described below),






modulate expression of genes
immunodeficiencies (e.g., as






involved in
described below), boosting a T






immunomodulatory functions.
cell-mediated immune






Exemplary assays for
response, and suppressing a T






transcription through the
cell-mediated immune






NFAT response element that
response. Additional highly






may be used or routinely
preferred indications include






modified to test NFAT-
inflammation and






response element activity of
inflammatory disorders. An






polypeptides of the invention
additional highly preferred






(including antibodies and
indication is infection (e.g., an






agonists or antagonists of the
infectious disease as described






invention) include assays
below under “Infectious






disclosed in Berger et al., Gene
Disease”). Preferred






66: 1–10 (1998); Cullen and
indications include neoplastic






Malm, Methods in Enzymol
diseases (e.g., leukemia,






216: 362–368 (1992); Henthorn
lymphoma, and/or as described






et al., Proc Natl Acad Sci USA
below under






85: 6342–6346 (1988); Serfling
“Hyperproliferative






et al., Biochim Biophys Acta
Disorders”). Preferred






1498(1): 1–18 (2000); De Boer
indications include neoplasms






et al., Int J Biochem Cell Biol
and cancers, such as, for






31(10): 1221–1236 (1999);
example, leukemia, lymphoma,






Fraser et al., Eur J Immunol
and prostate, breast, lung,






29(3): 838–844 (1999); and
colon, pancreatic, esophageal,






Yeseen et al., J Biol Chem
stomach, brain, liver and






268(19): 14285–14293 (1993),
urinary cancer. Other preferred






the contents of each of which
indications include benign






are herein incorporated by
dysproliferative disorders and






reference in its entirety. T
pre-neoplastic conditions, such






cells that may be used
as, for example, hyperplasia,






according to these assays are
metaplasia, and/or dysplasia.






publicly available (e.g.,
Preferred indications also






through the ATCC).
include anemia, pancytopenia,






Exemplary human T cells that
leukopenia, thrombocytopenia,






may be used according to these
Hodgkin's disease, acute






assays include the SUPT cell
lymphocytic anemia (ALL),






line, which is a suspension
plasmacytomas, multiple






culture of IL-2 and IL-4
myeloma, Burkitt's lymphoma,






responsive T cells.
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,







asthma and allergy.


120
HLWCF05
270
Activation of
Assays for the activation of
Highly preferred indications





transcription
transcription through the
include inflammation and





through NFKB
NFKB response element are
inflammatory disorders.





response element in
well-known in the art and may
Highly preferred indications





immune cells (such
be used or routinely modified
include blood disorders (e.g.,





as T-cells).
to assess the ability of
as described below under






polypeptides of the invention
“Immune Activity”, “Blood-






(including antibodies and
Related Disorders”, and/or






agonists or antagonists of the
““Cardiovascular Disorders””).






invention) to regulate NFKB
Highly preferred indications






transcription factors and
include autoimmune diseases






modulate expression of
(e.g., rheumatoid arthritis,






immunomodulatory genes.
systemic lupus erythematosis,






Exemplary assays for
multiple sclerosis and/or as






transcription through the
described below), and






NFKB response element that
immunodeficiencies (e.g., as






may be used or rountinely
described below). An






modified to test NFKB-
additional highly preferred






response element activity of
indication is infection (e.g.,






polypeptides of the invention
AIDS, and/or an infectious






(including antibodies and
disease as described below






agonists or antagonists of the
under “Infectious Disease”).






invention) include assays
Highly preferred indications






disclosed in Berger et al., Gene
include neoplastic diseases






66: 1–10 (1998); Cullen and
(e.g., melanoma, leukemia,






Malm, Methods in Enzymol
lymphoma, and/or as described






216: 362–368 (1992); Henthorn
below under






et al., Proc Natl Acad Sci USA
“Hyperproliferative






85: 6342–6346 (1988); Black et
Disorders”). Highly preferred






al., Virus Gnes 15(2): 105–117
indications include neoplasms






(1997); and Fraser et al.,
and cancers, such






29(3): 838–844 (1999), the
as, melanoma, renal cell






contents of each of which are
carcinoma, leukemia,






herein incorporated by
lymphoma, and prostate,






reference in its entirety. T
breast, lung, colon, pancreatic,






cells that may be used
esophageal, stomach, brain,






according to these assays are
liver and urinary cancer. Other






publicly available (e.g.,
preferred indications include






through the ATCC).
benign dysproliferative






Exemplary human T cells that
disorders and pre-neoplastic






may be used according to these
conditions, such as, for






assays include the SUPT cell
example, hyperplasia,






line, which is a suspension
metaplasia, and/or dysplasia.






culture of IL-2 and IL-4
Preferred indications also






responsive T cells.
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,







sepsis, neutropenia,







neutrophilia, psoriasis,







hemophilia, hypercoagulation,







diabetes mellitus, endocarditis,







meningitis, Lyme Disease,







suppression of immune







reactions to transplanted







organs, asthma and allergy.
























TABLE 2







SEQ



Score/





Contig
ID
Analysis

PFam/NR Accession
Percent
NT


Clone ID:
ID:
NO: X
Method
PFam/NR Description
Number
Identity
From
NT To























HCHAA63
456591
11
blastx.2
NEURAL
sp|Q9NQX5|NPD1_HUMAN
57%
130
867






PROLIFERATION

53%
242
676






DIFFERENTIATION

94%
952
1104






AND CONTROL 1

95%
827
952








34%
324
656








34%
627
731


HCNSP40
711868
12
WUblastx.
(AK008940) putative
dbj|BAB25981.1|
40%
106
744





64
[Mus musculus]


HCNSP40
456440
134
blastx.2
UNKNOWN PROTEIN
sp|Q29229|Q29229
36%
203
673






(FRAGMENT).


HBMXN79
460351
13
blastx.2
CDNA FLJ13110 fis,
sp|BAB14444|BAB14444
65%
75
647






clone NT2RP3002549,






moderately similar 1


HGBGV89
460353
14
blastx.2
tumor-associated
pir|JC6544|JC6544
99%
55
645






antigen TM4SF5 -






human


HDAAC10
456443
16
WUblastx.
(AK000029) unnamed
dbj|BAA90893.1|
99%
1082
144





64
protein product [Homo






sapiens]


HLHFP03
460467
17
blastx.2
(AX058598) unnamed
emb|CAC22514.1|
95%
390
43






protein product [Homo






sapiens]


HMELR03
456482
18
blastx.2
CG5604 PROTEIN.
sp|Q9VL06|Q9VL06
61%
1434
556








68%
687
22








77%
1489
1436


HKAFB88
460635
20
HMMER
PFAM:
PF01587
211.6
84
−566





2.1.1
Monocarboxylate






transporter





blastx.2
MONOCARBOXYLATE
sp|O15427|MOT3_HUMAN
99%
1368
448






TRANSPORTER 3






(MCT 3).


HSOAH66
456540
21
blastx.2
PRO2550.
sp|AAG35515|AAG35515
73%
13
177


HCRBR74
460694
22
blastx.2
(AJ224677) scrapie
emb|CAA12059.1|
100% 
507
214






responsive protein 1






[Homo sapiens]


HMSJW18
457182
23
blastx.2
CDNA: FLJ21463 fis,
sp|BAB15071|BAB15071
48%
716
1006






clone COL04765.

50%
500
679








57%
337
435








81%
1835
1659


HHTLF25
461438
24
blastx.2
KILLER ACTIVATING
sp|Q9UMT3|Q9UMT3
99%
142
474






RECEPTOR






ASSOCIATED






PROTEIN, ISOFORM






B.


HELDY05
456449
26
blastx.2
CDNA FLJ20635 FIS,
sp|Q9NWS6|Q9NWS6
92%
1217
846






CLONE KAT03466.

100% 
692
576








85%
1273
1193


HFPDE69
460450
28
blastx.2
CDNA: FLJ21463 fis,
sp|BAB15071|BAB15071
63%
2
295






clone COL04765.


HSRDE35
460538
30
WUblastx.
(AF176814) Ab1-philin
gb|AAG09269.1|AF176814_1
91%
1286
522





64
2 [Mus musculus]


HOFNY71
1152170
32
blastx.2
(AX068268) unnamed
emb|CAC27253.1|
78%
200
1042






protein product [Homo






sapiens]


HOFNY71
456517
136
blastx.2
(AX068268) unnamed
emb|CAC27253.1|
98%
344
499






protein product [Homo

78%
3
215






sapiens]


HOFMS34
704682
137
blastx.2
hPMSR6 [Homo
gb|AAA97460.1|
57%
324
605






sapiens]


HPMBR15
456527
33
blastx.2
CDNA FLJ11110 FIS,
sp|Q9NUV9|Q9NUV9
96%
2
193






CLONE

56%
18
335






PLACE1005921,






WEAKLY SIMILAR






TO AIG1 1


HCNSB01
460699
35
blastx.2
Secretory carrier
sp|AAB62723|AAB62723
92%
2345
1368






membrane protein.

100% 
2353
2261


HSDGR57
460599
36
blastx.2
KI-1/57
sp|O75804|O75804
96%
907
533






INTRACELLULAR






ANTIGEN






(FRAGMENT).


HJABB94
456466
37
blastx.2
(AL138875)
emb|CAC28082.1|
94%
329
33






bA103J18.1.3 (novel

100% 
1539
1306






protein, isoform 3)

38%
429
364






[Homo 1


HKIYO61
456471
40
blastx.2
CDNA FLJ13381 fis,
sp|BAB14576|BAB14576
43%
1153
914






clone PLACE1001010.


HMKCW19
740950
41
blastx.2
(AF220152) TACC2
gb|AAF29537.2|
99%
493
1560






[Homo sapiens]


HBIAS26
460709
43
blastx.2
CG14483 PROTEIN.
sp|Q9V849|Q9V849
38%
345
127


HNDAH54
460486
45
blastx.2
CDNA FLJ20378 FIS,
sp|Q9NX85|Q9NX85
86%
12
170






CLONE KAIA0536.


HTDAD22
456553
49
blastx.2
CDNA FLJ20378 FIS,
sp|Q9NX85|Q9NX85
61%
971
810






CLONE KAIA0536.

78%
750
682








72%
811
746








44%
1008
880


HSKYU29
460531
50
blastx.2
(AF060922) My020
gb|AAG43134.1|AF060922_1
91%
1026
586






protein [Homo sapiens]

100% 
619
575


HHGDU58
460717
51
blastx.2
TBX18 PROTEIN (T-
sp|O95935|TX18_HUMAN
89%
138
839






BOX PROTEIN 18)

74%
10
336






(FRAGMENT).

100% 
836
916


HUKBH05
457195
56
blastx.2
hypothetical protein
pir|T46306|T46306
86%
10
303






DKFZp434D2411.1 -






human (fragment)


HBJFU48
460392
58
blastx.2
PRO1722.
sp|Q9P195|Q9P195
64%
183
317








73%
31
132








63%
134
190


HCUDB38
460783
60
blastx.2
PRO1902 PROTEIN.
sp|Q9UHT1|Q9UHT1
55%
798
583


HBMWB01
460396
70
blastx.2
(AB055298)
dbj|BAB21923.1|
77%
313
92






hypothetical protein






[Macaca fascicularis]


HNGAX58
460488
74
blastx.2
HYPOTHETICAL 15.4
sp|Q99770|Q99770
60%
11
232






KDA PROTEIN.

75%
235
282


HCFCE10
456437
77
blastx.2
ENV PROTEIN.
sp|Q9YNA9|Q9YNA9
28%
1287
799








37%
688
464








27%
1582
1289








48%
801
682


HCFNC26
456439
78
blastx.2
LINE-1 ELEMENT
sp|O62658|O62658
30%
646
47






ORF2.

48%
1237
1076








33%
1304
993








29%
399
73








66%
1356
1312


HORBI81
456519
81
blastx.2
ESTROGEN-
sp|O95361|O95361
95%
111
1067






RESPONSIVE B BOX

87%
1046
1282






PROTEIN.

55%
844
870


HOSCY73
456520
82
blastx.2
SUPPRESSOR OF G2
sp|Q9Y2Z0|Q9Y2Z0
98%
1382
2002






ALLELE OF SKP1

98%
79
477






HOMOLOG.

24%
826
975


HNGIK21
456500
85
blastx.2
intrinsic factor-B12
pir|T09456|T09456
81%
269
415






receptor Cubilin

88%
430
456






precursor - human


HE9FE83
460431
86
blastx.2
PRO0898.
sp|AAG35479|AAG35479
60%
21
290


HNGEM24
460493
88
blastx.2
PRO0898.
sp|AAG35479|AAG35479
72%
52
162








80%
5
49


HTNBR95
457175
92
blastx.2
U3 SMALL
sp|O00566|MP10_HUMAN
93%
1121
651






NUCLEOLAR

96%
266
114






RIBONUCLEOPROTEIN






PROTEIN MPP10 (M1


HUKEX85
457196
93
blastx.2
(AB055293)
dbj|BAB21918.1|
65%
923
636






hypothetical protein






[Macaca fascicularis]


HUKEX85
457176
144
blastx.2
(AB055293)
dbj|BAB21918.1|
65%
923
636






hypothetical protein






[Macaca fascicularis]


HWTBM45
456569
94
blastx.2
CDNA: FLJ21463 fis,
sp|BAB15071|BAB15071
74%
987
814






clone COL04765.

82%
820
701


HADFK68
683272
95
blastx.2
probable transposase -
pir|S72481|S72481
58%
127
234






human transposon

58%
26
97






MER37

31%
345
497


HSDZM95
460524
97
blastx.2
(AY012159) virion-
gb|AAG42155.1|
100% 
439
356






associated nuclear-






shuttling protein [Mus






musculus]


HSNAA55
460535
98
blastx.2
LIPOIC ACID
sp|O43766|O43766
100% 
293
192






SYNTHETASE (LIPSYN)






(LIPOATE






SYNTHASE) 1


HAEAV45
554653
106
blastx.2
ribosomal protein L10,
pir|A42735|A42735
99%
683
135






cytosolic - human


HLNAB07
460469
109
blastx.2
CDNA: FLJ21463 fis,
sp|BAB15071|BAB15071
65%
11
184






clone COL04765.

73%
175
297


HSZAF47
456551
111
HMMER
PFAM: Collagen triple
PF01391
 54.4
299
478





2.1.1
helix repeat (20 copies)





blastx.2
(AX054802) unnamed
emb|CAC21966.1|
92%
98
976






protein product [Homo






sapiens]


HTADX17
457172
112
WUblastx.
(AK009505) putative
dbj|BAB26328.1|
67%
548
778





64
[Mus musculus]

53%
490
585








58%
165
488


HFTCU19
735139
113
blastx.2
CG11488 PROTEIN.
sp|Q9VPL3|Q9VPL3
25%
32
763


HTLDP69
456560
117
blastx.2
hypothetical protein
pir|T12538|T12538
97%
96
485






DKFZp434I114.1 -






human


HNFIU96
460611
120
blastx.2
PVA1 GENE.
sp|Q26195|Q26195
54%
280
134








61%
139
86








66%
126
91


HGLDE38
460647
121
blastx.2
CDNA FLJ20489 FIS,
sp|Q9NX17|Q9NX17
65%
6
161






CLONE KAT08285.


HNGAC63
460491
123
blastx.2
Hypothetical 12.9 kDa
sp|BAB12124|BAB12124
69%
331
543






protein.


HCUFZ62
461448
125
blastx.2
SYNAPTOGYRIN 2
sp|O43760|SNG2_HUMAN
96%
124
375






(CELLUGYRIN).

88%
883
1125








96%
806
889


HSSJN64
460596
126
blastx.2
PRO2852.
sp|AAG35505|AAG35505
60%
81
248








54%
250
339


HTEHH53
456555
127
WUblastx.
(AK006829) putative
dbj|BAB24760.1|
64%
85
639





64
[Mus musculus]


HSLBF69
456539
132
HMMER
PFAM: Sulfatase
PF00884
107.7
941
1225





2.1.1





WUblastx.
(AK013194) putative
dbj|BAB28703.1|
81%
1476
1715





64
[Mus musculus]

77%
161
1480


HTEDS39
745350
133
blastx.2
C-type lectin
sp|AAG00516|AAG00516
100% 
142
435






(Fragment).

89%
511
627


HTEDS39
456554
150
blastx.2
(AF133299) lectin-like
gb|AAF22159.1|AF133299_1
84%
198
374






NK cell receptor LLT1

72%
80
283






[Homo sapiens]

88%
738
866








78%
450
590




















TABLE 3








SEQ






ID

EST Disclaimer













NO:
Contig
Range
Range



Clone ID:
X
ID:
of a
of b
Accession #'s















HCHAA63
11
456591
1–1128
15–1142
AL523472, BF313764, AL523471, AL530333, AL521468, BF311636, BF530286, BF966244,







AA480091, BF316117, BE882586, BF314941, AI879485, BF340062, BF966904, AV726609,







AW157080, BE262566, BF568133, BF207161, BE378380, BF528979, BE272500,







BE314276, BF338232, AI800618, BF315847, AI951795, AI799722, BE799574, AL513715,







BE311751, AI361036, BF476717, AI805156, BF347594, AI888307, BF529289, BF313281,







AI878983, AW163049, BF678944, AW651699, BF725309, BF314648, BF435522,







AW593199, AI889480, AA621805, BE047119, BF346663, BF343191, AW612912,







BF195662, AI801367, BF858451, AW338273, AI742494, AA044254, AI683381, AI289074,







AW673160, AA397622, BF092386, BG056125, BF094832, BF092567, BF912620,







BF092571, BF094666, BF912623, BF094744, BF092351, BF092398, BF094619, BF092384,







BF092333, BF092564, BF094557, BF092536, AI569761, BF094617, BF888230, BF092413,







BF092579, BG222535, AI921223, AI569988, BF094634, BF094798, AL513716, BF825635,







BF092531, AI954055, AW518921, BF377293, AI811191, AA609522, BF888222, AI683749,







AI581035, W57690, AI889033, BE795631, BF092757, AI926831, AI598080, AW243932,







AI498981, BF092541, BF825634, BF912637, AW591212, AI433980, BF092373, AI369785,







AI858952, BF094749, AA040108, BE383488, H46483, N52689, BF092538, AA435740,







BF222275, AA877657, BF092756, AW955234, AA069342, BF092407, T05793, BE168727,







AA877463, BF092540, AI139320, BG180787, AI805420, W32237, AI370294, AW604489,







AI761161, AA661720, BF827029, AI826358, AA847765, BF094716, AI358099, AW753436,







AI936090, AI433260, AI678478, AW051375, AI969161, AA773192, AI094479, BF092405,







AW604488, T05288, BF589555, AW769427, BG165620, AI624185, AA069343, AW963939,







AA040109, AA631244, AI631324, BE895880, BF094783, BF340201, AI688195, AI872060,







BF206472, AA719907, AA044278, AF272357, AF283247, X67209, AL050221, and







AX015351.


HCNSP40
12
711868
1–1020
15–1034
AW001557, AA524870, AW962331, AI339658, AW603823, AI339800, AA327382,







AA514534, and AI269776.


HBMXN79
13
460351
1–1260
15–1274
BF127526, BF968547, AA528121, AW973043, AI147569, AI273717, AI309014, AI050673,







AW078851, BE393420, BF575449, AI290263, AI079315, AW593082, AA758714,







AA758616, AI784589, N98290, BE885364, AI809580, BF744842, AA100605, AW059741,







AA371090, and AI192275.


HGBGV89
14
460353
1–954
15–968
BF435758, AA694177, AW665746, AI052584, AA910944, W84524, AI242043, R95924,







AA844492, AI284409, H49258, W84512, AI245850, W86027, AA918870, H49257,







AV698843, AA782492, BE677728, T55097, AI262459, AA772665, AV725321, AV702671,







AV707589, AV701858, AV705234, AV702673, AV709356, AV707649, AV654035,







AV703417, AV653353, AV708024, AV707401, AV702164, AV708786, AV729473,







AV727822, AV728404, AV651897, AV725031, AV725618, AV725582, AV726194,







AV727238, AV707311, AV726628, AV703214, AV702625, AV726755, AV703632,







AV706407, AV701874, AV706223, AV652528, AV709580, AV652001, AV661286,







AV726590, AV703761, AV708809, AV706718, AA740678, AV703367, AV726156,







AV687342, AV652547, AV701873, AV709935, AV704144, AV704785, AV725991,







AV703168, AV705504, AV727618, AV729388, AV704954, AV727468, AV702498,







AV704756, AA631650, AV709494, AV707948, AV708520, AV706410, AV706076,







AV702851, AV707654, AV701183, AV729076, AV707628, AV706882, AV706133,







AV658436, AV725255, AV704590, AV660258, AV725181, AV709596, AV701704,







AV682997, AV708858, AV691615, AV705416, AV707686, AV705655, AV727126,







AV703525, AV702787, AV707794, AV655577, AV650843, AV725369, AV702817,







AV709776, AV686417, AV706162, AV709880, AV701880, AV701728, AV705299,







AV694871, AV709273, AV706165, AV704116, AV702071, AV706814, AV727459,







AV701876, AV728715, AV701184, AV725577, AV705343, AV703090, AV702339,







AV729366, AV707009, AV702958, AV705525, AV728546, AV727343, AV702854,







AV727314, AV656283, AV702954, AV704611, AV706025, AV693117, AV704401,







AV729129, AV725596, AV704879, AV709932, AV703505, AV706683, AV726337,







AV702783, AV727469, AV706851, AV729411, AV706991, AV709939, AV706527,







AV698270, AV707088, AV706989, AV726830, AV725386, AV645545, AV708991,







AV728997, AV726653, AV703137, AV701754, AV726559, AV727103, AV702280,







AV706104, AV702760, AV729597, AV656240, AV702187, AV692972, AV728455,







AV652156, AV727337, AV729077, AV729220, AV655901, AV685688, AV689800,







AV709733, AV704974, AV687909, AV727032, AV708087, AV702728, AV703591,







AV706560, AV707882, AV701499, AV701538, AV706741, AV707510, AV702738,







AV704740, AV706734, AV701879, AV705020, AV646736, AV725152, AV728289,







AV707322, AV709692, AV707798, AV724987, AV705684, AV702146, AV707368,







AV708320, AV705263, AV705189, AV729555, AV709244, AV701844, AV725387,







AV703086, AV726787, AV707822, AV704981, AV656004, AV727189, AV707556,







AV709075, AV645768, AV705814, AV705267, AV727017, AV725645, AV702800,







AV685955, AV702537, AV707658, AV729424, AV726781, AV704626, AV706891,







AV709025, AV726681, AF027204, AX020190, A25909, AR088705, AR062871, AR038855,







AX028305, AR079804, AR091393, AX009487, A85395, A85476, AX026824, AX026823,







AX001322, AR062872, AR062873, AX001082, AJ244004, AR017907, AX021518,







AJ244005, AX006816, AR037157, AJ244003, A20702, A20700, AX006825, AX006826,







AX006822, A43189, A43188, AX008555, AX042372, AR038762, I63120, A98767, A93963,







A93964, A98420, A98423, A98432, A98436, A98417, A98427, Y16359, AR008429, D78345,







A86792, I44681, X83865, A84772, U94592, AX035462, A84776, A84773, A84775, A84774,







AR054109, AX006821, AR067731, AR067732, A58522, A91750, AJ244007, I18895,







A18053, I13349, M28262, AX011024, A93016, AX009712, AX003207, A60212, A60209,







A60210, I15717, A60211, I15718, E03627, AR096545, I48927, AR069650, Y09813, A02712,







A77094, A77095, AR080470, AR077142, A81878, I84553, AX033488, AX033489,







AX033490, A95051, AR095492, I84554, A18050, AX012337, A23334, A75888, I70384,







AX033474, AX033486, AX033487, A64973, A60111, A23633, AR007512, I08396, I00682,







A11623, E00609, A11624, E13740, A11178, E01007, AX035980, A10361, I49890, I62368,







AR073846, AX006823, A35536, A35537, A91965, A02135, A04663, A02136, A04664,







I06859, I08395, AR043601, A11245, AX032758, I03331, A02710, E12615, AX018504,







AR035193, E14304, A07700, AX027811, AX027809, A13392, A13393, AR031488, I13521,







I52048, A27396, AX027813, AX030369, AX030368, AR069374, AR027100, I44531, I28266,







AR069375, AX042377, AR083151, AX027812, AX027810, AR091518, AX027816,







AR072535, AX042375, AX027817, AX027818, AX027814, I21869, AX042373, I44516,







A70040, E16678, A82653, AR093385, E16636, AR074365, AR093392, A58524, A58523,







A24783, A24782, AX032992, AX032993, A92133, A95117, AR085082, AR085089,







AR085091, AR095490, AR095491, AR085079, AR085083, X81969, A90655, AF149828,







I01995, I08051, AR038066, AR031566, I25027, AR069426, I26929, I44515, I26928, I26930,







I26927, I60241, I60242, A20699, E00696, E00697, AX001330, E03813, AR093384, I66482,







AR009151, I66485, I66483, I66484, AX004550, AX036660, AX036661, I66498, I66497,







I66496, AR027099, I66487, I66486, AX023553, AX003194, AX023548, AR072540,







AX003206, AR093383, AR051652, AR051651, Z32836, D50010, AJ230935, AX047064,







AJ230902, AJ270780, AJ230972, I05558, AX047063, AX027636, A70872, AB043473,







W86026, AC016135, AC002518, AC022305, and AC018512.


HARAA15
15
460398
1–787
15–801
AA317225, and AA317195.


HDAAC10
16
456443
1–1184
15–1198
AW073901, AA573809, AA573925, AW029086, AI948957, AI660866, AW340058,







AW628242, AI193160, AI187143, AI445861, N79237, AA573898, AA483813, AI248965,







AI318094, BF084254, AI076155, AA552031, AW451534, AA551795, R06009, AA552527,







W03754, AW960587, AA588124, AW840805, Z36760, T31354, AI673158, T24507,







AA588114, C00989, AA573864, AX016706, AK000029, AB036706, AB016496, AX016708,







and AL354714.


HLHFP03
17
460467
1–599
15–613
H46196, AI421986, H19572, H46195, BF947135, H19490, BF738481, BF994257, BF127477,







AW139949, BF947011, AF141377, AF169202, AC011976, and AC011976.


HMELR03
18
456482
1–1607
15–1621
AW853219, AW997066, BE243270, AA587619, BE711235, BE711238, R92130, AF114048,







AA885705, BE867296, AA631738, AA573593, BF088850, BE242758, AW629955,







BE243140, AW275658, BE243090, and AL136418.


HNGJJ65
19
456504
1–1108
15–1122
AL096710, AL096710, and AL096710.


HKAFB88
20
460635
1–1354
15–1368
AL513917, AI346905, AI188571, AI300172, AI924557, AW190979, BF058735, AW081916,







AI889056, BE731954, BF058331, AW152435, AI831568, AW273379, BF432470, AI828946,







AW070806, AW269473, BG056360, AI696939, AW262601, AW517431, AW338247,







BG056160, AI887577, AI887270, AI200262, AW241554, BF107179, AA411305, AI089787,







AW337188, AI814972, AW191885, AI148501, AA129777, AW265062, AW264993,







AI138479, AW044434, AI085751, AI819917, AI346889, AI348064, AI280971, AA832210,







AI369677, AI206844, AI278708, AA521070, AA505353, AW194948, AA293664,







AA832335, AI360658, AW970750, AA765683, AW076017, AI806875, AI086842,







AW083772, AI817006, AA627943, AI219190, AI304420, AI160697, AI367482, W46383,







AA832334, AI221077, AA527578, AI417425, AI375918, AI219396, AW135952, AI149452,







AA285161, AI375151, AI262227, AI660563, AI400808, AA639863, BE858635, AA457698,







AA743228, BF939697, BE274053, AW204473, AI474200, AW385016, AI215947,







AA479826, AA480795, AW135744, AA848027, AA812576, AA829620, AI149503,







AW516782, AI380474, AW082780, AU139188, AA918694, BF896273, AA864697,







AI290433, AA741211, AI123950, AI220924, N93936, AA291604, AI246014, AL513918,







N27015, AA932153, N40020, AW086005, AI342099, AI032257, AI214752, AA449724,







BF765417, AI200028, BE045994, BF763032, AI209038, BF765125, AI073359, BF765421,







BF765708, AA887785, AI886251, BF800377, AU156601, BF516178, T59382, BE207860,







AI085877, BF764971, AA429578, AI991676, AA192884, W46772, AI956153, AI221033,







AU147377, H87158, AA643165, BF994266, AA133273, N67739, BF766053, BF766051,







BF594416, W40436, AI344579, AA192093, N40138, AA411598, W03557, H30281,







AA720977, AA025261, AI766871, BE551602, AA976638, BF304900, N99655, AA227863,







N71705, AI313892, AA931200, AI073362, AW969458, BF309094, AW839848, BE301150,







AW859101, AW862111, BE733798, AA069211, AA448579, D20249, F26295, AI243265,







AI200881, AA121404, BG168026, AU121590, AA745672, AU139677, BE615010,







AI659960, AI656179, AI339414, AI151203, AA432329, H87414, H87660, AA025339,







AI219572, AA483826, BF594845, M91486, W19224, AI128892, AI038898, W19219,







BF108180, AI286251, AA069170, AA227806, BE293064, AI970111, BE614783, BF569212,







BE273264, AA746333, AA490374, AI024775, BF765128, BG025141, BG255800,







AA809985, BE272680, BG035031, AI650708, AI699651, AI952840, AI572319, U81800,







U87627, AF178954, and AF204397.


HSOAH66
21
456540
1–1174
15–1188
AA935659, AW070446, AI336306, AI636549, BF812696, W27084, AA351868, BF817511,







AI281622, AW169469, AI640905, AA196287, AV754525, AA368329, AW028376, H50031,







N49540, AI940546, AA935827, AA847341, AW953437, AW467781, AI114755, AV735017,







AW029626, AI888050, BF724416, BG253317, N49298, BE747923, AI174827, AA364082,







AI520925, AA230024, AI475294, AA640563, AU145155, AW516058, AV732057, R79396,







AW837273, AA831801, AW837267, BF815277, AV730440, AA101744, BF892846,







AA828840, BE880448, BF881869, H44137, AI819419, AA832016, W28786, BE465696,







AA715508, AA385094, AL046487, AI884404, AU121702, BF849123, AI133083, BF739035,







BF773356, AA827383, AV708459, AA015948, AA507990, AI819391, AA496941,







AW152451, AW366198, AW850985, BE706737, AI799421, AA629939, BE888431,







AA569220, AA661554, AW970856, BE675831, BG005873, AW970570, BF773082,







AW880986, BE045167, AI160875, F23268, BE049409, H59856, BE042324, AA665102,







AI752977, AA084320, AW129188, BF880160, BF724945, BF056684, R31264, BE139230,







AI318548, AI344906, R66407, AA364147, AA767571, AI620666, AA218684, AA446931,







BF882222, AW614862, AI805699, AW837219, BF841410, AA420531, AI982994,







AW157128, AA219166, AU157259, H05066, AA171400, AI499376, AV764119, U85195,







AE000658, AC007686, AB042425, AF207550, AF113694, AP000255, AC020916,







AP000213, AC006360, AP000135, AC026776, AC007404, AP000031, AC024075,







AC025264, AC008985, AC010458, AL117336, AL353748, AC005486, AL355476,







AL391839, AL031674, AC006974, AP001712, AC005288, AC008536, AC009004,







AC024571, AC019187, AL034420, Z93015, AL049743, AL133355, AC008543, AC006370,







AC004148, AC022013, AC018507, AC017087, AL035699, AL035420, AC008537,







AB023048, AP000511, AL138688, AL117339, AC006329, AC005529, AL121586,







AC006261, AC011495, AL157384, AL035658, AC009060, AC007358, AC010519,







AC006538, AC016602, AC011811, AC006013, AL031662, AC007383, AL109613,







AC004960, AC008379, AC005330, AC005744, AL033378, AC007537, Z83844, AL157702,







AL049694, AL137009, AL136137, AL138716, AL117329, AC000038, AC004846,







AC027644, U07562, AC004213, AL080243, AC005530, AC000046, AC008264, AC020629,







AF109907, AL133548, AL110280, AC005598, AC005484, AC073323, AC005668,







AC006075, AP001748, AC006390, AL031296, AL133174, AL008987, AP000099,







AC005696, AL031904, AP000030, Z23878, AL136228, AL133517, AL022320, Z49258,







AP001725, AC005522, AC004099, AC079844, AC003102, AC002404, AC005377,







AC006599, AL022721, AC009405, AL121905, AC004626, AL118520, AC003071,







AC010521, AP000514, AC005751, AC004006, AL022723, AC004257, AC004991,







AC010553, AC006241, AC007283, AL031591, AF190464, AC009087, AF235097,







AL008582, AC020913, AC005837, AC008745, AL031121, AC005102, AL080316,







AC007993, AL121934, AL035588, AL353804, AL035461, AC020956, AC040163,







AL033533, AC004081, AL158159, AC073898, Z69375, AC005066, AL031681, AL050335,







AC011540, Z75744, AP000036, AP001717, AC011742, Z98752, U95742, AC007597,







AF200923, AC011469, AC010223, AC007546, AC005089, AC007900, AC005091,







AC005519, AC006088, AC005527, AL353777, AC005080, AC005859, AC015550,







AF258547, AC025436, AL139286, AF241735, AC008753, AL050318, AL121928,







AC018738, AC011477, AJ003147, AP000127, AP000205, AL031284, AL138832,







AL020993, AC002073, AL049547, AC005411, AC008848, AC004883, AL117187,







AL139351, AC006970, AL158141, AC005937, AL136445, AL096840, AC003957, U65590,







Z82248, AC008626, AL163248, AC007956, AC005200, AC005586, AC005003, AC002430,







AL121601, AC004812, AC008395, Z82176, AL049562, AP000503, AL008731, Z83838,







AL050403, AC003047, AP000244, AL109804, AC005624, AC006344, AL136300,







AC007216, AL163194, AL034421, AC018647, AC010271, AC005736, AL133326,







AC007963, AC007907, AL133321, U89337, AC011480, AL133286, and AC018994.


HCRBR74
22
460694
1–907
15–921
AI588970, AV723350, BF590203, AA444139, AA393569, AA399071, AV723460,







AV728395, AV722361, AI718370, AA418665, AI379612, AI129258, AI658841, AI685542,







AI435359, AI005196, AI885864, AI800686, BF475906, AI018515, AA401420, AA448520,







BE218367, AI375426, BF439199, BF223410, AI094019, AA447539, BG055041, BE858894,







AI374902, AI005649, AW136117, AA400285, D79837, D79860, AA620952, AI028787,







AA759022, AI341750, D61885, AV728636, AA127018, AA435498, AA460975, AA443959,







AA707516, AI129874, AW008672, AI274085, AI494379, D62374, AA128221, AV709002,







D79883, AI474174, R24595, Z40803, F02215, Z45077, AV723524, R44400, R40285,







F05970, AA910488, T23467, AI953018, AA782944, AA774444, AI431623, AI473290,







AI379646, D79872, D79372, D62484, AI919153, AA418611, AA412171, D62671, D79233,







AV726229, AV726605, AW582828, AA977656, AA364473, AI867742, AJ224677,







AX002935, AX002933, AX047646, AJ223206, AJ132434, and AX047662.


HMSJW18
23
457182
1–1824
15–1838
AA336580, BF897247, F33126, BF816497, BF514629, AA547979, AL519565, BF983228,







AL044489, AV714931, AW081165, AW188427, AA572983, BF679554, BG164602,







BG164455, AI833293, AA397411, BF769608, AW063912, AL079869, AA070456,







BE541237, BF874751, BE675240, AW970987, BE538259, BG056362, BF901147,







BF968850, AW814632, AW962194, BE794230, AU122388, AI281401, BE867712,







BE396049, BE963665, AU121918, AI433104, AV754399, BF741126, BF676525, BG032943,







H87818, AV661138, BF982222, BF769607, AA309362, AA744068, AA745459, AW182499,







AV731938, AL138756, AF196969, AC005488, AC006211, AC005098, AC004166,







AL158040, AP001725, AC008569, AC005280, AF207550, AC005585, AP001726,







AC005236, AC005081, AL033529, AC004965, AL136137, AF196779, AC007055,







AL096840, AC083863, AL157838, AC004019, AC005531, AL356801, AC020904,







AC004491, AL162615, AC007225, AL161670, AC008958, AL035458, AL109935,







AC005089, AC007676, AC004876, AC004638, AL136418, AC073898, AC004815,







AC002300, AL049780, AC002310, AC005295, AL034372, AF205588, AC006011, U91318,







AC010412, AC007597, AL122020, AC004000, AC011500, AC004826, AC008474,







AC006480, AF111169, AC013429, AF047825, AL117382, AC005015, AF030453,







AC004217, AL136172, AC002477, AC008736, AC011462, AP001711, AC004983,







AC009314, AL354864, AJ400877, AC008521, AC005666, AC004966, AC005103,







AC011465, AC005057, AL162430, AC007216, AC002350, AC005011, AC005399, U95742,







AC004905, AC020916, AF168787, AC005102, AC009123, AC002314, AL049829,







AC008015, AC005632, AC008543, AC009953, AC008372, AC005844, AC005755, U63721,







AL121656, AC007151, AC011449, AL121890, Z85987, AC005189, AC005088, AL049830,







AP001718, AL109743, AC018720, AC005736, AL356299, AL161731, AC011495,







AC004895, Z98050, AC007722, AL354720, AL109797, AL133453, AC004971, AC005914,







AC021036, AL050349, AC005911, AC012384, AC006449, AC011484, AC008635, L44140,







AP001712, AC008753, AC023490, AL024498, AP001717, AC008551, AC027319, U91321,







AP000689, AC005231, Z98884, AC004832, AP000557, U62293, AC000025, AC005971,







AC006509, AC006064, AC010271, AC008760, AC004953, AC007021, AP001724,







AC010605, AL121926, AC000052, AC007685, AL138836, AC006337, AC010789,







AC008745, AC003070, AC003665, AL117258, AC006023, AL109825, AC007957,







AC004967, AC004089, AC002565, AC005288, AC011455, AC004834, AL035086,







AL080243, AC003036, AP001759, Z85986, AL117381, AC006013, AP001714, AC005071,







U80017, Z95116, AL133545, AC004963, AC006077, AL049760, AC008403, AC022148,







AL035683, U47924, AC006515, AP000512, AC021016, Z98742, AF001548, AC005529,







AC004253, AC005995, AL050318, AC004890, AC008537, Z93015, AF312915, AC010422,







AL137039, AL031255, AC008623, AC012442, AP000045, AL117348, AL022320,







AP001753, AL022721, AC005730, AL136305, AL022322, AC005052, AL158830,







AL050335, AC004882, AC004859, AC007637, AC011475, AL132653, AL049759,







AC007066, AC007226, AL096791, Z95152, AL138752, AP000501, AC004084, AL355385,







AL450226, AC011811, AL035079, AC008083, AL137787, AL160231, AC007956,







AL159168, AC000353, AC015651, AC005696, Z99128, AC006345, Z95113, AC027821,







AC027821, AC021317, AC021317, and AC021317.


HHTLF25
24
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, I25670, X78928, and







AF072845.


HMKDD07
25
460481
1–614
15–628
AI921875, AA170835, AA866613, AI310163, AI349001, AI310017, AI224790, AI252834,







AC011040, AC011040, AC011040, AC024107, AC024107, and AC024107.


HELDY05
26
456449
1–1408
15–1422
AW841744, AV719467, AL046264, AI986442, AV714313, BF828542, AI276932,







BF001902, AI888828, AV654613, AL046263, AI439852, AI239525, BF507380, AW071751,







H55089, F08626, BF353044, BE875082, AW468989, AK000642, and AL008718.


HFIVB57
27
460449
1–781
15–795
AI765327, AI391585, BG149914, AA972654, N94621, D25640, AI992349, AA234468,







AA565409, W23604, AC002394, AC002394, AC002394, AC002394, AC022278, AC022278,







and AC022278.


HFPDE69
28
460450
1–563
15–577
AI445914, AW502949, AA630476, AA493808, AA809125, AI357807, AI310670,







AW439224, AV744179, AA280886, AW973541, AI310787, BF832074, AW275432,







AI733856, T05834, AI349130, BF853009, AW962006, AA084439, AI961983, AI444575,







AI417469, AI732682, AW069227, AI144055, AW192199, AI587583, AV695478, AI587565,







AA468271, AI753904, AI003068, AA502175, AW238341, AW510403, AI568376,







BF897648, AV756663, AW104161, BF527620, BE676019, AI473995, AL079553,







AV760014, AW063541, AL038842, AW303872, AW473160, BF987139, AI246796,







AA557945, AI869797, AA177061, AW517561, AA533660, AI365988, AA570255,







AI247101, AL043745, AW192419, AW238137, BE676900, AW007980, AA584489,







AI859906, AI049630, AA493546, AA507692, AI343986, AV758849, AA101744, AA507623,







AI336771, BE139139, AA525998, AA631915, H86725, AW970571, N66775, AI434653,







AI355224, AL134216, AA524800, AW979200, AA555232, AI199816, AA741301,







AA654038, AW574899, AA507499, BG112362, AL041381, AA449923, AW063526,







AA878492, AV711203, AI251203, AI251034, AI623899, AA599712, AA834816, BG152753,







Z83826, AL096701, AL121926, AC005899, AC006449, AL354720, AC005632, AP000116,







AL139100, AL080243, AC005694, AL136418, AC011470, AC003007, AP000049, Z85986,







AC009516, AP000501, AC011500, AP000426, AP000311, AL139099, AC004884,







AP000248, AC020908, AC006064, AC006536, AL132639, AC005037, AL133453,







AL139092, AC005538, AC012005, AL354696, AP001695, AC006515, AC010092,







AC002470, AF254983, AC005529, AL031733, AL022311, Z98036, AL118505, AL359763,







AC007773, AC004832, AC005283, AL163201, AC005332, AL031431, AF165926,







AL359382, AC068130, AC009499, AF001549, AC004453, AC003108, AL021937,







AC005098, AC002563, AL023693, AC004166, AC021019, AC005874, AF134471,







AL109952, AP001717, AC005971, AB023048, AC011475, AC006329, AF001550,







AC021016, AL136365, AC003665, AC011495, AC005291, AC004448, AC005832,







AC007055, AL161670, AC009086, AC004890, U85195, AC018663, AC016683, AJ229042,







AC010618, AL021391, AP001711, AL133246, AL034405, AP000031, AC018644,







AL135787, AC007774, AL162831, AL079342, AB020868, AC009530, AE000658,







AC004821, Z97054, AL121586, AC005730, AC006011, AL049870, AJ251973, AL049780,







AL157713, AC008745, AL031277, AC017100, AC007637, AC005484, AC004938,







AC003029, AL109804, AC002117, AB042297, AC004967, AC004526, AL023807,







AC011464, AL356748, AP001720, AC004019, AC010553, AL022721, AP000163,







AC002302, U91318, AC005815, AP001713, AP001716, AL031674, AC005358, AC008482,







AL109614, AC004841, AL135901, AF190465, AC010271, AF129756, AL158040,







AC010267, AL445483, AC002350, AC008569, AC022201, AP001715, AL022316,







AL139384, AP001760, AC008635, AL391684, AL109963, AL121890, AC006088,







AC005088, AF235097, AP000553, AL031772, AC005058, AF134726, AC026431,







AC004867, AC027319, AC004840, AL021877, AC007690, AL031659, AC007371,







AC005522, AC005056, AC004826, AC005969, Z84477, AC005274, AC002543, AC007276,







AC006483, AF053356, AL121929, AL136139, AC004777, AC005288, AC002565,







AC004148, L76568, AK021820, AL021528, AC011465, U47924, AL096712, AL117382,







AL133332, AL163204, AP001731, AL033529, AC005527, AC005049, AL031589,







AL078477, AL391839, AL357129, AC005914, AC004477, AC005209, AC005519,







AL365214, AC002996, AC004990, AC008623, AL132713, AP000226, AL138759,







AL049712, AL354864, AC015651, AC002492, AC005081, AC010223, AC011462,







AC005264, AL117328, AC007226, AL035416, AL022165, AL133163, AC004583,







AC020906, Z95115, AL391114, AL031657, AL133245, AC002347, AC002301, AL031283,







AC010473, AC005225, AC009312, AJ400877, AC007404, AC009244, AC004895,







AC007963, AL121656, AC022046, AC022046, AC011127, AC011127, AP001584, and







AP001584.


HMSHM14
29
461897
1–742
15–756
AW817008, AW817118, AW951170, AL078634, M60510, and AR040601.


HSRDE35
30
460538
1–1282
15–1296
BE742604, BE621651, BF793948, BE299600, AL525510, BE739428, BE621032, BE300271,







BF037910, BE617818, BG121594, BE409316, BE646590, AI085965, BE962230, AA075546,







BE300434, AA075675, AW170271, AW051776, BE879163, AL523028, AI492240,







AI986434, AA417668, AI276289, AI091507, BF027269, AI148079, AW168126, AI925426,







AA604521, AA609132, AI200642, AW439781, W46825, T50022, AA775791, T31817,







BE740085, AW884462, R77171, AA747906, AI927129, H55807, AA576766, F28444,







BE315244, BE208178, R81379, BE302077, R61827, AL523899, BF998272, BF857276,







AW090678, BF998267, AW952610, BE154540, AA714715, AA669090, AI978746,







BE154534, R81378, H55806, AW576851, AW081405, AI190823, Z41500, AW382992,







AW510416, BE065740, T81932, BE154539, T65941, R98506, BE154542, BE300466,







F37137, AW135144, BE154536, AA622979, BE272000, AI590743, AW382978, BF917045,







AA532521, T31351, BE965319, T50085, AL523029, AW383783, AW383787, AA337175,







BF203427, BE314830, BF312136, BE154528, AW383007, AW383026, BF916762,







AW383799, AW372038, AA649006, AI497840, BG000993, AA738432, BE617342,







AL521264, BF313357, AL523049, T81931, T85124, BE019859, R98462, BE383794,







AW139705, BE728133, BE743407, AA248720, BE544193, AI873732, T64516, BE905918,







AW361146, BF917265, AI039642, AW372020, BE384312, BE897132, D45709, BE707989,







BE871970, BF972259, BE185668, BF881322, AL525557, BG055957, BE746377, BE389790,







AI696261, AW372024, BE272520, AF176814, AL359388, AL359388, and AL359388.


HNFID82
31
456494
1–1546
15–1560
BF855161, and T81602.


HOFNY71
32
1152170
1–1448
15–1462
BE379381, BE378584, BE378500, BE736746, BE780958, BE378075, BE515303, BE614826,







BE392462, BE393601, BE394088, BE388690, BE378758, BE735483, BE394406, BE378901,







BE394632, BE378357, BE615872, BE387695, BE392780, BE563033, BE392047, BE395788,







BE394179, BE395181, BE389364, BE392832, BE389345, BE392472, BE515219, BE615406,







AA464826, AA453149, BE735871, BE392729, BE387682, BF210381, AJ403128,







AA442802, AW877209, AL119457, AL119399, AL119324, AL119511, AL042544,







AW971745, AL134902, AW804686, AL119443, AW392670, BE695785, AW384394,







AW861889, AW858455, AW363220, AW861944, AW604723, AW858526, AW858525,







BE705903, AL119497, BE705906, AW577135, AL119483, AW372827, AL119391,







AW604726, AL119335, AL119319, U46347, AL119396, AL119464, BE705905, U46341,







AI499491, Z99396, U46351, AL119484, AL119363, AL119341, AL119355, U46350,







U46349, BF868697, AL119444, U46346, BE705904, BF868687, AL119439, BF868684,







AL119522, AL119418, AI432654, AL119496, AL134527, AL134533, AL134528,







AW861954, AC007078, AC004980, AX030435, AJ251859, AB026436, AX046357,







AR060234, AR066494, AR080280, AR054110, AJ279014, A81671, and AR069079.


HPMBR15
33
456527
1–1258
15–1272
AW793687, AW793670, BE217751, AI743730, AI632249, AW341058, BF110107,







BF693860, AA885501, AI025396, BF974662, AA926639, BE467604, AA406363,







AU157056, AA406419, AI735749, AW135649, AI017864, H44563, AU156233, AU156349,







AW044138, AA669403, R68742, BF438885, AI735367, W38756, AI680553, R68687,







AW193147, N92644, AI288896, H44564, BF093968, BE767183, BE247766, AW300237,







BF849215, AW836736, AK001972, AJ227872, AC069304, and AC069304.


HSAVD46
34
456536
1–759
15–773
AL519778, AL519564, BE549977, AW293350, BE673994, AI479992, BE467490, AI802131,







AA903062, BE218785, BF111390, AA527216, AI269065, AW022008, BE817446,







AA328288, AA904434, AW394125, AL037626, AC026491, AC026491, AC026491,







AC012385, AC012385, AC012385, AC012518, AC012518, and AC012518.


HCNSB01
35
460699
1–2441
15–2455
BE727563, BG248792, BF984368, BE271284, BE735923, BF527732, BE559957, AI797287,







BG115092, BF317212, BE396639, BE729397, AV734956, BE260465, BE276441,







BG255138, BE409355, AW963436, BE397161, BE546083, BE781522, BE562425,







AU139797, AW409857, BE782551, BE267961, BE734764, BE312935, AV761704,







BE904711, BE270658, BE312359, AV724774, BF342762, AW578006, BF312468,







BE047487, BF741486, BG169917, AI417037, BE206393, AW963831, BE812360,







AW614852, AW409599, BE645429, AI031970, AI570957, BE390003, AW085167,







AI752162, AI342088, AV707918, AV685296, BE278325, BG059639, BF028602,







AW752726, AI091080, BE296867, AI670117, BE296667, AI375955, AI674218, BE908294,







AA406138, AI307214, AI824094, AA583346, AI278555, AW027523, BF527302, AI804252,







AI308199, AA576393, AI246637, BE390473, AI143247, AA835834, BE218423, BF034042,







AI129466, BE857958, BE561075, BE675926, AA829819, AI039708, W74766, AI796976,







AI041997, AA761551, AA179217, AA148562, AV705360, T60490, BF953246, AA179218,







AA831975, AA629019, AW269719, AA479955, AI089680, AI203687, AW148934,







AW300738, AI627451, BE276171, AA477543, AI028397, AA310733, BF926592, AI932633,







AW005238, AW000812, AW732230, AA453114, BF376689, AI751392, AA806549,







AA171826, AW139810, AI752161, AW083398, AW800289, BE327918, BE397225,







AA535593, AA171534, BF819153, AA927753, AW888756, H30473, AA297784, W73904,







AA968854, H81763, AI290336, BF700298, BE543777, AA297277, AA632814, BE274582,







BE311529, AA527897, AW800327, AW276025, AW888764, BF849941, AA705564,







H44302, AI751393, AA467994, AI762765, BE242929, BE271076, AW629605, T66192,







AA302994, AA825494, BE019889, AI990013, H39789, F12041, R49957, AW571867,







BE269825, AA477659, AW957023, N50982, AI990689, AI656978, AA018213, AI150679,







AW025641, AI381847, W01877, AA478857, R59453, AI633898, T61334, BF060950,







AI082544, AA234882, AA505342, R59393, AA468054, AV748070, AA297899, AA018203,







AW205263, AA342570, BE241908, BE242057, AA196205, AA235309, AA853470,







AA298718, R32801, AA335553, AA853469, R28140, R50303, AI282443, AA639623,







BF854405, AA341701, BF767415, AA375885, BF792351, AA628000, AA434129,







AA157024, AA430443, R50304, R33937, AA350589, AW978249, AA299638, AI707957,







BE409949, AA319615, AA954287, AI223839, AA298947, BE830781, BF759397, Z40331,







AA335692, BF819718, AI123938, W32368, BE242111, BG122377, BE774043, BF819693,







BE384715, R28025, AA810959, AI623914, AI906396, AI906347, AW799410, R47809,







AA699990, AW799408, AA865846, AA935525, AA302955, AA196147, AR077724,







AF005038, AF295402, AF295405, H81762, and AA227218.


HSDGR57
36
460599
1–900
15–914
AL521196, BG119163, AW872751, AA129439, BF793666, AV751753, BE350904,







BE177088, BG059636, BE885154, AA425398, AI381416, R17127, AW951978, AW515563,







AI418660, AA314750, F32787, AI590092, AW021547, AA151302, Z42142, BE762720,







AA904204, AL533463, AL530516, BF475746, BE072823, BE762723, BF154572,







AW954129, AV707405, AK000610, AK025144, U77327, AF241831, AF227684, AF064105,







U38894, AL133477, AL133477, and AL133477.


HJABB94
37
456466
1–1541
15–1555
BE905356, AI026821, AA503776, BF114724, AI435527, AL036946, AW298357, BF240642,







AA969442, AI767392, AI142574, AI094514, AW073866, AW241144, AA206595,







AA040034, AA354909, AW972134, AA814156, AA933895, AA040828, C01416,







AA457220, and AL138875.


HOFAF39
38
456516
1–1753
15–1767


HTPCS60
39
456565
1–1565
15–1579
AL538348, AW196877, AW160841, AW161283, BE150392, BF333925, AA227689,







AA127575, N57582, AA357020, AW578713, AW162380, AW578712, AW753614, and







AC008585.


HKIYO61
40
456471
1–1529
15–1543
AI742541, BE145059, AI820089, AI422815, AA054182, N52432, BE141338, AW812893,







AA229226, AW937304, AU155285, AL037440, BE551779, AV719324, AA487956,







AV718692, AC009501, AC005880, AL109666, AC040171, AC006556, AF001552,







AC005887, AP001660, AC010000, AL078615, AC005368, AC006461, AF130358,







AL136090, AL356503, AC007253, AL139112, AC018927, AL031599, AL159980,







AC006324, AC006101, AC007501, Z82176, AL391983, AC000114, AC009405, AL034406,







AC005261, AC008444, AL391683, AB020870, AC006287, AC008082, AL162293,







AP001720, AC016144, AP001672, AC022316, Z84466, AF265340, AC005186, AC007463,







AL138828, AL023580, AC007557, AL159988, AC007630, AL133512, AL021451,







AC004460, AL133238, AC005210, AC008613, AP000101, AC007741, AC008045,







AC007538, AL009030, AL031116, AP000240, AL121866, AL079352, AC004743, Y10196,







AC005680, AL163249, AP001705, AC006973, and AL158072.


HMKCW19
41
740950
1–2081
15–2095
AW131855, AW103791, BE786125, BE907341, BE741764, BE875000, BE779562,







AW168069, AI683159, AV751548, AI564711, AW674714, BF854041, AI591098, BF878946,







AA769694, AA779380, AA909179, AI633482, BE707213, AW380346, AW582296,







AW856115, BE265523, AA704275, AI801508, AW856067, AW856267, AW582282,







AW582306, AW380347, AW856286, AW861031, AW582313, AW861030, AA133029,







AW380331, BE149717, AA176435, AW815392, AI299389, AA635668, AI298684,







AI525534, AA176449, AW856270, AW605995, N57107, AW815391, AW605988, D61446,







AA872748, AA935751, AA534776, AI539158, AW391288, AW815359, BF942285,







AI350167, AI679077, AA632939, AW815535, AV745565, BF942259, AW379905,







AW605992, AW391278, AW605996, BE062982, AW391319, BF350206, BF999200,







AW362630, AI344011, BE149712, AW362647, AW815553, H39909, AI677929, AW605991,







AW815565, AW815543, AW815526, BF109465, AA593098, AW815555, AW605994,







AI245733, H23488, AA678366, AW362634, BE349315, BE930127, AI537774, AA132953,







AW815533, T54480, BF437358, AI865714, AW753790, BE046140, BF999191, F24023,







BF999193, AI219007, AW380323, H23487, AW362635, R81352, BE711690, BG010970,







AA197191, AI216736, BE062972, BE219070, AW815517, H14895, AW104094, BG010606,







AA132922, BE712214, AW362606, AW379901, R81609, AA506062, AW364970, R38679,







AI151455, F10797, T35069, AW364972, AW364946, R46074, Z40370, F31439, AA132865,







BE825746, AV750348, AA878673, AL048731, R38243, AL048738, AL048728, R20572,







BE711888, W25677, AI214322, BF741198, BF999196, F01663, T29399, F13192, BF375252,







H90276, AI335122, H91375, BF942195, Z44440, T58343, T58387, Z42121, AI299390,







BF758951, BF999195, BE502773, H14836, BF999199, AW238260, AA232632, AA935720,







AI299381, AI299401, AA091810, AW194490, BG008582, BF857409, AW675126,







BF810957, AW371584, AW582268, BG013709, AW380354, AF176646, AF220152,







AK027066, AF095791, AB041546, AR083279, AB050421, AL122045, L25851, I33984,







AP000340, AP001761, AF004162, AK024974, AB045997, AF119908, AL136979, AJ250403,







AK025391, and AF116698.


HNFIG36
42
456495
1–1078
15–1092
AI248457, AA677952, AI122702, AI436618, AI091491, AA706750, AC000400, AC002121,







AC006405, AC006405, AC006405, AC068936, AC068936, and AC068936.


HBIAS26
43
460709
1–399
15–413
AV718233, AV758342, AW950203, BF215185, BF673269, AV708608, BG261013,







AV708625, AV761517, AW009489, T49448, AW970025, T49449, AV757113, AI292038,







AA541537, AV740956, AA834645, AI244426, AI191342, AI628613, AV763049,







AW004753, F30948, AA885458, AW955082, F25552, BF054813, BE645802, AV713018,







AA356955, AW957305, H40428, BF245708, AA355112, AA552169, AA187017, AA846878,







AI910358, AI684655, AA918022, AI245274, AI244721, AA181303, AA974888, AI633587,







AA522725, AA873880, AA977363, R93237, AA352551, AI204631, AI198700, AI015353,







AI201852, AA029544, AA662656, AW001837, AA829667, AA369573, H40429, AA626833,







F13651, AV738396, AV712538, AV754074, AI339186, BG031008, C04983, AA522987,







AA029543, BF886930, AA573674, AA909099, AA585220, F34453, AA324454, N56270,







AW151714, AI335208, BF872365, AI582932, AV656595, BF854113, H89138, BF037484,







AW081036, AI863191, AI445992, AW148356, BF812960, BG112239, AW079336,







AV658585, AW983832, AI499285, BE544111, BG180273, AV741327, AI866469,







BF895953, AW983691, R81679, AV681858, AI623941, BG166654, AW151893, AW806761,







AV743631, AI288285, AI702301, BF812936, AI923370, AA833760, AI886123, AI537261,







BF812938, AI241923, AI863321, AW020095, AI624293, BF970652, AL036673, AI963846,







AW983703, AW051088, AI433157, AI345745, BG029829, AI952278, AI702073, AL036802,







AL042191, AL037582, AW130930, BF812961, AL037602, AW149925, AI801325,







AL079963, BG109270, AW163834, AI445990, AI688847, AV703585, AI874166, BG031664,







AI500061, AI434741, AI249877, AI633125, AI269862, BE018334, AI612750, BF794025,







BG036479, AI698391, BG165051, AI538564, BG256090, AV702117, AI868931, BF970768,







AI915291, AL045626, AI620284, AW152182, AW089275, BE907357, BG164558,







AA557245, BE905087, AI889189, AI866770, AI538116, BG253614, BF092710, AI927755,







AI623682, AI884318, AI636619, AW162194, AL036638, BF726207, BE910373, AA580663,







BG249582, AI521560, AI890907, AW118518, AA641818, AW104141, W74529, AV681618,







AL120700, AL036396, AL041562, AL039086, AL118781, AW026882, AI699011,







AI872423, AV692389, AI829990, AI874004, AI538342, AA761557, AI890507, AV682803,







AI580435, AA502794, AI251221, BG025209, BG113299, AI637748, AI872154, AI288305,







AI859464, AI859991, AA614183, AW238688, AA983883, AW880037, AI500706,







AI494201, F37439, AL119791, AV758053, BG001235, BF526020, BF680133, BE536058,







AV721325, AA579618, AI973152, AI174394, AW117746, BF875941, AV682488, AI610690,







BG105099, AI624938, AI648408, AW161579, AI344817, AW021588, AJ299431, A77033,







A77035, I89947, AF090934, AF183393, AF008439, AK000718, A08913, AK026528,







AK000432, AL389939, AF230496, Z82022, AX006092, I48978, AR038854, AL050024,







A08916, A08910, A08909, AK024538, AL122100, AL137463, AL389935, X82434,







AK024992, AL137550, AX040974, AF118094, AK026642, AF061795, AF151685, A08912,







U91329, AK026550, AK026885, AK000647, I89931, Y14314, AL359615, AK026855,







AF113019, AR087170, AB048919, A18777, AK025084, A08908, AK026542, U80742,







AL080148, AL050138, AK026593, AB050410, AL049452, AL080154, AF153205,







AK026630, AF090903, AL353940, AF155148, AL133558, AK026959, AL137521, I00734,







AF113677, AF217966, AL390154, E00617, E00717, E00778, AK026629, AK026534,







AF285167, AF130100, AL133665, AK026551, AF130110, X65873, U35846, AK026462,







AK026532, AF116688, AF017437, AL122049, Y16645, AF159141, AL122050, Y10080,







AB048974, AK027146, AF113222, AK025632, AF262032, AF125948, AL137533,







AF090901, AF139986, AL389982, E12747, AL137476, AL050277, AK026464, AL137478,







AK025339, AK026744, AF217987, AJ238278, AF185576, AF113691, X93495, AF207829,







AK026533, AL359622, AK000418, AF119899, AF087943, AL137560, AF119865,







AL133640, AL359583, AL117416, AK026762, AK026784, AK025435, E07108, AK025254,







I09499, AL117435, AL137480, AF067790, AX040958, AK026647, AK027114, AK027081,







AF132205, AF113699, AK026164, Z72491, U58996, AK000083, AB050431, Y09972,







AX046842, AK000450, AL157482, AL137488, AF106657, AL137548, AF032666,







AL110280, AK024594, AL162002, AL080086, AF119337, AR020905, AF067728,







AX042059, AF118070, I48979, AB052191, AX045627, AB047904, AL157431, AL117440,







AF155827, AF030513, AF113013, AF106862, AL080074, AK026865, AL137271,







AF116691, AL133067, AL137557, AF130055, AL049466, AK025391, U67958, AF225424,







AB050534, AK026526, AK025414, AF130099, AF116649, AK025524, AF138861,







AL442082, AJ012755, AF116631, D83032, A08915, AF119883, AB033881, S36676,







AB048953, I03321, E06743, AF130092, AK026452, AL050116, AF130082, AF177401,







AL050092, A03736, X63574, AB051158, X72889, AF012536, AF100931, AF130066,







AF126247, L19437, AK024588, AL049300, A65341, AK026480, AF143723, E02349,







AB048954, AF130105, AF155221, AK025798, AB007812, AF026124, AF116602,







AL359601, AK027144, AL133016, AK026506, AK025484, AL133565, AF116682, Z97214,







AF061943, X53587, AL133560, AX026824, AX026823, A58524, A58523, AR013797,







AK026504, AF119875, E05822, AL049430, AF218014, AL049382, X80340, and AK026741.


HE2EC79
44
460671
1–721
15–735
BF515951, AU144114, BF514646, AI278618, AA425749, AI651734, AI888258, AI371154,







AW593395, AI334335, AI803458, AA446628, AI767188, AI370597, AA628659, AA453128,







AC007554, AC007554, AC007554, AC020899, AC020899, and AC020899.


HNDAH54
45
460486
1–761
15–775
AI174703, AA640305, BF530611, BF525663, AW963489, AI446424, AW021674, AI803771,







BE155250, AV755245, AI754064, AV712092, AL048969, AA515165, AA632821,







BE677227, BF541501, AI889345, AI086603, AI613459, AA602105, AW820011, R22196,







N73337, AA557945, BE245594, AW275640, AW869495, AV757180, AW888719,







BF031507, BE970216, AW850448, AI445616, AA524604, BF928877, AU147869,







BF132387, AW901135, BF958025, AI355246, BF346854, AV710839, AW630599,







AW820030, T96105, H66812, AA743445, AA883595, AL133861, AW769859, AW815585,







AW815584, BF680082, AW501278, BF949151, U52112, AL020997, AR022305, M14949,







AC005911, AF217403, AC010524, AL133545, U80459, AL080250, AL049872, AL035249,







AC005358, AC012072, AC004929, AC004598, AF153836, AL135844, AC009247,







AL096763, AF128525, AC006958, AL137800, AK026675, AC005837, AL049758,







AC005632, Z73979, AC004884, AL023584, AL035588, AL355497, AC007912, AC007458,







AC003665, AL031767, AC007093, AL109797, AC024561, AP000228, AL138976,







AC007057, AP000140, AC007679, AC011462, AL136135, AL445248, AL356575,







AL356747, AC006468, AC011500, AL161781, AC008072, Z98949, AC009003, Z93015,







AL034422, AC007899, Z95114, AL353152, AL353804, AL157952, AL035458, U91318,







AC004913, AC025588, U80460, Z82178, AC004213, AC007151, AC004779, AC006024,







AL161670, AF165926, AL138721, L78810, AL118499, AC018720, AC004820, AC009314,







AL355499, AC004882, AC004973, Z99570, Z93017, AC006237, AL157818, AC006011,







AC009516, AC010326, AL022149, AL031846, AC005377, AC002316, AF042089, Z86090,







Z93928, AC005740, Z93018, AP000553, AP001065, AP000088, AC005529, U47924,







AC005746, Z82190, AC011450, AC002312, AC004104, AL161937, AL022322, AL022723,







AC018755, AL133465, Z97989, Z99128, AC005537, AC002366, AP001052, AC011451,







AC005800, Z93244, AL031295, AL138702, AL022163, AL035530, AL138752, Z96074,







AL359555, AC016644, AC018751, AC005410, AC010081, AC005520, AL118502,







AC009004, Z84466, AC006236, Z98044, AC024247, AP001243, AL121914, AC006204,







AD000812, AC005839, Z49155, AP000226, AL117694, AF228703, L78833, AL139327,







AL121586, AC008733, AC011495, AL035423, AL096828, AC002126, AP000087,







AC005670, AL136418, AC005039, AC006345, AC008102, AP001752, AP001694,







AC007066, AP001754, AL035460, AL161621, Z83844, AC006162, AL109743, AC003037,







AL117381, AL022577, AC006511, AP001753, AC003692, AP001726, AC005215,







AD000833, AC068499, AC008088, AC003956, AL132718, AL133515, AC007365,







AC006539, AC004125, AJ009610, AL445490, AL121829, AC067969, AC007907,







AL138756, AL136179, AL138958, AC005828, AL022315, AL117334, AC005736,







AC006084, AC008784, AC009086, AP000694, AC005726, AC011311, AL117336,







AC023344, AL031602, AC006344, AF124523, AC007124, AL050324, AC002133,







AC008518, AC005102, AC005551, AC003041, AB032485, AC004887, U89337, AL390755,







AL079341, AL133294, AC007956, AC007739, AL022237, AL354948, AC023792,







AL049570, AC009073, AL049762, AL353748, and AC073127.


HNHFU59
46
460498
1–492
15–506
D80258, C14389, D80164, D59627, D59787, C15076, T03269, D59467, D80522, F13647,







D80038, D80195, AV723927, D58283, D81030, C14227, D51423, D80227, AV719783,







D80193, D59619, D80014, D80210, Z21582, D51799, AV718800, D80391, D80240, D80253,







AV719822, AV720211, AV719324, D80188, AV720464, AV720203, AV718489, AV718692,







AV720812, D80196, AW959570, D59927, AV724520, D80043, AW978634, AV718770,







AV720731, D80219, D59889, AV699927, AV718844, AV699447, AW975618, AV722801,







D80212, AV701004, AV718707, D80366, D80022, AW965158, D59275, AV699550,







D50979, AW949641, AV721386, AW949645, AW949629, AW949632, AW949653,







AW949633, AW973447, AV700889, D80045, D81111, C75259, T11417, D80268,







AW949657, D80378, AV700229, D80168, D51213, AW966062, AW949642, AV720791,







C06015, AW949656, AW949643, AW966013, AV718530, C14014, AW966053, D50995,







AW960465, AV742001, AW966534, AV742667, AV701125, AV701335, AV701166,







AV701043, AV701332, AV701017, AV701248, AW966022, AW959597, AV701431,







D80949, AV719758, D81026, AV719468, D80166, AW965175, AW949658, AW966531,







AW966041, AW975621, AV701130, AV701419, AW966050, AV701154, D59859,







AW959799, AW966054, AV719188, D59502, AV700357, AW978648, AW949646,







AV718440, D80269, AV720028, D51250, AV701422, AW978661, AW973307, AW960553,







AW949654, AW965177, AV718633, AW959628, AW975605, AV719557, AV723097,







AA285331, AW973485, D80024, AV718931, AW753053, D57483, AV701149, AW752082,







AW959582, AW964488, AW973541, AW949631, AW949618, AW959202, AW949655,







AI557774, AV699715, AW973334, AW178893, AV719049, D59610, AV718681,







AW966029, AV719913, D51060, AW964756, AW965197, AV718938, AW964737,







AW956434, AW958992, AW949634, AW965185, AW966065, AW964477, AW966043,







AW959062, AV699669, AW965163, AV701443, AW753067, AW959136, AV738928,







C14331, AW973474, AW966075, D80241, AW956397, AW958993, AW959469, D59503,







AW960473, AW177440, AW973482, AW965184, AW973488, D58101, AW962245,







AW962082, AW975613, AV699746, AW975623, AW949630, AW965196, AW960454,







AV720878, D80064, AA305409, AW966023, AV699866, AV720654, AW966059,







AW960532, AV700086, AV700495, AW973330, AW960504, AW973470, AW753064,







AC007489, AX027925, AX033851, A62300, A62298, X68127, AX047063, Y17188,







AX020191, A67220, AJ132110, A84916, AX047064, AR070327, AR092424, AX047062,







AX021518, AX020190, A25909, D26022, D34614, X67155, AR018138, AR077702,







AR060385, AR025207, D89785, AX035434, AB028859, A78862, AJ302649, A82595,







AR087649, AR008278, AF058696, AB002449, D88547, I14842, I50126, I50132, I50128,







I50133, X82626, AR054175, AR038669, AX028130, AR016514, Y17187, AR060138,







AR016808, A45456, AR074139, A94995, A26615, AR052274, A43192, Y12724, A43190,







Y09669, AR008277, AR008281, AR066488, AR066487, AR074136, A30438, AR008443,







A63261, A70867, I79511, AR091537, AR062872, AR093385, AR016691, AR016690,







U46128, A64136, A68321, D50010, and AR060133.


HE8CV18
47
456447
1–1433
15–1447
AI201812, AI418194, AI814457, AI829948, AW131757, BF953329, AI422043, AL137061,







AF107044, AL137061, and AL137061.


HFKEB72
48
456456
1–1406
15–1420
AI627782, BE463414, AI243445, AC020960, AC020960, AC020960, AC008515, AC008515,







AC008515, AC044801, AC044801, and AC044801.


HTDAD22
49
456553
1–1206
15–1220
AI816833, AI808975, BE328641, AW961358, BF978566, AI148832, AW628704,







AW194010, AA719072, AW002020, AI003646, AA829505, AA719089, AI301815,







AW241894, AW023122, AI962608, R64453, AI564265, AW298337, AA317045, AA632106,







R02147, T69830, AW194454, AI983693, AA436182, AA584489, AI587583, AI587565,







BF765191, AW503420, AA604843, AA714110, AA086318, BF796375, AA503392,







AI623563, AI473851, AA100540, AA570632, AI354243, T16427, AA302970, AU146736,







AI302994, AL035086, AB033035, AC020906, AC004150, AC005776, AC008736,







AC004821, AC005921, AL139353, AC008474, AC009086, AC004166, AC008379,







AL049748, AC008649, AC083863, AP001711, AC005920, AL135839, AL121585,







AP001412, AP001714, AF001549, AL033529, AC010458, AL109743, AC027644,







AP001670, Z93015, AP000152, AC005690, AC003041, AP000252, AL132639, AL161670,







AL050318, Z81314, AP001725, AL133229, AC010311, AC009892, AC008747, AL138688,







AC007731, AC002544, AC004531, AC004824, AC005500, AC008764, Z85987, AL158040,







U95742, AC011742, AL024498, AC005522, AP000509, AC007216, AL022323, AP000030,







AP000212, AP000134, AL080243, AC005209, AC005098, AC011464, AC005080,







AC005901, AL049832, AL109797, AP000547, AP000116, AC005052, AC009756,







AC010553, AL021918, AC000055, AC009244, AC002316, AC004859, AC009060,







AL121601, AC005295, AL133448, AC004867, D84394, AP001728, AC005081, AP000963,







AC021036, AF134726, AP001745, AL138787, AL354720, AC005057, AP000503,







AC005632, AC009953, AC008115, AC020916, AC007225, AP001717, Z98884, AL353653,







AC005231, AL354864, AC008675, AC018757, AC006345, AC008760, AC007226,







AL359700, AC005037, AC006468, Z84466, AC010285, AC006312, AC005972, AL138717,







AC008753, AF001552, AC005236, AL109935, Z99716, AL133453, AP000365, AL121586,







AC006449, AC008155, AP001748, AC008567, AC011470, AC003070, AL136179, Z85986,







AC005480, AC003664, AF045555, AL121658, AL049829, AL031311, AF129756,







AC004815, AL009181, AF107257, AL121981, AC004099, AC005379, AL008582,







AC006271, AC010422, AL034429, AC005529, AC007739, AC006251, AP001752,







AC005531, AC008124, AC004953, AC003029, AP000103, AC007308, AC002558,







AC007881, AL133480, AC016940, AL137783, AC005899, AC006559, AC008745,







AC004253, AC002470, AL096840, Z95152, AF219991, AC016995, AC011442, AC006023,







AC006538, AC013434, AF117829, AL096814, AC018758, AL034548, AC005940,







AL133399, AP000091, AL022100, AC008521, AC008892, AC007880, AC006329,







AC011495, AL031427, AC008569, AC010358, AC005399, AL133243, AJ246003,







AC004466, AC006160, AL121652, AL021393, AL356299, Z86090, AC005102, AC005011,







Y14768, AL035681, AL133232, AL133545, AL078621, AC005409, AC006211, AL356747,







D87675, AC008897, AL359638, AL359638, AL035086, and AL035086.


HSKYU29
50
460531
1–1034
15–1048
AL531681, AI375202, AI375278, AV715681, AA837401, AI796142, AI560855, AL531682,







BE816886, AI926594, BE866670, BF724932, BE439400, AI560005, AA460512, BE897244,







AI754662, AA236467, BE567753, BE621951, AL048935, BF593909, AI735275, AI829453,







AI126040, AI720353, AI042066, AA812318, AA459869, AI750120, AI278030, AI880427,







AA505788, AA777085, AI951210, AA191177, AI925159, AI568853, N46987, AW189441,







AI032998, BF678667, AA503316, BE781431, AA418306, AI074343, AA234625, AA158099,







AW168180, AI223849, AI249778, BF515073, AW176444, AW374317, AA465697,







AI927126, AW374306, AU132275, AI189848, AI650244, H97747, BE548165, BE889623,







AW193415, N58277, AV712219, R93228, H25924, AW957681, H80319, AA678161,







BE889140, AA610090, BF212263, BG166417, AW194165, T52806, D60502, AA330456,







H09631, H13930, AA815440, AI241336, AW451102, BE844181, AI074631, H59105,







BF516387, H16980, D54133, BE798028, R97671, AW374310, AA699674, AI151162,







AA745623, AA988969, AV740686, N34735, BE567465, AI694312, BG036771, AV741433,







H25925, R98375, BF667258, R45077, R67571, AI373903, AV714897, AI242878, H67773,







BF977202, BF977469, AI760225, H80318, H61430, R97672, AV763885, H93650,







AA479406, AI469631, AI244636, H61634, BF573643, R93236, BF891879, R91684, D54550,







BF244798, AA316701, R91685, BG249327, AW954463, AW992196, BF210243, T96127,







AA002186, BF977611, AA348497, W28706, W01672, N71645, R42945, T52805,







AA479305, N55745, D79952, AW176445, AI559762, AV661028, AA136245, AA663815,







BF822096, BF239078, AI394137, AI421608, BF885193, BF185283, H13929, AI886341,







W27179, H61069, AI985389, F07700, BF211695, AA336654, BE244661, BF184173,







AA382068, AW374296, AI802298, AW503970, H62431, BF571054, AW374298, C00763,







AA113136, BE965559, AA084938, BF219170, BF692088, C17702, AL036992, AV756429,







AI081942, R19975, BF027970, H09630, AA158098, AV747620, N77648, AF067396,







AL132665, AB004788, AF079221, AF255051, AF067395, and AR064426.


HHGDU58
51
460717
1–954
15–968
BF513643, BE764446, BE764487, BE764480, AL035694, AJ010278, Z78884, AL035694,







AL035694, and AL035694.


HMDAA66
52
460472
1–572
15–586
AL022160, AC006231, AL049781, AC023514, and AL022160.


HNHFW22
53
460350
1–737
15–751
AC021605, AC021605, AC025839, AC025839, AC021895, and AC021895.


HSQFP66
54
460537
1–463
15–477
BE465277, BF593260, AI765036, BE181153, BE181155, AA834498, and BF365438.


HNGEV29
55
442573
1–1139
15–1153
AA977582, AC055739, AC068978, and AC068978.


HUKBH05
56
457195
1–1227
15–1241
AI400278, BE313085, BE383358, AA298993, AL530935, AU126961, BE314879, BF317174,







BE259667, BG253760, BF927739, BE728354, BE409263, BE729268, BF918936, BF918942,







AL048903, AW797208, BE297579, BF847648, AA304712, AW961340, H67555, BF869582,







AL048902, AL137599, AB033000, AK001356, and AK001651.


HATDL27
57
460380
1–894
15–908
AC004903.


HBJFU48
58
460392
1–835
15–849
BF674706, AA657543, AV757289, BE139139, AI250552, AI251284, AI251203, AI284543,







AI251034, AW674277, AI254770, AW303098, AA582073, AI249853, AC005696,







AF045555, AF243527, AP001725, U91318, AL121897, AC008155, AC005081, AC011470,







AP000692, AL109976, AL135928, AL033529, AL353807, AC020916, AL158830,







AP000501, AL008637, AC006270, AC011464, U95742, AC011555, AL049795, AL033383,







AC005921, AC004922, AC007934, AL121653, Z82215, AL049636, AC007216, AC005971,







AC005058, AL135783, AP000355, AL121928, AL162724, AC004906, AC006312,







AL359272, AP001666, AP001716, AF111169, Z95116, AL159168, AC006023, AP001626,







AC007383, AL121658, AF109907, AC004770, AP001667, AL354720, and AP000044.


HCUBN59
59
461443
1–664
15–678
AI479885, AA489095, W80862, AA492564, AI174766, AI306324, AI436601, AI435258,







AI886472, BE277210, AI249573, BE276480, BF869744, BF918928, BE409047, AI271126,







H18354, BF853867, N64697, AA654781, AI269490, BF945880, AI306191, AI305894,







BF667587, AI343148, AI311684, AW879550, AI886654, AI630176, AW089101, AI932967,







AA572773, BF343686, T52103, BF918979, AI536900, BE154678, AI088768, BF197650,







BF673596, AI421841, H23653, BG002349, BF858167, AA704393, AI250472, H94556,







BF918950, AW860980, AI433008, AA376014, AI309375, AA478355, AL038190,







AA809516, AW853005, AW264934, AW963750, BE274150, AI476089, BF348316,







AI254913, AI080505, AA572953, BE836705, AA493894, BF854267, AI124610, AV710626,







AI366993, AA278250, AW303008, BF197670, AA167055, AA702729, AI344812, AI334464,







AV759675, AA828227, AW449173, AI572924, BE709662, AV757425, AW973291,







AA573748, AI207465, AA779718, AA902215, AW850537, BG177202, AV733830,







AW975273, AV729809, BF674715, AC006345, AC005052, AL049742, AP001709,







AC009312, AC008760, AC008403, AL352984, AC008551, AC002480, AP001207,







AC008155, AC008518, AC022432, AL137780, AF109076, AC006275, AC006967,







AL137782, AC017067, AC006387, AC004554, AC011559, AL049776, AF111167,







AC007488, Z95116, AL035423, AL445248, AC004485, Z72519, AC007358, Z98051,







AC005231, AC023510, AL096803, AC009516, AC016637, AC005005, AL137179,







AL138706, AF001905, U47924, AL355112, AC007376, AL136962, AC011310, AL159970,







AL133289, AC005799, AF064865, AC004998, AC016681, AC018641, AC018637,







AL021155, AC010081, AL121897, AC004840, AC011811, AL158830, AL137139,







AC004821, AC003084, AC007324, AC008101, AC004832, AC009228, AL022326,







AJ277662, AL163249, AF243527, AL138752, AC005000, AL360227, AC008079,







AL109914, AC004216, AC003007, AC022206, AC005049, AL079300, AL139289,







AL135927, AC007227, AC004408, AL035405, AF163864, AC005086, AC005585,







AC004099, AC009044, AL035045, AL022721, AC004791, AL162390, AL020995,







AC011530, AC004605, AF053356, AB016897, AL137162, Z97353, AC003684, AC007845,







AL022155, AC016395, AC044787, AC006958, AC009196, AF001550, AL158832,







AC005881, AC004782, AL021326, AC018764, AL160231, AC004686, AC012078,







AC004002, AF152364, AL035448, U95739, AC055740, AC016025, Z97181, AC016026,







AP000558, AP001962, U91325, AC005071, AJ400877, AC004933, AL121869, AC005722,







AL133294, AL049780, AL138919, AC020906, AC007055, AC005215, AC010596,







AC018751, AL138976, AP000555, AL121981, AC005632, U52112, AC020934, AP000142,







Z85987, AL163247, AC007350, AP001695, AL359700, AC010605, AC008074, AP000455,







AL023513, AL121751, AC009086, AC003046, AC074112, AC004448, AL136223,







AC004030, AL136311, AC003119, AC004547, AJ246003, AC012081, AC005914,







AL391693, AC007136, AL096775, AC002549, AL137064, AC004913, AC009073,







AL049762, AC004858, AL356094, AC021016, AC002432, AC009134, AL139318,







AL161670, AL353807, AL138741, AL161449, AL009179, AP000245, AF001549,







AC005884, AL132821, AL031427, AC023473, AC007192, AC007850, AL118497,







AL121658, AL034395, AL021977, AC005907, AC005412, AC025436, AC068313,







AL133405, AL138894, AC009468, AC004895, AL133339, AC006028, AP001716,







AC008134, AC021068, AC078889, AC077690, AP001819, AL137073, AC005962,







AC011479, AC008764, AC009319, AL136304, AC009315, AC009600, AL353748,







AL132987, AL135928, AC007425, AC011490, AC006038, AL096840, Z96074, AF053645,







AL133445, AC002041, AC063976, AC005546, AL031054, AC005411, and AC005011.


HCUDB38
60
460783
1–843
15–857
AI791857, AC009228, AF024533, Z95115, AL096763, AC005952, AC005137, AL096888,







AL136300, AC008738, AF001549, AC006456, AC005086, AC004953, AL049829,







AL354861, AC003684, AC020904, AC024561, AL118520, AC012330, AC007325,







AC002985, AC023491, AC002073, AC020550, and AC007981.


HDPCO25
61
460682
1–753
15–767
AI193249, AI809829, AC055725, AC055725, AC055725, AC069244, AC069244,







AC023797, AC023797, AC023797, AC023415, AC023415, AC023415, AC048331,







AC048331, and AC048331.


HDPHI51
62
460679
1–714
15–728
AC005946, and AC018755.


HSDAG05
63
460523
1–930
15–944
BE735967, BG028960, N30151, AW194704, BG104863, AW874573, AI334393, BF057674,







BF593830, BF744833, BE856414, AI949076, AI890882, AW027820, BF594266, AI632175,







AI356379, AA203630, AA594117, AI823467, AI651286, AI276677, BF222575, AI370022,







AI356428, AI493393, BE501312, AI288570, AW172483, BF508777, AA036755, AA831078,







AI027633, W84550, AW977869, W28230, N40442, AA906113, AW076062, AA256336,







AA458607, AA524825, AA812137, AW972891, R80312, D20096, AA236380, AW137712,







AI956006, BE782754, AI611671, AA256337, AA844452, AI040458, AA988565, AA057371,







T97621, BF058685, AI825118, T97573, AI886103, H87501, AA236379, AI457303,







BG149517, R97822, R80208, F20270, BF969075, AI083695, AA091887, N35763,







BE674002, AW858522, AW601637, AL134524, AL038878, AW577201, AL045327,







AW577199, AW858525, U46344, AW971982, AW971734, AW972279, AW374052,







AW972273, AL045328, AW968336, AW971725, AW971737, AW974096, AW975037,







AW972897, AW971168, AW972845, AW970619, AW970977, AL042898, AW979127,







AW974791, AW971738, AW972686, AW975048, AW970886, AW974926, AW979047,







AW971301, AW972357, AW970558, AW971072, AW972717, AW974393, AW971126,







AW974800, AW972642, AW971060, AW979134, AW976498, AW975628, AW976024,







AL134110, AW971198, AW975391, AW971149, AW970079, AW968212, AW973652,







AW975104, AW971108, AW968164, AW971290, AL047163, AW974098, AW972153,







AW974390, AW974346, AW972293, AW970070, AW974795, AW972867, AW973774,







AW972649, AW973377, AW975044, AW971114, AW971299, AW968347, AL135012,







AW969911, AW970973, AW972810, AW972314, AW969784, BE927373, AW971300,







AW969793, AW970612, AW970556, AW979070, AW972292, AW979105, AW577192,







AW969791, AW975965, AW974801, AW971191, AW973968, AW971099, AW969702,







AW971146, AW979287, AW970576, AW970856, AW975002, AW979234, AW972220,







AW971038, AW974810, AW975152, AW974104, AW971342, BF084778, AW971187,







AW969988, AW970034, AW973397, AW970950, AW970916, AW969716, AW979204,







AW976043, AW972317, AW975443, AW975017, AW973157, AW970580, AW975380,







AW971403, AW971244, AW979098, AW972909, AW974083, AW976022, AW969928,







AW975028, AW979136, AW979138, AW969974, AW969733, AW975142, AW976010,







AW974889, AW970050, AW970953, AW974127, AW974093, AW974128, AW970540,







AW972667, AW971328, AW972936, AW971116, AW975169, AW974092, AW975410,







AW974666, AW976014, AW979080, AW979002, AW972665, AW974945, AW975657,







AW975984, AW971375, AW975140, AW974118, AW975954, AW975236, AW975105,







AW979184, AW975032, AW969781, AW971363, AW975031, AW974378, AW970586,







AL139349, AR066494, A93923, A93931, AL133053, AL122101, A93916, Y17793,







AX030435, AL133074, D17247, A85203, AJ276251, AL133049, AJ276253, AJ276255,







AJ276256, AR096545, AL133082, AJ276254, AF019249, AL133076, AL133068, and







AR023813.


HMWGY01
64
672222
1–768
15–782
BE062342, Z59258, and AC011276.


HSVAQ28
65
460592
1–428
15–442
AL117344, AL117344, and AL117344.


HGLAM53
66
615771
1–819
15–833
BE747964, BF338150, AI457481, AI458684, AL519858, BF341518, AI291292, AU152841,







AW139401, N78405, AI458990, AW139402, AA644231, AA928861, R15203, AI656348,







AA973701, Z39616, R44374, AA738248, AA991427, BF791764, BF791631, F08992,







F08996, AA293895, N62634, BE392523, R56283, BF447914, BF341045, T58591, T58522,







BF939563, BF732685, BE220757, and AK001610.


HLTAI94
67
456476
1–1248
15–1262
BF242486, AA233821, AA215805, AA662913, AW611703, AW866630, AA360596,







AA251825, AI701594, AA215640, AW589351, AW971408, AA506213, AW589361,







AI262873, H96685, AI942406, AI761712, AL357565, and AL357565.


HMKAH10
68
456609
1–907
15–921
BF507527, AI218188, BF813944, AA992778, BF837401, AI916419, N25033, AL041587,







AV750565, BG249496, AI934065, AL513879, AV761267, BG030785, BE879336,







AV696866, AV726694, BF970436, AI961371, AI538850, C21335, AI370322, AI440444,







AV705864, AI828806, AI338802, AI687568, BG251629, AW168503, AI356868, AW161202,







AI312040, AI673140, AI493576, AI266652, AA810767, AI659989, AI934011, AI698987,







AA070777, AI702527, AI888480, AI521005, T69241, AI421149, AA575922, AA019328,







AI799766, AA761557, AI289101, AI699175, AI470717, N21402, H89138, AI282679,







AI818065, AI453328, AI521634, AW085393, W45039, AI803740, AI560004, AI434038,







AI862976, AI440088, AA830994, AI399977, AI271822, AA830709, AI683270, AF124396,







AF000167, AF141289, X99971, L19437, AK026408, S54890, AF084644, AF084645,







U49908, AF030513, AR038854, AF115392, AR050959, AK000432, AL133653, AF061981,







A21103, AL389982, E01614, E13364, AK000636, AL110228, AL133607, AK027102,







AK026549, AF090900, AF208850, J05277, A41575, S36676, AF054988, AF082324,







AF130056, AL080159, AL137292, AC008897, AK026534, AK027081, AF111849,







AL357195, AL080148, AB048964, AL137476, A15345, AL136884, AL133084, AF082526,







A83556, D44497, AR029490, AB047878, AF124728, AL133016, AF208026, X93495,







AL080140, AF111851, AR083279, AF029750, AK000559, AC018638, AC018638, and







AC018638.


HSVBH58
69
757227
1–464
15–478
W69468, AI954159, BF591122, BF593057, BF807940, AI871746, AI807580, AI922524,







AW956377, N80292, AI676213, BE467086, AV656311, AI984255, AA132452, AI271476,







AW780241, AI954063, N53166, AA769325, AI375545, AI436321, BF063926, BF798303,







AA767391, AA836991, AI923968, AW519209, AA476935, AW273346, AA423804,







AA860230, AW129462, AI400578, AA724396, AA748499, BE677845, AW020788,







AI017695, AA889495, AI028269, AI860419, AI051533, AW339455, AI678172, AA806940,







AV645398, AA609569, AW452389, AA778650, AL040747, AI949278, N92405, BF430946,







AI624860, AL043589, AL041618, AL040967, AI439198, AL040910, AI356952, AI811516,







AA749268, AI206880, AI990273, BE501264, AI650706, AI872882, AA339537, AI123785,







AI431957, AI784422, AW194576, AI187038, AV762653, AA016267, AI678429,







AW392066, AI273421, AA419002, AI358604, AI961623, AA897667, AA586499, N93437,







AI417772, BF798258, AA524576, AW799936, AW378085, BE241923, AI691155, F37181,







AW602710, AA013428, BE827449, AW168519, BE164987, BF526319, AA908333,







W24630, F28686, AA757772, AW015132, AW015964, BF030752, AI933540, AA657684,







BF091761, BF529959, BF529891, AJ400845, AC005071, AJ400846, AF217981, AC005073,







AC005073, AC005073, AC005071, AC005071, and AC005071.


HBMWB01
70
460396
1–705
15–719
AI284640, AA526787, AL046205, AI334443, AW274349, AV762395, BF677892,







BG249643, AA521399, BF311000, AV740801, BF241967, AI270117, AV762098,







AF330238, AL042853, AL138265, AI890348, BF680041, AV760777, AV728425,







AA587604, AV733830, AA720702, AU147104, AA490183, AW970915, AV762397,







BG109996, AF074677, AL037683, AV725431, AW303196, AL120269, AI754955,







AI559705, AI625244, AW662543, AW472872, AV725423, AW193265, AW301350,







AI267818, AI696962, AL046409, AW502975, BE139146, BF991286, AA521323,







AV713243, AV729809, AV763449, AW963497, AI064864, AV761489, AW501386,







BG236735, AV652936, AW975987, AV760395, AL044940, AI305766, AV761106,







AV764329, AW419262, BF679256, BF816072, AA515829, AV762959, AV702857,







AV735495, BF965007, AV757607, AL041690, AV760057, AI963720, BF337291,







AV759505, BG036665, AI281881, BF668217, AI613280, AI564185, AW407578, BE967369,







AW410400, AI537506, AI350211, AA665330, AW327868, AV762111, AV735370,







AV742057, AI969436, AW088846, AA581903, AV761362, AW028429, AI457397,







AL040921, AI133164, AI345157, AV760937, BE562953, BG058664, AU145314,







AV759172, AI972203, AV762535, AW969629, AV760042, AI254615, AI623720,







AW265385, AV762009, AI434695, AA682912, AV763354, AI798473, AV760371,







AV763255, AV764578, AI345654, AA577906, BF793766, AA468131, AW956640,







AV759274, AV763971, AV761786, AW021583, AI859742, AV764530, BF679304,







AV710066, AA491284, AV718260, AV762139, AW872676, AV734666, BG036337,







BF592311, AA468022, BF697673, AV760571, AI821494, AA525190, AW974109,







AV735239, AV764307, AI708009, AI076616, AI538433, AI345518, BG057168, AI754658,







AA723017, AI679782, AW088202, AV762050, AW276435, AW518220, AI499134,







AI924251, BF592200, BF475381, AA491814, BF725504, BG171422, AV763122, AL119691,







AI312309, BF347791, AA126035, AW072923, BF347740, AV763847, AW600804,







BF940837, AV728928, BF964720, AW079135, AV760486, AC011500, AL096840,







AC004638, AF029308, AC005387, AC008403, AC009245, AL121601, AC004453,







AF217490, AC003003, AC020906, AC005393, AC007225, AC006241, AP002026,







AL022163, AF265340, AF001549, AC022432, AL031650, AC007216, AC006132,







AC005821, AC020934, AL135749, AC020893, AC007488, AC004814, AC025264,







AL356244, AL139331, AC022027, AL118520, AP000501, U95742, AC005940, AL353807,







AC004019, AC007384, AP001710, AP001670, AC008537, AF067844, AC008265, Z82244,







AC007541, AC016637, AC010643, AL049762, AC007345, AC007404, AL008718,







AC010328, AL049692, AC006277, AC006251, AL034351, AL049757, AL031311, U85195,







AP001753, AL135927, AC007227, AL158830, AL031432, AP000962, AL049795,







AL137073, AL096776, AE000658, Z99495, Z99716, AC004975, AL163247, AC011462,







AC002310, AC009516, AP001671, AC004253, AC006449, AC002477, AL079340,







AL033392, AL133517, AC006501, AL135783, AP001717, AC002094, AL121653,







AC004987, AC008770, AL022323, AP001715, AC053467, L78833, AC004832, AL132987,







AC004690, AC009060, AC008760, AJ009615, AP001596, AL117341, AC008115,







AC007676, AL080242, AC018758, AC004659, AC006130, AC006441, AC008865,







AC006285, AP001667, AC008745, AL353739, Z97054, AC002369, AC005839, AC005019,







AC007057, AC007899, AC008474, AC079610, AC004808, AC027689, AC011495,







AC007041, AL138755, AL162430, AC005799, AC020916, AC007011, AC004650,







AC008279, AP001688, AL159977, AP001331, AL049872, AC003664, AC005250,







AC002430, AL136981, AC087093, AC005000, AC007620, AL157915, AC004998, Z93241,







AB017654, AC008651, AC007878, AP001718, AL121905, AL096701, AC009470,







AC004815, AL033383, AC005212, Z98051, AC068713, AC005257, U62317, AJ400877,







AC005209, AL035411, AC003102, AL136980, AL136223, AC004865, AL163853,







AL136300, AL359457, AP001346, AC022137, AC006028, AF017104, AP001711,







AP001696, AC007091, AC007308, AL354720, AC011510, AC004933, AP001728,







AC006960, AC007283, AC069080, AF060568, AC000025, AC018644, AC007919,







AL096712, AF053356, AL133332, AC006111, AC008068, AL022238, AC011479,







AC008519, Z98046, AC010458, AC005901, AC000353, AL136137, AL138759, AL080243,







AC010326, AL022315, Z75744, AC005057, AC019227, AL133551, AL109623, AC005104,







AP000280, AC008567, AC003007, AP001748, Z93930, AL136381, AL031577, AC007688,







Z95114, AF190465, AC018639, AC008812, AP001697, AL133174, AC006539, AC005969,







AL035659, AC020552, AC009466, AC066590, AL109865, AL049766, AC010311,







AC024239, AC005670, AC004547, AC002059, AL354696, AC008469, AL445483,







AC004796, AJ003147, AP000692, AL391833, AC010422, AC073219, AC023864,







AC023864, AC016142, AL353659, AC025975, AC011036, AC027544, AC068755,







AC068682, AC024475, AC073446, AC073446, AC023309, AC008470, AC027584,







AC027584, AC009097, AC009899, AC031987, AL162727, AC069362, AL356976, and







AC022696.


HBMXP84
71
460397
1–505
15–519
BE676678, AI041986, AW138792, AI538684, AI457698, AI366797, AI671214, BF515279,







AI688981, AW519152, AI476423, AI433346, AI079260, AA628758, AW024403,







AW235566, AI193554, AI871617, BF593585, AI824190, AI082294, BF940272, AI635312,







AA813988, AW340444, AW664250, AI962200, AI424891, AU144866, AI339229,







AI089412, AW771554, BE857016, AA055519, AW263408, AI003957, AI242128,







AW183948, AI382873, AI201284, AA971967, BF437180, AI199535, AI339355, BE206308,







AI147400, AI687190, AW978061, AW972007, AA496541, AI806607, H10935, AA693833,







AW026275, W80915, AA625056, AW190472, AA702471, AI935367, AA514277,







AW196346, N20032, AI827791, AL040734, AW518723, R84959, BE176371, BF765252,







AA564997, AW665091, BF766112, BF766116, AI687226, AA852967, AA055610,







AA885153, BF810533, H20823, AA669860, R08197, AW872411, T05891, W80914,







AI765293, AA770100, AI687321, BF765227, BE176498, AI620029, BF766101, AA653362,







H59446, BF765234, AW294508, AW274283, AA644156, AW293234, N92954, BF438263,







BE048757, BF807688, AW166685, BF247956, AI525856, AC007956, and AB002315.


HLYAF80
72
460622
1–812
15–826
AA972709, BG111199, AI499555, AI872315, AI924051, AI494201, BF812963, AI804505,







AI815239, AI500659, BE883591, AI866465, BG167830, AI815232, AI801325, AI866691,







AI500523, BF812438, AI538850, BE885490, AI887775, AI582932, AI284517, AI923989,







AI872423, AI590043, AI500706, AI491776, AI445237, AI926593, AW151138, BF811804,







AI289791, AI521560, AI889189, AI500662, AW151974, AI285417, AI623302, AW172723,







AI539800, AI284509, AI582912, AI538885, AI440263, AI889168, AI927233, AW058275,







AI866573, AI633493, AI434256, AI431323, AI866469, AI805769, AI434242, AI888661,







AI500714, AI284513, AI888118, AI285439, AI436429, AI859991, AI889147, AI355779,







AI623736, AW194509, AI581033, AI371228, AI798359, AI590024, AI491710, AI431307,







AI440252, AI687588, AI440238, AL047422, AI567971, AI866786, AI860003, AI610557,







AI431316, AI242736, AI828574, AI539260, AI887499, AW151979, AI539781, AI431238,







AI537677, AI702065, AI539707, AI885949, AI285419, AI559957, AW089557, AI521571,







AI469775, AI866581, AI567953, AL047398, AW074057, AI815150, AI446495, AI889191,







AI952433, AI867068, AI225248, AI358271, AI698352, AI282249, AI371229, AI440260,







AI474699, BE612681, AI687607, AI932620, AI890907, BF811802, AW129310, AL515185,







AI866458, AI432644, BF815930, AI273179, AI499478, AI371251, AI866510, AL134524,







BE897632, AI866461, AI923046, AW151132, AI049859, AI539771, AL048403, AI955221,







AA878808, AL119511, AL513655, BF814072, AW151136, AL043152, AI273116,







BG252929, AW858243, AI371237, AI561170, AI554821, BE895765, AI690946, AI469764,







AI358612, BE877142, AW191003, AI648567, AI433157, AI433155, BF814447, AW081103,







BE909406, AI538883, BG029667, AI801286, AL048375, AW021373, BF527274, AI867066,







BG113493, BG260144, AI362495, BG110517, BG168696, AI371243, AI500683, AV736134,







AA857847, BF892773, AV736402, BE963035, AI635331, AV743129, BE896091,







BG257535, BF911528, BG033906, AL039390, BF795712, AI493559, BF796402, AI049850,







AL045626, AW858522, AI274759, AI355008, BF036448, AI433976, BG254745, BE875243,







BF726204, BE779152, BG169065, AA437293, AI049851, AI440236, AI282247, AI249936,







AI963849, BE537531, AI539863, AI582910, AL040459, AI366900, AI537943, AI559976,







BF814504, AW020095, AI366910, BG259737, AI521566, AA715307, AL513725, AI561177,







AA809974, AW197139, BE618455, AL514283, AI250646, AL047611, BG113712,







AI888022, AL133391, AL355136, E13998, AX040958, AX040974, AL133084, AL133070,







and AC022867.


HNFDS53
73
460489
1–897
15–911
AI041543, AI810369, AA171925, AI573239, AA196756, AA425895, AA454552, AA860221,







R60501, AW166127, AI050663, R51736, R41735, AW206994, R41563, R38697, AA071093,







R44618, AI125305, F04677, R06426, Z40621, F10728, R06481, AA456444, AC011871,







AC069260, AC067779, and AC067779.


HNGAX58
74
460488
1–708
15–722
AA843450, AW088616, AI539563, AI570261, AL048616, AW008317, AL038785,







AI873916, AW872676, AA226584, BG236735, AI537506, AV761127, BG029216,







AV713243, AW268300, AA501809, AW630298, BE872393, AU147922, AA482711,







AI367975, AV763540, AA515224, AI144055, AI250019, AA629992, AI017024, AI287485,







AI613419, AI963720, AW794809, AI890348, N64279, AV710066, AV728425, AI270117,







AL118991, AI445516, AV725423, AI064952, BF304325, AI291124, AV764389, AI912322,







AV762781, AV764523, AI824562, AI192631, AV729947, AV759685, AA587604, AI431303,







AW467362, AW969866, AL031282, AF015158, AF015167, U95743, U95742, AF015160,







S75201, AC004638, AC007216, U12582, AL049552, AL159168, X55925, AC005225,







AC006483, AC002119, U12580, U57009, AL139099, U12584, AC007620, U18398,







AC005730, D83989, AC024158, AC020908, X54176, AC011479, AC009498, AC004257,







AF015170, AC008747, AC007537, AL136218, AL021392, AL121886, AC004971, X53550,







U18395, E01888, E02193, AC009229, AL033544, AC022407, X54178, AL049759,







AL050341, AC005261, AL022322, AC004024, AL034420, X54181, U18391, AF077058,







AF123462, Z99716, U18394, AL133544, AC006249, AC012599, AC009516, AL122001,







AC004894, U57005, AP001477, AC005838, AF232229, U57004, AC004166, AC008764,







AC004824, Z85986, AL109936, AC005224, AL035587, AL163279, AL159997, AL121825,







AC011742, AL096791, Z97181, Z97832, L29074, AC004821, AC008892, U50570,







AC003109, AL136298, AC008482, AL137123, AC005520, AC008372, AL121963,







AC021752, X14008, AP000555, AC002470, AL353812, AL354893, AC018642, X55926,







X54180, AP001067, AC005822, AL121989, AL160413, Z98941, AL050318, AC009228,







AC074331, U57008, AL136179, AC008745, AC022816, AL136103, AL135901, AF019413,







AC007436, X55924, AC004019, AC005015, AC004701, AL359457, AP001730, AL355499,







AL391119, AL353653, AL158832, AL049646, AC010251, AC005971, AL022323,







AC007565, AL121899, AC087225, AL033533, AC006576, Z84480, AL139378, AL135818,







AP001709, AC006207, AC024057, AC087187, AL135924, AP000311, AL138836,







AL138752, AL157906, AC005923, AC024603, Z93241, AC003041, Z74739, AC000052,







AC006334, AC003119, AC005060, AC005602, AP001760, AC024085, AF159056,







AL031295, AC006312, AC004962, U52112, AL031848, AL135906, AC023344, AL157776,







AC005207, AL109825, AL137060, AL021154, AC021015, AC018637, AL034451,







AC005046, AL023494, AC027319, AL121575, AC011310, AC008543, AC009477,







AC002543, AC011475, AL049715, AC002316, Z69705, Z69921, AL080315, AC006033,







AL133286, AL121655, AC016554, AC004067, U18387, AC006430, AC002347, AC008813,







AL034380, AL389887, AL160237, AC002301, AL160392, AL096870, AC016922, and







AC016922.


HOECO90
75
460505
1–831
15–845
BG253227, BG034360, BF115177, AI632191, AA121546, BF676520, AI654697, AI492921,







AW469342, BE503347, AI570945, AA256226, AI285298, AI246762, AI859013, AA256168,







AI273122, AA995514, AL514471, AA127683, AA915978, BG110425, AI686453,







AA876658, AA150857, AA642423, AA150548, AA167031, AW674667, BG179078,







BF185961, BF791217, BF695949, AW963236, BF756684, AF116612, AC069028,







AC069028, and AC069028.


HSVAY16
76
460595
1–868
15–882
AL530241, AL047471, AI264179, AI637796, AI637979, AA804739, D79782, D62805,







AI680570, BE242554, BF089929, AA993094, H23633, AV722809, D79325, H09111,







H06798, D62033, AI372797, H10032, AA350131, D81766, AA740292, BG055352, D80713,







D80714, AA351205, AA449752, AV724345, AL049784, Z74739, U57961, and U50534.


HCFCE10
77
456437
1–1576
15–1590
BE064394, AW469439, BE157228, AA398986, BE064386, AW474349, AI951306,







BF995651, W60721, AI743287, AI050825, AA552514, AI418815, AI651550, AI311783,







AI949802, AA461571, BF766707, AA922652, N91112, AI571732, AW298143, AA454942,







AI911509, BF432341, AA457055, AI936370, AI087071, W60681, AA861649, T67280,







AW852413, AI472074, AI140878, AW852440, BF764727, AW242797, H21175, AW852402,







AW852407, AW852424, T86449, W67519, AW852386, AI494473, N68468, T78901,







F04821, BE148405, AA291948, AW630328, BF438136, AW852412, AW852435,







AW852417, BE675366, AA478908, H52791, AA026555, AI090619, N71108, AI288072,







AW852404, R86767, BF764717, AW237540, AI690255, AI359839, BE467234, AI356396,







AA668598, AI373771, AI274634, AW271466, AA039909, AW207210, AI568788, W05031,







BE183282, AI439197, BE241583, BF941662, BF732367, BG001051, AA926952, AA460283,







AI817206, BF764852, BF056421, AA843945, AI380499, BE010038, AA383115, AA776046,







AI650332, AA342903, AA658035, AA041532, T23749, AW852388, T23746, AW271395,







AW299265, AW852455, AW301214, AI382392, T54604, AW852422, AA165581,







BE072243, AI675065, BE072227, AA343020, BF764664, AW852376, T23747, AI468154,







T23748, T23743, T23744, T23732, BF762199, H59423, AW139704, H27972, AA302043,







AV654161, AW301194, AI638246, AI052296, AI970975, BE080693, AL046961, AI522208,







AI582834, AW025282, BG119153, BG058137, H97314, AI934742, AI659625, T54627,







BF771746, BF935806, AV660791, AW955710, W46955, AA828129, AW117471,







AW979002, R86678, R22182, AI369156, AW275195, AI198193, W68151, AI015493,







AA486606, AI039135, AI985357, BE350924, AW955711, AL137013, AL121755,







AL136130, AC010877, AC005820, AL031000, AC021107, AC022432, AC003686,







AL022345, AC002464, AC011493, AC008173, Z83745, AL139163, AC007326, AL136984,







AC009952, AC017089, AP002026, AC011455, AL096775, AL078591, AC004074,







AJ003147, AC015979, AL356131, AB014607, AC006017, AC008175, AC007359,







AC007965, AL354857, AC011451, AC008567, AP002027, AC008770, AC010133,







AC007379, AC006970, AP001960, AL359457, AL157931, Z83841, AC006596, AC025577,







AL138976, AC004985, AC005318, AC016752, AL031259, AC017016, AL109954,







AL096801, AL049551, AC073898, AC068948, AC010889, AK024638, AC008813, U65960,







AF327083, AC005913, AC007542, AL445669, AL391294, AB045361, AK027110,







AP002534, AL353706, AC078847, AC009276, AL136295, AL022727, AC004636,







AC007283, AC011751, AC019058, AC002485, AC021070, AL161415, AC007966,







AC006048, AP000509, AL031785, AL138733, D84394, AP000270, AL157775, AL121944,







AC006023, AL391376, AP000032, AC007570, AP000103, Z69722, AF297093, AP001714,







AL391595, AC034207, AC022027, AL354811, AL135902, AL022401, AC018448,







AL163642, AC005951, AC019179, AJ011930, AL163300, AC005598, U63630, AC012067,







AL445195, AJ009615, AC002519, AL359924, AC002288, AC004205, AC005406, U36755,







AP000566, AL096868, AC004817, AL023581, AC004072, AC005377, AL353678,







AL132708, AC010598, AL121969, AL132990, AC009494, AL133321, AL109942,







AC005618, Z82182, AC004929, K02402, A22491, AL365505, AP001676, AF265342,







AL132994, AC005242, Z83827, AC002467, AC007948, AC007631, AL033403, AL353732,







AL356118, AC020553, Z98049, AC005297, AC010737, Z49155, AC009415, AC009947,







AC008752, AC006991, Z84481, AL137843, AC004912, Z99916, AC006355, AC009318,







AC011309, AK022218, AC009235, AL353667, AF165926, AL023580, AC008462,







AC011550, AF007544, AL158830, Z96811, AC005516, AF146367, AF124523, AL133328,







AL024458, AL157372, AC004826, AC004065, AB019438, AC002115, AL354943,







AL162872, AC004130, AL353765, U34879, AC006001, AL353807, AC007068, AC008040,







AL133241, AL355382, AC006050, AC004210, AC007322, AL109809, AC008555,







AC004896, AL136520, AL359238, AL359763, AL137123, AL049861, AP000508,







AC004899, AC006539, AC026723, AC005626, AC002472, AC025467, AL137013, and







AL137013.


HCFNC26
78
456439
1–1359
15–1373
AA557489, AA524909, AA493682, AA661943, AA558724, AA525044, AI491828,







AL078605, AC015971, AC011742, AC007002, AC004253, AC009487, AL035422,







AL109614, AC004520, AL137222, AL359457, AL133244, AC004859, AC002040,







AP000350, AP001748, AC007052, AP001728, AL078605, and AL078605.


HELDZ32
79
456450
1–1093
15–1107
AI822137, AI821793, AI141174, AI742807, AI376221, AW008096, N70665, AI147430,







N66810, W05747, AW894967, AI708189, AI792140, N75004, AI597655, AI140241,







AC025019, AC025019, and AC025019.


HODAZ26
80
456514
1–1115
15–1129
AI797353, BG116864, AW665873, AI885785, W60844, BF740039, AI982900, AA010869,







AW444960, AI266484, AA927017, H18446, AI830505, W20375, T11084, AA486911,







AL514473, BF921103, BF970652, AI418681, AI378080, AI564723, AI445829, AL513999,







AI628217, AL514359, AI802542, AI583065, AW050522, AA848053, AA641818,







AW169604, AI659795, AL513977, AL514627, AW051056, AW983832, BF727091,







AW129659, AI824576, BF814527, AI567582, AI919500, AI469532, BF812960, AL514867,







AL514087, AL513901, AL513597, AI699865, AI613038, AW983783, AI698391, AL513907,







AW079159, AI624293, AI254727, AL514691, AL514015, AI537273, AL514791, AI656412,







AI280747, AI249962, AI951950, AW075484, AI884318, AI680162, AI866162, AL515373,







AW149227, AW085786, BG179993, AI570807, AI648458, AI468872, AI699011, BF968910,







AI627988, BE966990, AW051258, AI499890, AW983829, AL513865, AI352497, AI624693,







AI624548, AV743962, AL513553, AV741327, AL119399, BF920893, AI648509, AI587606,







AW083778, AI923989, AW087934, AI270183, AI889189, AI862024, AL514919, AI440284,







AI636588, AV760409, AI934147, AW080746, AL110306, AI352274, AI953767, AI929108,







AI554821, AI890223, BF971336, AL515235, AI933903, AI241923, AW162194, AL043355,







AW983691, AI288050, AI873604, AI702406, AI520809, AW129230, BF814516,







AW079572, AI335426, AI348777, BG027280, AI932949, AW198075, AI499285, AL515195,







AL513631, AI539771, AL135065, AI224027, AL531436, AL514025, AW117919, AI619502,







AW168802, BF725868, AI633125, AI367210, AW088628, AI916419, AI635492, AI569309,







AW051088, AI860783, AI539847, AI963763, AA806720, AI798456, AI591310, AV761267,







BG030785, AI280732, AV728157, AI520862, AI677796, AI917963, AI858827, AI537024,







AW026882, F27788, AI923370, AL037582, AL037602, AI537261, BF812938, AI673363,







AI824746, AW151714, AI537677, AI670009, AI440239, AI433157, BE543089, AI702073,







AI801766, AI610115, BF766535, BF812961, AL515191, AW880037, AI591228, AI612913,







AI452560, BF868489, AI249877, AW191844, N29277, AI249946, AI934011, AI538564,







AI273901, AI498067, AI915291, AW152182, AI673785, BF055899, BF792961, AI280751,







AI473536, AI554818, BE544111, AI475139, AI568114, AI174394, BE965129, BF814449,







AL514085, AW161156, AI344817, AI435268, AI473451, BG259944, BG181094, AA938092,







AA427700, AI471227, AW118518, AI452993, AW088134, AI679891, AV721325,







BG112239, AI885982, AI567846, AI567769, AL513779, BF054877, AI886181, AW081255,







AI476478, AW827119, AL513911, AA835801, AI636456, AW103628, AI803778, AI539071,







BG058150, AI745684, AW303152, AW006032, AL119863, AV709679, AC002081, Z82022,







I92592, AL137533, U68233, AL050366, AL137271, I89947, I48978, E02221, A77033,







A77035, I68732, AF067790, AK026480, AF119909, AK026630, AK025708, AB047904,







AK026462, AF056191, AF159615, AL137538, AF061981, AR020905, AL133067,







AF132676, AL049382, AR070212, AF061836, AF155148, AR038854, AK000432, I33392,







AF217966, AL050138, A18788, AF119899, AF130055, AF158248, AL080159, AJ299431,







AL359583, AK025491, AK026608, AL162083, AK024992, AF090900, AF116654,







AF116646, AF116631, AL122050, AX040974, AK025092, AL137488, AL390167,







AL080124, S61953, AF119878, AK025435, AL110280, A21103, AF130104, AK024524,







AR083266, I48979, AL137476, AK024538, Y14314, AL122093, AL133565, AF113019,







A65341, AL359601, AL133075, AF030513, AF139986, AL122121, AK026408, AF113677,







Y16645, AL137550, AB048954, AL122100, M27260, AL050116, A45787, AX027129,







AK026597, AF177336, AL389939, AL050149, I09499, AL080148, AL133560, AF061573,







AF106862, AF116691, AF119337, AF100931, AL137557, AL133558, E05822, Z72491,







AF111851, AK025209, AK027182, AK027164, AK027160, A08916, AK025465, AL133072,







U35846, AL389982, AL137480, A18777, Y11254, AK025414, AX006092, A08913, U80742,







I89931, AK026583, AL162003, AF183393, X84990, AF026124, AK000250, AF026816,







A08910, AR087170, AK000418, A08909, AB041801, AK025312, AL353940, AF119883,







AL359941, AB052191, X62580, AK027146, U96683, AF061795, AF151685, AB049758,







AF208026, AL050277, AK026533, X79812, AK026959, AK027193, AF113222, AF111849,







AL110221, AF177401, A15345, S68736, AF130110, AF104032, AL389935, AF314091,







AL162002, AF097996, AR019470, AF087943, AL133640, AB048919, U58996, AL133665,







AK025632, AL080074, AL133016, AF225424, AK026885, AF262032, AF113691,







AF116682, AL137292, AF111112, AF230496, AK027113, AF090934, X87582, AK026506,







AL117460, AX020124, Y09972, AF175983, AL353956, AK024594, A03736, AK000718,







AF008439, AL122110, AF116688, X82434, AL122049, L19437, AK024588, AK026504,







AK027096, AF067728, AX042059, AK026528, AL110196, AF143723, AF079763,







AB048974, AK027204, AK026542, AF130105, AK025798, AB007812, AF000301,







AK027144, AF125949, AF217991, AL133113, AX019230, X72889, AL080060, AF119875,







AK026651, AK026744, AB050431, S78214, AK000323, AL137463, AF078844, AF017437,







E06743, AL137478, AF119865, AL359618, AF130082, AF260566, AL110225, A08912,







AF057300, AF057299, AC002467, AL353625, AF118094, E04233, AK026057, AK000137,







AL157479, AF116602, AL117457, AK026452, AC011953, AC025036, AC026985,







AC002081, and AC002081.


HORBI81
81
456519
1–1973
15–1987
BF183268, BG117779, BF973603, BE543674, BG034909, AW378496, BE786924,







BE788087, BE787610, BE786929, BE787323, BE786984, BF036748, BE788815, BE785694,







BF540864, BE619213, AW378489, BE789261, BF031451, BF038867, AW378503,







AW378487, AW966535, BF382065, BF349850, BE868611, AW966540, AU152317,







BG248747, BE964114, BE301111, BF381331, C03298, AI745151, AI539776, BG059523,







BF920931, AA082677, AI811460, AI745144, AA837560, AW958945, BE789166,







BE545882, H04082, BE964000, AV763721, BF196490, AV710772, H02259, AA826857,







BE273600, BF183498, AU150250, AA351846, N52796, BF092307, AA351845, BF508479,







H02356, AW368401, BE544626, BE149510, AA747644, BE619762, AW304170, H03387,







AA346881, AA229163, AU157333, AA669840, AI801505, AI284640, D45542, AA584201,







AV735087, AW872676, AA828704, AW103758, AA503258, AW505125, AW277174,







AW500064, BE350475, N53150, AI085719, AA177061, AW270768, AV760378,







AW511743, AI291037, BF724372, AI431303, F36273, AW406755, AA535661, BE047069,







AL037683, AW872575, AI753488, BE502107, AV762957, AW193265, AL041706,







BG109996, AI613280, AV762050, AI350211, AI471481, BF668217, AI814735, BE676915,







AI345518, BG249643, BG169853, AI434695, AI285696, AL046409, AW193432,







AW021116, BF806176, N35896, AI688846, AW407578, AI358501, AL119691, AI358229,







AV763354, BE206443, AV759362, AV701116, BF805094, AW327868, AW504224,







AV710482, AW419262, BG236735, AV760774, AI246796, AL134330, AI619997,







AI590958, AI279037, AL042753, AI133164, AW073470, AA747276, AA584082,







AV728425, AW662490, AK002035, AF096870, AC005324, AL080124, AL157817,







AC007051, AL031668, AC004971, AC007316, AL049795, AC007919, AL133174, Z69666,







AC005778, AC027319, AL353657, AF091512, AC004166, AC004932, AC008372,







AC005098, AC001228, AC004686, AL133284, AC008760, AC016652, AJ003147,







AC005081, AL022315, U91326, AL353194, AC002316, AC007881, AC000025, AL158196,







AP001730, U47924, AC016656, AP000116, AL138706, AC007272, AL391260, AL161892,







AL031054, AC009484, AC004760, AC011473, AL136300, AC007541, AC005091,







AC004584, AF196779, AL121601, AL163203, AC011475, AC007620, AC006345,







AL133516, AC004477, AL353802, AC003688, AC006011, AC007034, AC005015,







AL022165, AB023049, AC021999, AL031346, AC002538, Z95152, AL049757, AL121928,







AL031286, AL445189, AC007666, AC005538, AC003684, AC018637, AL033377,







AL031393, AC000353, AC005911, AC019215, AP000961, AL133418, AC005156,







AP001748, D84394, AP001346, AC009405, AC004230, AL117381, AP001717, AC005786,







AC002404, AC011811, U07562, Z82180, AC024067, AL117345, AL049643, AC010596,







AC002544, AP001688, AY007154, AL121652, AC006046, AC006571, AL022163,







AP000031, AC078899, U62317, AL008735, AL022316, AC007043, AC005295, AC006480,







AC020916, AC009079, AC005069, AC008474, AC004485, AC008537, AC006205,







AC005038, AC005519, AC006329, AC025430, AC005378, AC005080, AL121751,







AL050349, AF088219, AC004836, AL163249, AC007358, AL031667, AC006285,







AC004878, AL121905, AC010789, AL139099, AL122023, AL445248, D83989, AP000552,







AL353807, AL022311, AL357052, AC008897, AL163208, AL137016, AL031662, U85195,







AL031283, AL022726, AC009530, Z85997, AC004826, AL049557, AP001711, AF077058,







AL034379, AL138976, Z93096, AJ006998, AL049833, AL049758, AC004965, AC004638,







AL132639, AP000517, AL121753, AC015550, AC011470, Z98046, AC007686, AC002430,







AL020995, AL355385, AC021998, AC007488, AC005280, AC002565, AL080241,







AC016026, AL161788, AL138836, AC004675, AC020898, AL355103, AC006208,







AP000426, AJ400877, AC019046, Z68273, AC006501, AC017019, AC002289, AF129756,







AP001671, AP001675, AC009044, AP000032, AP000103, AE000658, AC005250,







AL117373, AC009516, AC004846, AC021036, L44140, AC003101, AC006111, AC008062,







AF227510, AL445493, AC005291, AC005793, AC005225, AC012502, AL118520,







AC022013, AF042090, AC007384, AC018507, AC010553, AC006002, AC017082,







AL021453, U95742, AL356354, AL158832, AL031661, AC002122, AP000556, AC007676,







AJ009615, AC011452, AB023054, AL031391, AL050318, AC018641, Z82201, AL031432,







AC008123, and AC008562.


HOSCY73
82
456520
1–2039
15–2053
BF345673, BE732149, BF684799, BF973626, AA922195, AW873081, AW194609,







AW197591, BE732105, BF341027, AI185740, BE537520, BF310138, AI149726,







AW965809, AL521968, BF694016, AA305846, AI800098, BF341904, AA416876,







AA640513, AW675226, BG117398, BF341862, AA935807, AA713593, AW449858,







BG249168, AA312842, BE876387, AA704143, BF110474, AI628310, AA470048,







AA176983, AA737820, AL521967, AA702002, AA397353, AA983700, AA975245,







AA863436, AA983708, AI471343, AW965126, AI282285, AI913509, BE081596,







AW376741, T32544, BF570805, AW505291, AA046362, AW363086, AA724214, AI610316,







R84594, AA714758, AI915421, N47199, BE081889, AI670874, AA442519, AI954659,







BE266231, D19789, AA773604, BE264023, BE082127, AL538302, AI479754, AI678909,







AA577812, N57913, AA081883, BE154945, F30845, BE154911, AF132856, AF068289,







AL157877, AL139008, AL137707, AB040962, AC008044, AF001548, AC003101,







AL034420, AC006088, AL049610, AC024085, AC006146, AC005049, AC008738,







AC005103, AF047825, AL034548, AC005015, AC025594, AC008569, AL163280,







AP000552, AC009516, U82668, AC073316, AC004491, AC004840, AC004851, AC012351,







AL035685, AC027644, AL080243, AC005520, AL121771, AP001754, AC010485,







AL031657, AC004953, and AC002389.


HADFF38
83
460706
1–1179
15–1193
BF529960, BE513699, BF971923, BF967593, BE514136, BE396317, AI950930, AI963407,







AV727387, BE207779, BF684969, BF350416, AW327780, AI913172, AW294972,







AI278243, AI191611, AI961363, AI285899, AU148374, BE260248, AI312609, W49592,







AI686738, BF673598, BG057573, AI085547, AA827086, AI023458, H95492, R72704,







BF000627, AW297531, AI348391, W49593, BE312078, AW129050, AA748714, R72632,







AA669790, AA565564, AA830495, AI686406, AA868665, AA565120, BE928359, H94972,







AW511899, AA326273, AW961220, BF513032, AA724683, R47808, AI033449, AI203796,







BE837582, AV741543, AW074582, AW505178, AA809832, T70553, AC015651, U43187,







AR008278, U78876, AC018571, AC018571, AC018571, AC015651, AC015651, AC015651,







AC046185, and AC046185.


HFXHN31
84
427118
1–527
15–541


HNGIK21
85
456500
1–971
15–985
AI243728, AW269742, AF034611, AL365215, AL365215, AC019248, AC019248,







AC053510, and AC053510.


HE9FE83
86
460431
1–875
15–889
AW934950, AV740801, AW406447, AW276827, AW438643, BE150580, BE205860,







AV759382, AW088058, AI672135, AI281903, BE047069, BE350475, AV760774,







AV761317, AW088846, F29989, AV682003, AI053672, AA177061, BE046438, AW673241,







AA468022, AV760571, AA610491, BF813686, AA631507, AA621858, AI963720,







AI345654, AI284640, AW778859, AA806796, AI431303, AL046409, AW406755,







BF056371, AA661948, AW020141, AI364020, BG056088, AW193265, AW517377,







BE042649, AW833862, AI697208, AI344844, AI287528, BF668217, AA581903, AA577906,







AA528516, AA559290, W79504, AA669840, AI061334, AI289067, AV719506, AW193432,







AI688846, AI350211, BF939954, BF241967, AI613280, AI814735, AI471481, AV760624,







AI365988, AV759204, AI619997, AI281881, AA744031, AW975425, AW886169,







AA579063, AI270117, AA350859, AI375710, AV733830, AI580652, AW088224,







AW265385, AI654525, AW511743, AA491814, AA630925, AW886165, AA535661,







AW500125, AL037683, F25135, AV710066, AA228420, AW339568, AI801600, R98967,







AI565581, AW419262, AA070867, T53128, AV760937, F18974, AI198376, AI311557,







AV764307, AW273218, AW973496, AA654771, BF109360, AI379719, AA714453,







AV760207, AV758994, BE327924, AA610271, AW769399, AV764523, AI298710,







AA488432, AV761188, AW261871, AV761608, H70615, AV763354, AV761498,







AL119691, AI890928, AI744827, AI053429, N25296, AV762139, AI286356, AA602528,







BE044854, T91187, AA630030, AA569284, AV763971, AA134367, AW662543, AA192740,







AW238278, AI049634, X55922, AF015148, X55930, U67221, X54180, AF015155, U67825,







U57007, U18391, U18392, X55925, X53550, AF077058, U18394, D83989, X55931, U57006,







I51997, M87919, X54181, U67826, U57009, U18395, U18393, U57005, U57008, X75335,







U67827, U18390, X55926, X54178, X54175, U18399, U18387, AF015153, X55924, U18398,







X55928, X55932, M77848, X54179, U18396, U67801, M37551, X54176, A39972,







AF015157, AF015154, U57004, U18400, X55927, S70707, S70699, AF015151, AC011480,







AL109935, AL133387, AC004263, AC016025, AC008474, U63721, AX000035, AC007249,







Z30979, AC011464, AL137074, M96329, AL118520, AC005484, AC006026, L47228,







AL031427, U67832, AC022201, AC011895, AL136295, AL031276, AL138733, AL008709,







U91322, AL008726, AC006432, AF015156, U95740, AC005924, AL031255, AC006211,







AL121870, AP001666, AC001231, AF215937, AC008071, Z97632, AL034376, AL132794,







X76070, AD000684, AC005282, AL163282, AC005815, AC005632, U67831, AC003957,







AC003684, AF015149, AL096734, AC010422, AL390023, AL022323, AL109965,







AC006205, AC010326, AC005081, AC005775, AL049762, S70706, AC006483, AC008039,







AC005900, Z82244, AC011500, Z22650, AC006433, AC020906, AC005154, U67234,







AD000092, AL445217, Z82901, AL136231, U62292, AL162040, AC005257, AC006475,







AC005625, AC004841, Z93023, AC000026, AF123462, AC005000, AC007243, AC009470,







AC002070, AK025812, AL035072, AL078477, Z68870, S70689, AC004848, AL136308,







AL096712, AC006597, AL121601, AL080242, AC005778, AC002299, AL109743,







AC004257, Z83844, AC002543, S48196, AC006285, AC007899, AC012596, AC004647,







AC006449, AL022476, AP000117, AL355144, U67231, AL139384, AC005104, AC004672,







AF042090, AL022302, AF053356, AL121653, AB045361, AC022154, AC026693,







AC008443, AC007132, AL136300, AC010271, AL049795, AL050341, Z99716, X14448,







AL078581, AL049870, AF088219, AC008764, AC004638, AC003104, AC005911,







AC010203, AJ275021, AL136304, AL096700, AL021878, S70692, AC009233, AL357153,







AC022148, AC005785, AC007350, AF302689, AC008079, AC005962, AC004894,







AC006275, AL049757, AC005837, AC008760, AC006115, AC008745, AC007666,







AL354891, AC009533, AL031542, AP001718, AL133500, AP000697, AC011497,







AC006312, AP000509, AL031685, AC006001, AL445189, U73684, AC004773, AL035404,







AL023803, and AL353734.


HMKDS08
87
460477
1–544
15–558
AI635659, AW058323, and AI955959.


HNGEM24
88
460493
1–917
15–931
AV695577, AV695579, BE044854, AW851028, AV691142, AW023672, AV683587,







AV687695, AI672135, BG167743, AV686551, AW836456, AA557686, BF036387,







AW472872, AW129001, AW472847, AI471543, AW274349, AW265385, AW820914,







BF131362, AA341228, BE046438, AA469451, AV760817, BF094375, AV699574,







BG104686, AV760571, BF805334, AI537955, AI284640, R98967, AA177061, AV757607,







AW501687, AI358571, AA338486, BF217299, AW303196, AV735370, AW301350,







AI859251, AV731284, AA318652, AW438643, AV761941, AL042214, BF940517,







BF820332, AA649642, AW833862, BF792685, AA523843, AA594145, AW515401,







AA515224, AW799230, AW503415, AA441788, AI358343, AA714140, AA533725,







AW102955, AI184226, AW674955, AA449088, AA553465, BF940837, AI679706,







BE677384, AA635067, AI568678, AW151884, AV761786, AW500226, BF668217,







AI439360, BG249643, AI633007, AI253797, AL050312, AC009145, Z98743, AL031431,







AC002563, AL160175, L78833, AL356299, AC003043, AC010792, AL122020, AC007014,







AL133229, AC007686, AC068799, AC002528, AC002059, AL022323, AL117382,







AL008582, AL353812, AL121832, U02050, Z99916, AL136124, AC000134, AL049776,







AC011484, AC007653, AC007687, AP001699, AC002045, AC005211, AC005356,







AL354889, AC011489, AL080245, AC002299, AC010271, AL035420, AL021154,







AL133344, AC008269, AL032821, AL355365, AF243527, AL035079, AL162571,







AL391358, AC024086, AL133321, AC011495, AL031680, AL035249, AC021037,







AC011500, AP001605, Z93016, AC005988, AC022392, AL354824, AC004912, AC004382,







AC006241, AL049694, AL132794, Z95115, AL096703, AC006965, AL035703, AC002300,







AF217403, AL031662, AL034429, AC010326, AC005341, AL121901, AC004947,







AC005781, AC006205, AL022162, AC083874, AC006958, AL023284, AC010854,







AC005725, AC005231, AP001712, AC006048, AL139109, AC073316, AC005264,







AC006254, AL031283, AC005089, AL009174, Z98200, AL078590, AC005837, AC005015,







AC007298, AC004932, AC020908, AL023553, AC008623, AL023799, AL022311,







AC009002, AL031848, Z84488, AL117381, AC005822, AC005696, AL035415, AC007637,







AC005056, AC005874, AF134471, AC022173, AC005256, AC002316, AL135939,







AP000532, AC009263, AF168787, Z93096, AL031577, AC006231, X75335, AC006236,







X54486, AC008124, AC002418, U02054, AL096794, AP000509, AP000128, AP000206,







AL161938, AL121978, AP000245, AP001709, AC004089, AC005599, AL354861,







AL355492, AC009358, AC073504, AC004770, AC009483, AL133545, AC004806,







AC006126, AP000466, AC007327, AC006347, AC003666, AC006030, Z97635, AL096814,







AF042091, AC008068, AC084693, AL163282, AC004072, AF253314, AC007537, Z82194,







AC005080, AL049569, U91321, AC006019, AC073249, Y14768, AP000505, AC005048,







AC007666, AL139092, AC005237, AC005229, AC000052, AC004019, Z74617, AL138997,







AL133286, AC016623, AL050337, AC011491, AL133305, AC005020, Z99755, AC008372,







AL356354, AL035458, AC010293, AL355499, AC007707, AC005924, AC002070,







AL157789, AC007358, AC006125, AC002039, AC006312, and AC064796.


HNGFT78
89
460359
1–574
15–588
BF905928, BF906197, AC004518, and AC004518.


HNHDL85
90
461815
1–798
15–812
AL139082, and AL139082.


HCEIA77
91
456433
1–1868
15–1882
AL120374, AL120375, AW299700, AL535680, AI703170, BE048345, AW628698,







AI697576, BF943213, BE300188, AI827735, BF090136, AI027741, AW468728, AI539767,







BF952216, N62986, BF962939, AA325319, R20619, BF984207, H17680, W27595,







AA774545, N51897, N34812, AI700597, AA809692, AI096982, H18789, AV729511,







AA826272, AW450112, H89490, AI004507, AA918185, Z44415, AA630039, H89637,







R13498, N79892, W22875, BF476215, T99474, H42169, Z41688, AI809309, H17065,







AA322769, AL535681, T98880, BE834373, BF816583, BF055592, and AI612712.


HTNBR95
92
457175
1–1377
15–1391
AI742680, AI760199, AW971436, AW130117, AW044708, BG236448, AI222419,







AW384883, AU147072, AV708960, AA992199, AI862023, BF739779, AI263606,







AU147071, BF971287, AI955575, AI867639, AI681988, AA847842, AA731098, AA992130,







AA770202, AA810790, AI056034, AU147153, BG142174, AI420376, AI087795, AA025657,







AW592097, AA909134, AV731318, AW369791, W80758, AI357730, AI284853, N68965,







AW971435, AA504113, AA083314, AA347116, AW608731, AI094541, BF378463,







AA668325, AW961699, AI244315, AA047545, AI809694, AA837327, AW591044,







BE180982, T97061, AI271912, AA888598, H88801, AV757996, AA385505, AI806559,







AW606827, AI000365, Z25156, F00393, AW961694, BF735155, C02251, BF432720,







Z41368, T96950, BF337008, AI884600, AI679865, AI679289, AA947202, BF735713,







BF735541, BE620199, BE160828, BF735781, H89025, BF084679, AC007881, and X98494.


HUKEX85
93
457196
1–916
15–930
AA452738, AA452553, AI821070, AI821839, AI821093, AI821817, AA657420, AA630910,







AA650198, AA630911, AW973757, BF832074, AW970896, AW805547, AA524616,







AW029515, AW805539, AW024018, AC005098, AC009032, AC007066, AL138787,







AP001705, AC005971, AC004638, AC007435, AJ009616, AC006530, AC005080,







AC005353, AC007308, AC004166, AC068658, AC006965, AL035669, AL138836,







AL096701, AL135927, AC007227, Z85986, AC007263, U95741, Z99128, AC008403,







AC005004, AP000689, AP001709, AL137119, AL133458, AP000244, Z83845, AP001670,







AC007537, Z84469, AC006483, AC008474, AC004531, AC007371, AL121890, AL109801,







AC004019, AC004971, AC007686, AC018719, AC018684, AL360078, AC011479,







AC002991, AC009516, AC005529, AC007956, AC004933, AD000864, AL049872,







AC007620, AC005103, AF047825, AP001725, AC010271, AC002115, AJ003147,







AL161445, AL163249, AL133387, AL132639, Z99756, AL160414, AC006028, AC008687,







AC020552, Z93017, AL022326, AL353898, AL353701, AC005527, AC004525, U85195,







AP001694, AC013434, AL049569, AL139384, AC004491, AE000658, AC004883,







AL034405, Z94801, AC020898, AC007912, AP000117, AC004929, AC003663, AF243527,







AC011475, AC073968, AC005225, AL135787, AC006241, AC083863, AC005412,







AC007226, AC004882, AC005330, AL034429, Z83844, U07561, AC000052, AC008664,







AC005914, AC007277, AC009756, AC007597, AL135749, AP000140, AC013436,







AL135978, AC005368, AP000228, AL078461, AL135960, AJ131016, AC020917,







AL035422, AF190464, AC008753, AC006006, AC009244, AC008379, AP000088,







AL096870, AP000692, AL136162, AL022336, AP001718, AC023425, AC005933,







AC002091, AL031680, AL049776, AC006486, AF207550, AC011455, AL109935,







AC005071, AL023807, AJ277546, AL391259, AC008745, AC004797, and AC002477.


HWTBM45
94
456569
1–984
15–998
AL079734, BF668217, AW968341, AV762633, AA602906, AL138455, AW973992,







AI754653, AA468505, AW873061, AL079645, AW961160, AL537797, AW819125,







BE138594, AW504224, AV762430, AI801482, AW237905, AI669589, AW500684, N23504,







AI792499, AL040054, BE147833, AA644090, AW505253, AV763026, AV763058,







AI755214, T74524, N30291, AW500029, AI254779, BF922001, BG029528, H07953,







AW021917, AI754105, AI754567, BF681619, W96522, AV757289, AI380617, AW327624,







BE063437, AI345721, AA584489, AI040051, AA128592, AI267269, BF589824, AW975210,







AA129746, AA533054, AA714011, BE138509, BG236735, AI583142, AV764334,







AL042753, AW402784, AA904275, AI609984, AW263749, AW576251, AW974932,







AV759632, BF541258, AC012089, AC006312, Z85987, AC004922, AC005908, AP001711,







AL022316, AL133163, U91319, AL135927, AC007227, AC008064, AL136295, Z82215,







AC004985, AL391415, AL121873, AC004143, AC008649, AC004967, AL121578,







AL049597, AC024561, AC004973, AC007371, AC002509, AC004686, AP000252,







AC020750, AL133260, AP001714, AC025257, AC005736, AC009362, AL049830,







AC005015, AC004840, AC005723, AC009470, AC002488, AC018642, Z68276, AC009477,







AJ246003, AC008762, AP001630, AC027319, AL139100, AC004965, AC008764,







AC004531, AC004381, AL121658, AC004841, AL157938, AF141309, AL353807,







AC002455, AL049709, AC018751, AC006017, AC005261, AL122001, AC025593,







AC010422, AC011479, AC005952, AL139824, AL050332, AC020906, AC005098,







AC011455, AL035413, AC008733, AL133500, AC004815, AL121753, AL356299,







AC020663, AP001748, Z97196, AL121754, AC008265, AC011526, AC005704, AL121825,







AC006581, AL136124, AC005500, AC010094, AC009314, AC005225, AC006046,







AC005696, AC023425, AC008537, AL049796, AC009516, AC006057, AC006547,







AC005702, AC005102, AL080243, AC005728, AJ277546, AB023054, AL035422,







AC005071, AL121601, AL023583, AL138807, AL357497, AL035423, AC010311,







AL117344, AC006958, AL031293, AC005529, AC006023, AC003664, Z97181, AC006543,







AL137073, AL136179, AC002404, AC007731, AC007671, U91318, AC002302, AP000506,







AL353748, AL121586, AP001717, AC004089, AC008747, AF029308, AC011811,







AC007969, AP000302, AC008616, AL353777, AC002350, AL035417, AL008726,







AL356379, AC002312, AC008149, AL031587, AL136162, AC004865, AL121655,







AC008056, AL160313, AP001725, AL136304, AL022327, AL050349, AL121886, Z73965,







AP000555, AP000558, AC004166, U18398, AB000882, AC004217, AC005998, AL035089,







AC006544, AP000354, AL024509, AC002400, AL021878, AC005043, Z82245, AL031005,







AL049538, AC005600, AC008812, AC004867, AL023799, AC006965, AL049696,







AL078462, AL035249, AL360078, AC008806, AF111167, AL162424, AL137853,







AC011895, AC011465, AL109976, AC005067, AC006458, AC007021, AC004876,







AL139039, AC010200, AL031003, AL049776, AP000517, Z92845, AC005527, AC009531,







AL133411, AC000393, AC009319, AL023575, AF031078, AC006509, AP002534,







AL355392, AL031681, AL162272, AC006077, AC008569, AC007308, AC006483,







AP000114, AP000046, AC005304, AL022326, AL022312, AF030876, AC009032,







AL035703, AC005082, AC005781, AL163279, AC008556, AL139351, AL031594,







AC009247, AC012089, AC012089, AC031988, AC026674, and AC026674.


HADFK68
95
683272
1–816
15–830
T51619, AA768271, AC017097, AC017097, AC068301, and AC068301.


HADGG19
96
467821
1–853
15–867
AI344827, AI349017, AI249421, AI307463, AW074885, AW071363, AI252306, AW071404,







AI318581, AI307423, AI254690, AI249400, AI343038, AI336650, AI318125, AI251556,







AI344795, AW301381, AW302662, AI336506, AI254251, AA598503, AW303238,







BF054886, AI318301, AW268290, AI254503, AW301959, AI345410, AI378710, AI345630,







AI224771, AI223618, AI223628, AI344905, AI306076, AI309427, AI223943, AW075041,







AI340570, AI344901, AI349762, AL037521, AI469778, AW303165, AK025146, AL157706,







and AL157706.


HSDZM95
97
460524
1–531
15–545
AV724520, AV718692, AV718489, D80193, D59787, C15076, D80240, D80210, D80043,







D59467, AV718844, D59619, AV744770, AV744773, D80391, D80196, AV719822, D80253,







AV719783, AV720211, AW973447, D51250, D80045, AV699927, AV720464, D80219,







AV720731, C14389, D51423, D50979, AV720203, AV723927, D59927, D80366, AV722801,







D59889, AV742001, AV720812, D80038, C14014, D81026, C75259, D80022, D81030,







D50995, D80134, AV701125, AV699550, AV701332, AV701043, AV701248, AV701431,







D59275, AV742667, AV700229, AV701428, AV701017, D80195, AV719324, AV744768,







AV741012, AV701166, AV701154, AV699447, AV701021, AV718707, AV700889,







AV744771, AV699669, AW975618, D58283, AV741888, AV701335, AV701443,







AV701004, D80164, AV701149, AV700159, AV701419, T03269, AV718681, D80188,







C14429, D59502, AV744934, AV701344, AV743601, AV720150, AW959570, AV745080,







AV743654, D51799, D80378, AV742720, AV718770, AV721386, AV743008, AV701130,







AV719468, AV718800, AV699866, AV719049, Z21582, D59695, AW960553, AV701415,







AV720607, AW966053, AI557751, AV719913, D80212, C14298, AW965158, AW949632,







AV701151, AW966013, D80166, D80064, AW966531, AV720791, AW949641, AV701224,







AV719876, AW978634, AW966534, AV700622, AW949629, AV701261, AV745724,







AW975621, AV701055, AW949653, AV745723, D80227, AV701422, AW973541,







AV718440, AV720028, AW959628, AW965177, AW949645, AW949656, AW949642,







AW949655, AW966041, AV745369, AW966054, AW966050, AW978661, AW973485,







AV719188, AW973307, AW966062, C14331, AW959597, D59859, AV701118, AV719557,







AW960465, AV745847, AV701012, AW964488, AW959202, AV701163, AV718633,







D57483, AW949654, AW949631, AW949643, AW949618, D80269, AW975605,







AW978648, AV745366, AW949657, AW949658, D59610, AW949646, AW949633,







AV718931, AV699479, AW959799, AW966043, D80024, AW966022, AW973334,







AV742671, AV723097, D80241, AW959582, AW965163, AV701145, AW964756,







AW966029, AV718530, AV746385, AW964737, AV718938, AW959469, AW966065,







AW958992, AW959136, T11417, D59627, AW958993, AV699652, AW965184, AW959062,







AW964477, AW956434, AW962245, AV720654, AW965197, AW966075, AV700357,







AV742430, AV742048, AW960473, AW962082, AW965175, AW965176, AW973474,







AW965185, AW964468, AW956397, AV699746, AW965196, AW973482, AW752082,







AV720220, D51060, AV701123, AW960564, AW973465, AW960504, AV699682,







AW973488, C14227, AV719628, AW178893, AV645389, AV681510, AV681491,







AV645344, AA305409, AL137782, AX027925, Y17188, AX047062, AX021518, AX020190,







A67220, D26022, D34614, A25909, AR025207, AX020191, A62300, X68127, AX033851,







A62298, A84916, AR070327, AX047063, AX047064, AB012117, A85396, AR074141,







AR066482, A85477, AJ132110, A44171, AR088705, A86792, AX042372, AR018138,







AF260572, I19525, X67155, AJ302649, AX035434, D89785, A78862, D88547, AJ294956,







X82626, AF058696, AR092424, AB037923, AJ287395, AR008278, AB028859, AR087649,







AX028130, AX015396, A82595, Y12724, AR093385, AR074545, X93549, A94995,







AR060385, AB002449, AR008443, I50126, I50132, I50128, I50133, AF135125, Y17187,







AR066488, AR016514, AR091537, A30438, AR060138, A45456, A26615, AR052274,







AX035429, AX035428, AX035426, AR074139, AR066490, Y09669, A43192, A43190,







AR038669, AR066487, AR074136, AR008277, AR008281, I14842, I18367, AR054175,







AR062872, D88507, AR016691, AR016690, D50010, U46128, AR064240, AB033111,







A63261, AR008408, AX014811, A70867, D13509, A97171, A64136, A68321, A97217,







AR060133, AR087528, Y11458, I79511, U87247, U79457, Z32749, AB023656, AF123263,







AR032065, X93535, and AR008382.


HSNAA55
98
460535
1–708
15–722
AA772092, BF736774, AA377448, BF593948, BF056778, BE645178, AL528262,







AL528261, AI341095, W76202, AA725283, BE327213, W72965, N31804, N22662,







AI656430, AK026289, AJ224162, AC021148, AC021148, AC021148, and AC069481.


HMDAI51
99
456480
1–739
15–753


HBAFQ54
100
459927
1–682
15–696
AV701040, AV701229, AW960969, AI433668, BE748926, AI913673, AI368814, BE748406,







AA628780, AI692294, AI308055, AI950103, AI291586, R60690, BF902223, N25234,







AI758434, BE789415, AA324513, AW206412, F12190, BF791938, AW750769, AL045327,







AL134524, AW970053, AL045328, AW969911, AW970025, AW968212, AW968347,







AW969778, AW968207, AW970050, AW970070, AW968181, AW970101, AW970079,







AW974660, AW970107, AW968200, AW970540, AW970084, AW969626, AW858522,







AW968204, AW970108, AW968196, AW969848, AL047163, AW970032, AW975061,







AW970075, AW970020, AW969623, AL045817, AL042898, AW969843, AW976040,







AW974805, AW969628, AW970116, AW969653, AW968150, AW970076, AW969934,







AW969624, AW970115, AW969734, AW969864, AW969664, AW969862, AW969826,







AW970105, AW969648, AW969936, AW969961, AW970093, AL037295, AW969916,







AW970014, AW969621, AW969926, AW969958, AW969631, AW969651, AW970095,







AW970118, AL038838, AW970074, AW970069, AW970096, AW972375, AW968190,







AW970023, AW969698, AW970531, AW969632, AW970122, AW969748, AW969634,







AW969697, AW970003, AW969625, AW969677, AW968340, AW968147, AW970036,







AW970054, AW969642, AL037343, AI547295, AW968148, AW969888, AL037436,







AW601637, AW969932, AL038983, AW968126, AW969676, AL037335, AI142134,







AW969691, AW972220, AW970120, AW970092, AW968191, AW970013, AW970119,







AW976022, AL037323, AW970082, AW971172, AW970973, AW970535, AW577201,







AW968215, AW979227, AW577199, AL038878, AW969655, AW979059, AW971738,







AW972357, AL037727, AL037443, AL038822, AW968353, AW972594, AW970094,







AW970612, AL038532, AW978995, AW970068, AW968338, AL038761, AW975032,







AW969809, AW971184, AW975147, AW975031, AW979037, AL044201, AW968167,







AW974871, AW972309, AW970859, AL044125, AW979018, AW972724, AW974704,







AW975434, U46344, AW971207, AW969678, AW979138, AW971725, AW975364,







AW974639, AW974825, AW975231, AW975383, AW970605, AW970948, AW969966,







AW971038, AW979136, AW971989, AW979080, AW971726, AW975142, AW970608,







AW972829, AW972427, AL037435, AW971042, AW972325, AW975210, AW971012,







AW974353, AW971088, AW970085, AW975095, AW975443, AW969622, AW979002,







AW974384, AW968168, AW972596, AW974118, AW969988, AW975158, AW969823,







AW970596, AW971059, AW861914, AW971116, AW979024, AW973247, AW972759,







AL042135, AW969798, AW969876, AW971035, AW973717, AW979127, AW971187,







AW971033, AW972229, AW972212, AW970908, AW972218, AW974083, AW974795,







AW979301, AW970558, AW969785, AW975937, AW979212, AK025056, AR066494,







AJ276255, AJ276256, AJ276254, AR096545, AJ276253, A93931, A93923, A93916, D17247,







AJ276251, A85203, AL133074, AR023813, AL139255, AL139255, and AC020781.


HBGBA14
101
461874
1–441
15–455
AI683694, AI688317, AA523881, AW612950, AW075896, AI858933, AI394309,







AA524036, AW205191, BF197492, AI434019, BF902063, AW151530, AA128407,







BE620592, AW973472, AI582008, AW571727, AI371369, BE620052, AW512224,







AI674185, AI636139, BE043958, AA554717, AI683559, BE910647, AA291456, AI918634,







AW964095, AV727819, AV701880, AV706925, AV683233, AW960203, AW959871,







AW951774, AV702109, AW967325, AW955265, AV704767, AV728853, AW962388,







AW950373, AW966634, AW952306, AW950277, AV694524, AV687808, AV655667,







AV703421, AW957498, AV705635, AV653625, AV729077, AV702019, AV707123,







AV706975, AV705319, AV701657, AV703669, AV728403, AW958509, AW957985,







AV709939, AV708091, AW962651, AW954336, AV653153, AV706854, AV651281,







AV728652, AV705445, AW960538, AV728244, AV703262, AW955728, AV727916,







BF849476, AW962649, AV701184, AV726966, AV707163, AW960688, AV729129,







AW959933, AV706741, AW951732, AW959830, AV655661, AW965874, AV655638,







AV684762, AV702856, AW959796, AV709908, AW962924, AW962934, AW957653,







AW962408, AV704585, AW958225, AV705981, AV656256, AV701951, AW960545,







AV725550, AV702296, AW950194, AV728404, AW959055, AW964040, AW967280,







AV728312, AV702276, AV703090, AV707794, AV702736, AV707105, AV728425,







AV706891, AV702718, AV725387, AV727189, AV728874, AV651920, AW954439,







AV707401, AV658751, AW951235, AV706273, AV707329, AW949478, AW955088,







AW954889, AW960601, AW966970, AV687036, AW951269, AV707663, AV702954,







AV657617, AV727337, AV660258, AV706279, AV726091, AV703035, AV704607,







AV653872, AW955596, AW950443, AV725618, AW950434, AW952403, AW957102,







AV727822, AW952183, AW959806, AW950874, AV656903, AW962444, AV661704,







AW952751, AV697196, AW956075, AV645936, AV709587, AW955683, AW953788,







AV692600, AV650315, AV697880, AV659389, AV702794, AV727613, AV702071,







AV658334, AV655280, AV698765, AW956474, AV727787, AV659294, AV725745,







AV686060, AV703063, AV728148, AV686064, AW951239, AV660608, AV659322,







AV726156, AW951562, AV654908, AV704553, AV726590, AV702728, AV706311,







AV698545, AV656478, AV692176, AV709314, AV653353, AV704542, AV708381,







AV704124, AV660728, AV654070, AV702721, AV691080, AV702385, AV702345,







AW956640, AW949802, AV652001, AV651955, AW951882, AW962978, AV707979,







AV727003, AV709580, AV708786, AV659547, AV727526, AV703169, AW953394,







AV728518, AV725577, AV725033, AV706223, AW949451, AV728924, AV725617,







AV707863, AV696931, AW964421, AW951536, AV703062, AW952196, AV699089,







A62298, AX047063, and AR050070.


HCFMM26
102
460729
1–375
15–389


HOAAF80
103
460500
1–946
15–960
AW511261, AW510759, AW968608, AI913396, BG121775, BF030260, AI767608,







AW967783, BF241628, AA215384, AW769037, BF208908, AA287468, AA662240,







AW769560, AA465182, BF248364, BF209180, BE904505, AA283832, AA214513,







BF507741, BG250489, AA766028, BG111224, AB046790, AC022405, AC022405, and







AC022405.


HODCJ90
104
460501
1–1428
15–1442
BG255308, BF696327, AA769099, BF002402, BG170567, AV702602, AW051928,







AI701149, BF515347, AW166012, H14423, AA972142, AW590588, AI339332, BF105084,







N92764, AV749080, R59745, AV748762, AA100558, AA347767, AI383947, AA015757,







AV750792, AV682211, AI338203, AA347768, D81417, H72916, AA805417, AV747985,







D20390, AI025219, R52023, H14749, AA504717, BE540578, AC010088, AC006338,







AC025735, AC016698, AC006386, AC006366, AC010678, AC016728, AC010153,







AC010902, AL391867, Z55318, AC022202, AC022202, and AC022202.


HNGJU42
105
456507
1–584
15–598
AC022517, AC021618, AC021618, AC022517, and AC022517.


HAEAV45
106
554653
1–671
15–685
AL523067, AA631811, BE268495, AL537230, AA808947, AL520624, BG034078,







AW970052, AW162133, BG104951, BE299890, AW024271, BE735399, BF026622,







BG113569, AW275809, BG179727, BF791599, BF663253, AV713688, BE783378,







AW080276, BF344573, BE891021, BG171655, AA843136, AW156894, AW162669,







BF685774, AI707944, AU133101, BE410329, BF689750, AW157212, BE797183,







AA775858, AW161758, BE792002, AI879436, AA641026, AW150210, AI927563,







AI445180, AI191428, BF345481, BE327875, AI453269, AA775428, BE645873, AI816259,







BE901677, AI830702, AI972207, BG260296, BE254036, BG253017, BE267306, BE747399,







BG254737, AI983064, BE253703, BE378799, AI708007, AW162572, AI709295, AI872201,







BF568091, BF982857, BE378741, AA641014, AI796692, AW162608, AI434537,







AW276244, BG258804, BF969377, AI718184, BF340497, BE791257, BG108514,







BF797906, BE730669, BE139626, BF026659, AI816165, AI718877, AW072655, BE796518,







AI963719, AW439545, AI813374, AI829462, AI719589, BE741415, BE394239, BE796751,







AI510845, AI573051, BE888982, AI439304, BG119618, AI523175, AA564895, AW440491,







AI718011, BE619494, BG178515, BF663016, AI590667, BE562156, AI832807, BE789785,







BF663235, BG118670, BF034398, BF344270, AI972912, AW662410, BE786632,







AA582825, BE350705, AW026997, BG035579, BG034033, AI815937, BF315674,







AI978747, BE908842, AW027561, AI469124, BE549492, BE796456, BF025683, AA582003,







BE781196, BE741500, BG107147, BE964798, BE312880, BE732100, AV763012,







AW161041, AI735159, AA196035, AI635909, AI677799, AI564199, AI831490, AA704141,







AI015408, BG260750, AI954562, BF793991, BE783127, BE279194, BF026496, AI707999,







AI561187, BE258839, BE045297, AI192360, BF974699, BG119213, BG108059, BG181142,







BE728585, BE790434, BF796035, BE732259, BE902639, BE616496, BG107855, BE734551,







BE384565, AW075877, AW151662, AI683042, BE563821, BG029730, BE249928,







AV703077, AA564873, BG025016, BF305907, BF793172, BF340172, BF058949,







BE728750, AW411366, BE250056, BE618525, BE279200, AI672784, AI950848, BE888662,







BF128949, AA653821, BE736286, AA654404, BE407145, BE909484, BF690293,







BE733088, BF568180, AW103101, BE560294, BE783458, BE546623, BE736013,







BE260539, BE389377, BE780776, BE301823, AW080414, BF311961, AA666122,







BF971928, BF966023, BG178957, AW664615, BG109623, BG029033, BF034597,







BF305451, BE883061, BE280736, AI660650, BF683155, BE904396, AW008438,







AW874650, AW090121, BG035633, BF793618, BG250013, BF794965, AV764473,







BF346188, BF791403, BE781837, AW161532, BF337712, BG261028, BE789942,







AW262007, BF204086, BF203699, BG029984, BE785242, AI216087, AC010422, S35960,







AF218023, AK026568, M73791, M64241, L44140, AK027197, S64169, M81806,







AX015398, AP001605, AP001604, AP001699, AL121934, AF143815, X87106, M93980,







X75312, L13234, AC015550, AF280893, AC008752, AC019176, AP000107, AP000039,







AP001715, AP000281, U89405, AB007170, AL031276, AC007597, AC019181, Z98941,







D17268, S35959, AB019572, Z73963, AR070212, AL133084, AK026885, AL389978,







AF067420, AL080148, AL133072, AF225424, U42031, AL122049, AK026749, AL162062,







D44497, AF130100, AK024986, AF056191, I48978, AF159148, AB007812, Y14314, I89947,







A18777, AL359600, AB048881, AB047930, AL157878, AL133049, AL389947, AF019298,







E12579, S69407, AF044323, E02221, X62773, AK024992, X80340, AK025092, AF159615,







AF185614, AF118558, AF188698, AL050092, AL157433, AF016047, E03348, X66871,







E03349, AK026164, AF106657, AF207829, AL442643, A32826, A32827, AL049460,







AK000247, AL050172, AK027146, AL389935, U95739, AF126247, X66417, I29004,







AK024524, U92068, AK027221, S77771, AL137716, AF155148, AL133559, AF067790,







AX040958, AF113689, L10353, AL137478, AX040974, AK026793, AF047716, U30290,







AF311287, AL133014, M64936, AP001343, AP001666, AF111112, A30330, A30331,







X97332, Y11435, AK026504, S36676, AL133070, AK000432, E01314, E12580, AF185576,







AC009953, AL137298, AL162008, AF036941, AL050024, AL080154, I26207, AL133075,







AF061981, S79832, AF022363, AL080163, Y17327, AK024570, AR016469, AF113019,







AB048910, X83544, AF166267, AX015425, A08913, L13297, U80742, AF106934,







AL137641, X00861, AK024546, AF013214, AF116691, I89931, AF116688, X66862,







AB047248, AF130055, AK025958, AK025407, AK000212, AK025119, AR059958,







AF126488, AK000753, AL122100, U96683, AB050431, U87620, S69510, AF260566,







AF015958, AL353956, AF081571, A08912, AL133619, AK025465, AL137480, AL110280,







A08910, A08911, AC002464, AR087170, A08909, AK026480, AK000137, AB047878,







AK025541, U95114, I30339, X06146, I30334, X59414, AB052191, U36585, AJ001388,







AF140224, AF201468, L24896, AR038854, AF116631, AF217994, A08907, AF119336,







AL049283, A08908, AF119865, AX014095, AF218006, AF111849, I17544, AF026008,







AK025113, AL389951, AB029065, Z97214, AR073709, S76508, AC005353, AL008706,







AC012502, AK026608, I89934, AF161418, AF169154, AF132676, D83989, T47109,







T49332, T49333, T49876, T50114, T50166, T50182, T50234, T50491, T50495, T50626,







T50741, T50899, T51631, T52345, T52418, T52964, T53466, T54932, T57905, T72938,







T72946, T89117, T89206, R30809, R66232, H04340, H18203, H19746, H22434, H23800,







H24908, H25932, H25997, H26236, H39653, H41399, H43213, H43313, H43354, H43533,







H44010, H44518, H46702, H47349, R89216, H50527, H51416, H52041, H60635, H63573,







H65898, H66751, H68843, H68699, H79828, H91139, N23979, N24695, N32249, N32513,







N39202, N52466, N53029, N53041, N55358, N62657, N68617, N70301, N70489, N70534,







N70593, N70769, N72877, N77364, N80381, W01823, W02004, W05690, W17244, W24811,







W65440, W68618, W68608, W74295, W79166, W80476, W93657, AA022533, AA053734,







AA054326, AA054426, AA054245, AA057703, AA079213, AA079082, AA079408,







AA079458, AA081107, AA081294, AA081240, AA081181, AA084265, AA085821,







AA088233, AA088351, AA098923, AA099472, AA111988, AA126581, AA134201,







AA130413, AA130504, AA132320, AA143330, AA151230, AA151231, AA149977,







AA155788, AA156533, AA157674, AA167056, AA167057, AA180146, AA180165,







AA181839, AA182027, AA187395, AA187603, AA196818, AA458468, AC008590,







AP001604, AP001604, and AP001605.


HBJFV28
107
460387
1–491
15–505
AI032736, AI767490, AA630933, AA650147, AA010887, Z95704, AC006557, AL121983,







AL133244, AL357041, AC006500, AP001171, AP001678, AL136452, AL136967,







AL160392, and AC018716.


HDHMB42
108
460423
1–1135
15–1149
AV653554, BF110539, AW025103, AW804967, AV742421, AI202704, AA923079,







BF955234, BE351022, AI418402, BF955238, AI359393, BG012203, AW973663, W33011,







W35398, BG007082, BG012207, W27597, W28287, AI082272, N47292, N30737, W28307,







AA883245, AA631134, R42822, BG006618, AA558989, BE835360, BG006620, R17476,







Z44628, R91157, F05279, Z41604, AA056396, AW386203, C18454, C17382, F05412,







Z41975, AV741271, AV737624, BF093584, AA223657, AV735310, AW366020, R12919,







AI491710, AI538885, AI590043, AI702065, AI567953, AI446495, AI500659, BE883591,







AI866465, AI815232, AI801325, AI500523, BF812438, AI500714, AI538850, BE885490,







AI887775, AI582932, AI885949, AI923989, AI872423, AI284517, AI581033, AI500706,







AI445237, AI491776, AL514219, BF811804, AW151138, AI889189, AI521560, AI500662,







AI582912, AI539800, AW172723, AI284509, AI440263, AI889168, AI866573, AI633493,







AI434256, AI355008, AI866469, AI805769, AI434242, AI888661, AI284513, AI888118,







AI285439, AI859991, AI436429, AI355779, AI889147, AI623736, AI371228, AI440252,







AI431307, AL047422, AI866786, AI860003, AI610557, AI242736, AI828574, AI887499,







AW151979, AI539781, AI539707, AW089557, AI559957, AI521571, AI469775, AI866581,







AI815150, AW172745, BF812963, AI927233, AI635331, BG104735, BF726204, AI285419,







AI440238, AI494201, AI537677, AI289791, AL079960, AI567971, BG030516, AI432644,







AI431316, AI690948, BE897632, AI888665, AI932620, BE895765, AL514543, AI539771,







AL514359, AI866484, AI867068, AI539863, AL513983, AI702062, AW151136, BG252929,







AW858243, AI439995, AI554821, AL514089, AI690946, AI282268, AI648567, AI433157,







BG165043, AI828583, AC023790, AB040900, AL133051, AX040958, AX040974,







AF073993, E13998, AL133655, AC023790, AC023790, and AC023790.


HLNAB07
109
460469
1–671
15–685
AA360228, AA360227, AV764259, AA515728, AA658918, BG180976, AA134944,







AW021154, AV695478, AI791185, AW189068, BE145700, AV701116, AI537020,







AV762957, AI192631, AI267356, AI023375, AI267450, AA507637, AA743996, AA635433,







AW974996, AA618346, AA469327, AA584489, F25761, AL136000, AL354720, AL163280,







AL390039, AC008569, AC011462, AC013429, AL096701, AC006312, AL137818,







AC021999, AL121978, AL136979, AC005323, AC020754, AC005730, AP000555,







AL162615, AL139396, AC000118, AC004526, Z75887, AL138787, AL442064, AC009123,







AL133332, AC006511, AL157938, AC005822, AC008056, AL031276, AC010582,







AC013434, AL049830, AC008616, AF312915, AP001717, AF111168, AL031289,







AC000025, AC005969, Z85986, AC004985, AL024498, AC027644, AC004662, AC005520,







AC005225, AL355392, AL136295, AL020997, AC004851, AC005037, AC010458, Z84487,







AC005587, AC005180, AC002375, AL121586, AC002477, AL035658, AL391415,







AL391114, Z83822, AC007358, AC005057, AC010605, AL031311, AC020908, AC008738,







AL354696, AC021036, AC005332, AL049776, U85195, AC006329, AP000501, AP000065,







AL049796, AL022721, AL117381, AL022316, AC000026, AC004815, AL031432,







AC011444, AE000658, AC009362, AL096791, AL023807, AC004150, AL135924,







AP001726, AC022336, Z97054, AC002059, AL031658, AC006581, AC004685, AL009183,







AC005940, AC007546, AC011479, AC007639, AC004552, AJ400877, AC007551,







AC004408, AP001711, AP001684, Y18000, AL121873, AC010102, AL138752, AP000509,







AL159977, AC006241, AP000117, AC002395, AC022148, AL022320, AC004841,







AL137073, AL161445, AC005821, AC008011, AP001728, AC005088, D84394, AC009263,







U82695, AC025588, AL157838, AF001552, AC008623, AC016526, AL109627, AL050341,







AC000105, AC008039, AF165926, AL138836, AL031255, AC068948, AP001412,







AC004908, AC020916, AF196969, AL050335, AC008015, D87675, AF168787, AL365505,







AC004477, AC005529, AL158040, AC005736, AJ277546, AC009244, AC018751,







AC006345, AC008720, AC005015, AL035685, AL031666, AC005519, AP001754,







AC000393, AC005399, AC003982, AC010422, Z98743, AC009516, AL049872, AL031985,







AC006441, AP001718, AC005837, AC005696, AC007066, AC005553, Z97832, AC004876,







AL138756, AC004032, AC006057, AP000152, AL357153, AL353777, AL031602,







AC008379, AL035450, AC020898, AP000114, AP000046, AC011495, AC004882,







AC016543, AF207550, AC005632, AL031427, AF001549, AC004531, AL049762,







AC004231, AC008521, AC008848, AC009600, AC006255, AP000045, AC007371,







AC020626, AC002996, AL109797, AC011895, AC016831, AC010877, AC004883,







AC011450, AP001052, AL021391, AL161670, AC024085, AC008773, AL109935,







AC002126, AC003043, AL034417, AC011526, AC004098, AP001759, AL050349,







AC020906, AC083870, AC011510, AF024533, AC007041, AC021752, AD000812,







AL033528, AL024508, AC011475, Z97196, AC002288, AP000689, AC068499, AC018462,







and AC018462.


HMWDC28
110
460487
1–1132
15–1146
BF511110, BF511098, BF507863, W52839, AW194969, AI199267, R68505, AI521938,







R46033, W81166, BE000169, N47371, BE814496, W81165, AA086195, T64991, AI827849,







AI816972, BF592053, AI797732, AL158212, AL158212, AL133323, and AL133323.


HSZAF47
111
456551
1–1319
15–1333
AW298370, AI433823, AI239867, D62170, D61860, and AC007016.


HTADX17
112
457172
1–1126
15–1140
AA446344, AA612751, AA298785, AA298780, AA298784, AA446524, AA298781,







AA381170, AL357565, and AL357565.


HFTCU19
113
735139
1–1561
15–1575
AL522686, AL532414, AL528017, AL525764, AL525713, AL517601, AL532413,







AL517602, BE728855, BE728429, BE790819, AL522685, BF793544, BG035367,







BG250928, BG248668, BE620516, BG261214, BF527519, BE744785, BG027945,







BE875302, BG107554, BE272944, BE274274, BE728950, BG104319, BE272684, BF025761,







BF568694, BE408005, BE892043, BE259455, BE728885, BE786141, BE272952, BE298531,







BE263890, BF965267, BE786762, AW249086, AA773582, AW956716, BE896833,







AW248651, BG028978, BE272544, AI301926, BE514887, AW958043, AA707330,







BF116072, BE301937, AW328603, AW958038, BE546423, AI983484, AI336123,







BE409785, AI094652, AA857454, AA534610, AI301124, BE218758, BE391518,







AW026133, BE220235, AA039645, AI826760, BF132535, BG164328, W07685, AA143751,







BE217896, BE042731, AW800820, BE265135, AA316625, BF897135, R80333, AA143714,







BE965179, AI371146, AI299006, BE859048, AA181066, AI273416, BE265826, AW328604,







AI033743, AA209410, AI862384, AI989356, AA574155, AA576758, AA625711, AI274171,







AV749543, AA583179, R56497, AW664258, AI400307, R56369, AA084546, AW248447,







BF675036, AA805796, AI914573, AI183317, AA969588, AA516078, AA181067,







AW192448, AA976451, AA961148, R56104, H22843, AI264478, N80562, AI263748,







BE272880, AA352369, AW953396, AA325435, BF569001, R62408, AA904367, AA211866,







R62409, AA303270, BF095626, AA999773, AA917014, R38437, AW118274, AI150239,







AA354306, H22844, AA355150, AA353139, BF799722, R80227, AA039646, AA321806,







N40663, BG122562, AA321805, AI364445, R12864, AA295363, AA641448, AW901335,







AI001853, AI221522, AA303866, R49878, AW247358, T16973, BE206478, AW015905,







AA180189, AA862561, AA743072, AA346787, R56274, AA343417, AA180499, AA279741,







BE546075, AW007931, AW297178, AA179782, AI421063, R49877, AA279823, AI274516,







BF796689, AI525802, AA095578, AW855537, AC003665, AC008550, AA192915,







AC003665, AC003665, and AC003665.


HEBFL88
114
420660
1–320
15–334
AC010966, AC010966, AP001113, AP001113, AP001028, and AP001028.


HJMAV91
115
461886
1–852
15–866
AW972663, AA526403, AA886844, AC011331, AC011331, AC011331, AC019039,







AC019039, and AP001991.


HSDJJ82
116
460602
1–448
15–462
AW594636, AA610164, AL050309, AC011445, AL050309, AL050309, AL353698, and







AL353698.


HTLDP69
117
456560
1–1486
15–1500
AL040854, AL040853, AI359494, AA166837, AI925267, AA166662, BF197389, AI648525,







BG152643, AI243965, AW472821, AA224085, AL080154, AL109804, AC017113,







AC017113, AL109804, and AL109804.


HPEBT80
118
460513
1–346
15–360
AW955977, AW955982, AW949806, AV707155, AV707786, AV701953, AV706964,







AV706407, AI536138, AV728194, AV704601, AV727347, AW954104, AV728743,







AW960597, AW951555, AW953993, AV650843, AW949372, AW958849, AW964752,







AW951743, AV728741, AW954119, AV728426, AV729457, AW949517, AW964071,







AW956792, AW952582, AW952885, AW954153, AV728894, AW956059, AW967182,







AV708167, AV706459, AW950197, AW951750, AV701704, AW962136, AV702783,







AW960271, AV706060, AV703435, AV726756, AV657980, AV728428, AV649942,







AW949551, AW956781, AV706229, AW953080, AV707529, AV689804, AW956289,







AV725074, AV707218, AW958529, AV686231, AV687342, AV707528, AV648619,







AV661824, AV707526, AW967372, AW958858, AW962268, AW950594, AW959873,







AW960779, AV726716, AW957286, AW945159, AV701858, AW959149, AV656107,







AV702039, AV702625, AW950095, AV727468, AV703620, AW949729, AW952105,







AW956514, AW966737, AV705383, AV706104, AV650430, AV653325, AW952419,







AV708007, AV685536, AW955602, AV725260, AW956667, AW955605, AW951951,







AW963700, AV706745, AW952008, AW958002, AV652860, AW960297, AW959881,







AV706419, AW958568, AW963671, AW958569, AV703734, AV708809, AV702218,







AV709030, AV701596, AV701875, AV707769, AW952108, AV709248, AV702998,







AW967184, AW951719, AW963390, AW951772, AW949441, AW963354, AW961012,







AV706245, AV707649, AV706588, AV703152, AW963035, AV645504, AV705481,







AV708721, AV702222, AV701833, AW963231, AV729118, AV707197, AV706507,







AW963463, AV709348, AW951732, AW962713, AV729272, AW955632, AW950000,







AW961593, AW952005, AV724996, AV703950, AW963595, and AL021307.


HCNAV36
119
460695
1–809
15–823
AL519680, AL528106, AV703462, AL134430, BE966276, AI935666, AI310530, AA044672,







BF114799, AW954862, AA633864, BF061437, AL522511, AI608963, AL528107,







BF476072, AI828216, AI638071, AI283033, AW474027, AA770541, AI752992, AI079280,







AI469994, AI214200, AI073527, AA617801, AA083480, AW468167, AI366846,







AW102586, BE673391, AA044748, AW242267, AA476497, AI278917, AI214262,







AW190878, AI310353, W56187, AA922307, AI888954, AW513758, AA505952, H27999,







BE710496, BE614602, H26998, AA863144, H45347, AA083572, W56264, H45277,







AA482897, AI359218, BF115263, T33354, H30190, AI283900, AA315729, AW167352,







BE779235, AW082904, H08801, AA447258, AA634813, BE928714, AW515483,







AW515487, AI364881, F10427, AA747725, H97948, AA045015, F10291, R63835, T17138,







AA521158, AA961616, AW954863, AA452536, AW594459, N72159, BF642801, BE825666,







Z40618, AA447134, R43571, T65421, AI702525, R64447, BG111314, BF358234,







AW945396, AW949256, AA657615, AW996623, AW949208, AA045014, AI304637,







BG166516, AW375723, AW949212, BF372001, BF366001, AI431870, BF382893,







AA679694, AA379961, BF210755, AA585278, AK026850, AC004837, AC068993,







AC002486, AC004837, AC004837, AC002486, and AC002486.


HNFIU96
120
460611
1–442
15–456
BF062478, AC006238, AC002551, AC005529, AC005089, AC002301, AC020913,







AC008569, AC005562, AL020997, U91321, AP001718, AC009244, AF030453, AL163279,







AC006211, AC002299, AC006483, AC006012, AC011465, AC008635, AL122001,







AL008730, AL162458, AF053356, AC020934, AL009031, AC068799, AC005088,







AL137059, AC004967, AC004491, AC005682, Z92844, AC004983, AC006238, AC006238,







and AC006238.


HGLDE38
121
460647
1–539
15–553
N57703, BE968744, AV762454, BF513399, BF514803, AI253382, AA489689, BE179742,







AL109865, AL139318, Z85987, AC008155, AC025593, AC007878, AL442064, AF265340,







AJ011930, AL049766, AC002288, AF001549, AL133383, AL035419, AL079343,







AC010605, AP001596, AC027644, AC006111, AC004797, AL109657, AC005250,







AP001696, AL022721, AC004677, AC016751, AL121975, AC005529, AC016948,







AC009263, AC068799, AL136133, AL109865, and AL109865.


HODDB05
122
456515
1–1144
15–1158
BE220209, AI379772, AA663225, AA969100, AA449510, AI766832, AA449080, AI264822,







AA994523, AB048898, AL109932, AL109932, and AL109932.


HNGAC63
123
460491
1–540
15–554
AA904211, AA601674, T41134, AI268019, BF924753, AW270258, BF763954, AA564671,







AL120282, AI499954, AI719142, BG230549, AW337526, BF991881, H94979, AW149162,







AV709831, AW512300, AA156323, N23919, BF793051, AA584360, AA515728, BF991882,







AW662635, AI623364, AL134167, AA612727, AI915081, H22141, F31951, H63660,







F02399, F00688, BF902291, BE883977, AA523695, BE043997, BF744666, BE253949,







H24957, BG057631, AA508036, AA525293, AV728582, BE077105, AV763026, AV763058,







H02532, AW089016, AA573000, AW301483, AL138230, AA992877, AI801505, R81167,







C14698, AW754413, AI792092, AI821056, AI821805, BF906917, AA603264, AI336637,







AA523812, AW020150, AI537368, AI521525, T49933, AI298787, AI733856, AW512528,







AA397821, AA807704, BE616984, AA492105, BG028918, AA302817, BF906915,







AA557790, AV722030, AW511778, BG055738, H58393, AW467617, AA636077,







AV743799, AL045476, AA584493, AA768179, AI583291, BG003443, AI479068, AI984168,







AI285493, AA378489, AA584183, AA574286, AW069537, AI354484, BE177633,







AI590404, AV759632, AW102811, AW513905, AW275699, AA584655, BE063437,







AA862183, AI270120, AI002863, AI962030, AV722843, AW515437, AA610255,







AL041013, AW013787, AI762528, AI918419, AA831714, AC004099, AC004854,







AL049697, AF001905, AC002073, AC004797, AC005728, AP001710, AC007388,







AC008403, AL096701, AC007546, AL121833, AL050321, AC008745, AC005534,







AC005535, AC023478, AC003043, AC034195, AC010311, AC004638, AL109984, Y10196,







AC023905, AL133229, AC004985, U80017, U62293, AL031681, AC004858, Z81365,







AL031671, AL159168, AL021326, AC005071, AC005899, AC005057, AC004685,







AC009362, AL034423, AL034429, AC004796, AF030453, AC005800, AC002565,







AC005666, AL022238, AL162272, AL022320, AC005940, AC005291, AC005332,







AC022148, AL049872, AF001550, AL132996, AC009275, AL117337, AC005031,







AL121655, AC006241, AC005052, AC005066, AC004983, AC005520, AC007201,







AC011479, AC011299, AC006509, AF235098, AC002316, AC018663, AP001752,







AB009668, AL353688, AL121825, AL136324, AP002028, AC018719, AL031290, Z98885,







AC006023, AC008753, AL133342, AC021036, AC027319, AL050349, AC004383,







AC005839, AC024082, AL135924, AC007151, Z98743, AL008721, D88268, AL133035,







AL022476, AL031447, AC004382, AC018758, AC010527, AP000694, AC000134,







AF205588, AL161757, AL136418, AP001052, AL079342, AC004900, AC020626,







AC018514, AL008718, AL356652, AL049764, Z68269, AL031300, AC002549, AP001691,







AF109907, AL136105, Z82203, AP000547, AL109804, AC004230, AL138878, AC005971,







AF168787, AC005017, AC002551, AC007225, AC009134, AC011445, AL355094,







AC020629, AC024075, AP000501, AC020947, AL008636, AP001694, Z95115, AC005231,







AC004955, AL121652, AC006468, U73640, AC002472, AC008991, AC005367, AL109743,







AP000067, AC004743, AL121950, AC003089, AC009505, AC020552, AL136097,







AP000066, AL121900, AC008551, AL356299, AC007221, AC005081, AL022313,







AC004703, Z82217, AC012309, AC003101, AC004084, AP000246, AP000208, AP000130,







AC018764, AC006329, AC009263, AC004166, AC006017, AL034420, AL050318,







AC004409, AP000212, AP000134, AP000557, AL022326, AC005488, AL022316,







AC021016, AC005901, AC008736, AL121658, AC004525, AC020904, AC009086,







AC007570, Z95624, AL109940, AC009032, AF141325, AC005080, AC004079, AC006126,







AL138717, AL034405, AL139039, AC007376, AC008155, AL138881, AP001631,







AP001748, AX039602, AC005375, AC020663, AL021154, AL163249, AC006480,







AC007050, AC007664, AC005837, AF001549, AL121585, AP000688, AC004882,







AC005695, AC006501, AC009307, AL031003, AC006065, AC005516, AL049569,







AL022311, AP001725, AC018755, AL033533, AC005015, AC003003, AL021546,







AF219991, AD001527, AC006088, AC023490, AL118502, AC006101, AL136365,







AC007240, AL445465, AC003982, AL157789, AC004491, AC083863, AC004099,







AC004099, and AC004099.


HTADV27
124
456552
1–1241
15–1255
AC004227.


HCUFZ62
125
461448
1–1963
15–1977
AL527114, AL530639, AL525527, BE613157, AL527971, AL527662, AL525578,







BE744419, AL530640, BG260459, BE908482, BG170056, BE393924, BG118709,







BE275013, BE744815, BG170280, AW575072, BE264567, BE746927, BE741238,







BE730254, BG165516, BE264389, BE548524, BE898281, BE747690, BE617852, BE962359,







AI885402, BE617394, BG164178, BE298595, AW935158, BF034551, AW410641,







AW575125, AW819594, BF974904, AA402378, BF036070, BF124919, AW954693,







AI991184, AW387107, AW574756, AA873160, BF974176, AW170092, BE674969,







AW574792, BF115964, BE612995, BE676516, AI829944, AI921923, AW512364,







AA745265, AW008540, AW387090, BE410451, AI924538, AI074307, BE962247,







AL527127, AA477599, BE909093, BF439439, AW337968, BF220187, BE617598,







BF981222, AW028819, AA864636, AI829318, BE877307, AA402134, BF437001,







BE867863, BE208227, AI139793, BF219799, AA857987, BF976711, BG168005, BE901548,







AI479060, AI005213, AI091416, BG014395, BE735396, AI684266, BE617828, AW242087,







BE905461, BF875069, W95516, AW512623, AW512911, AI336176, AI362816, N54144,







W79630, AI075402, AI222764, AA456798, AI972124, AI161220, AA609827, AI538743,







AA443894, AW410640, AI090476, AW474208, AI446095, BF744898, AI096558,







BE870316, BF879837, AW245879, BF982815, AI086820, AA873436, BE617360, AI609956,







AI598268, AW194155, AI151109, AA536177, AI139048, AA488987, AA576443,







BG116465, AW473682, AA937584, AI028016, BF876032, AI805600, AI469710, W79486,







AW469206, AI074246, AI187204, AW117881, AA770528, AA431854, BE049246,







AI434504, AI619841, AI348212, BG106832, AI304813, AI241070, AW473135, AI872263,







AA838514, BF765177, AA394128, AA832041, AA477555, AI355882, AA704924,







BE396290, AI827052, AI635832, BF874815, AW583441, AA724420, AA460236,







AA463799, W81550, AW516942, AW438489, AI368869, AI187174, BF881820, N93383,







BG176553, BF034119, AI285018, BF037640, BF880959, BF033023, BF881809, AW591674,







BG115529, AI248658, BE891033, AW169451, AA399135, BF880954, F33861, AI365011,







AW572650, BG013080, AI346208, BF881616, AW401992, AW801679, BF976039, R72698,







BF880935, AA292270, AW517487, BE616937, AW628189, AA897757, BF744997,







BF880930, AI961866, AW771733, AI613224, AA488766, AI266343, AW192153, AI276959,







AA872815, AI247720, AI359194, AW006966, BE264829, AA555138, AI351451, AI620538,







AA632947, N95718, AA975671, F26639, N95198, W52694, AA305218, AI371332,







AI860610, AW105713, AI027063, AA468707, AA531066, AA973012, AI537125, F37944,







AA464262, AA465253, BF037848, AW583384, AA431853, AW934814, AW244117,







R72627, AI862935, AA587714, AJ002308, AJ002312, AJ002313, AR069079, AJ002307,







AF151985, AF039085, AR069086, AR069082, AR069080, AR069081, AR069085,







AR069084, AR069083, AR069087, AL133655, AL080148, AL133053, AL133049,







AX040958, AX040974, U30290, E13998, U48245, AL133076, U83172, AL133084,







AL133070, AR015970, AL049423, AK026626, AP000081, AK000074, AL122101, X59813,







AX041002, AL133051, AC004213, AC006531, AF032666, AL133074, E01614, E13364,







AF113222, AL133607, AL133608, AX015526, J05277, AC005519, M19658, AX030435,







AF002985, AR055519, AF119875, AR034821, AB038161, AP001623, AP001746,







AL389939, AF126531, X61049, AF017790, AL137266, AK000344, Z69719, S54890,







AF169154, AL122049, U89906, AB020592, AL022723, T47328, R08599, T96113, R01124,







H20537, H52662, H61205, N23034, W25402, W37484, and AA074639.


HSSJN64
126
460596
1–724
15–738
Z59747, Z59748, Y14768, U91323, AP000505, AF129756, AC005052, AL162615,







AC021396, and AC021396.


HTEHH53
127
456555
1–974
15–988
AI638623, AW340528, AA912681, AW082233, AI073399, BF966133, AW072250,







AA971343, AA383555, AI017073, and AL121901.


HSIDI15
128
460528
1–898
15–912


HE9HW52
129
460432
1–555
15–569
BG231973, W58459, AW974208, AI803690, AI673434, AW050614, AA908673, BE962188,







AW050632, BF979853, AI378033, AW001572, BF967768, W58347, BE931725, AI125225,







AI129923, AI214596, AA448177, AW131630, AW452081, AI200014, AI620164,







AA575928, BE386240, AI221658, AA877467, AA573435, AA809706, D80993, AA723851,







AI090712, AW974209, AI695504, BE302814, T49181, BE617792, AI422493, AW371789,







AW371791, BF375427, AW392371, AI361294, AA055320, D60924, AA864184, AI699226,







T66827, D80994, AW382253, AW371786, AA865889, AA906223, AC003042, and







AK026196.


HLWCF05
130
460619
1–632
15–646
AW673972, AA524980, AI961840, AW515257, AA877458, AI336752, AW070880, N66443,







AA528268, AI273991, N26777, AA004802, BF990906, AA700372, AI290414, AA906772,







AI243008, H17960, AA502507, AA860313, AW470183, H84037, BF348530, R54094,







BF764578, H84462, R30859, BF764695, AI864306, AW022917, AW970612, D45536,







AI908718, BF878700, AW999226, AK025020, AC009097, AC009097, AC009097,







AC026451, and AC026451.


HFGAL10
131
714960
1–1169
15–1183
AW948980, BE255767, T66050, T73758, F12097, T97529, T65281, Z43134, AB006631,







AC005805, AC005805, and AC005805.


HSLBF69
132
456539
1–2105
15–2119
BF036485, BE295686, BF437866, BE327182, AA565548, AI243677, BG035392, BF478009,







W38980, H87863, H87806, AA883670, AA314107, AL037910, BG222166, AI053445,







AW474152, BE891525, AW576287, AA579469, AA557486, AI446474, W23546, AI343143,







AW402458, AW502949, AI053786, AI623899, AI865213, AW962942, AI754291,







AA501614, AI433247, AA494038, BF475757, AI859834, AV682490, BF915516, AA834756,







AW970922, AW977540, AI064811, AW075979, AW973400, BF926429, AA857296,







F32808, AA984263, BG057491, AI952900, AA524832, AV713291, AV712414, BG060130,







AI370475, AI049996, AA553448, AV710245, AW955984, AI457597, AW675110,







AW975164, AI791189, AW973771, AA533408, AA053581, AW268277, AA425438,







AW327624, AA493641, AA584489, AA716755, AW472872, AW571493, AL045709,







AA994578, AI732157, AI732158, AW277109, AI889356, AW517716, BF766734, BF725347,







AA448838, AV763401, AA469327, AW955986, BF964699, AA745524, AA652813,







AI633021, AV759185, AI889361, AW419081, AU154948, AU153921, AW974932,







AW062724, BE302974, AW192330, AI753365, AI261913, BF693906, AU120769, H10143,







AA628627, AA812058, AW962713, C14692, BF681619, AI754037, AI174230, R92640,







T52478, AA515937, AW575714, AI696955, AI309384, AW513195, AW819626, AI538540,







BF991881, AI912514, AI056415, AV733057, AA457685, AW117976, BF832503,







AW271904, AA598649, AW238325, BF991882, AW591443, AV733352, AV762047,







AI679713, BF979071, AV762033, AA410788, AI538812, AI564508, AV764104, AI859946,







AV758989, AC008547, E07348, AL138725, AC018764, AC003037, AC007878, AF157065,







AP000114, AP000046, AC018720, AL133163, AL163853, AP001717, AC024082,







AL133324, AC008395, AC006950, AB016897, AL391840, AC024584, AL034423, Z96568,







AC009228, AC011495, AL121754, AC006013, AP001724, AL109802, AC008623,







AC005409, AC009307, U35114, AP000302, AL031680, AC002044, AL035659, AC008733,







AC006039, AC005680, AC010205, AC007385, AC009194, AL359700, AC004590,







AL021937, AL137073, AC012076, AL031283, AC008149, AC004909, Z82190, AC018546,







AL391114, AL138735, AL034420, AC004019, AC008626, AC018644, AC005179,







AC006138, AC003051, AC004813, AC008403, AL139095, AC005243, AC004929,







AC008056, AC004596, AC025593, AC005800, AC004510, AL035454, AL121965,







AC008952, AL139339, AL121657, AC008724, AC008893, AL133338, AL356379,







AC007057, AC002064, AC006023, U91326, AJ229041, AC008265, AC004024, AP001232,







AC006347, AC020728, AL137005, AC004508, AL391122, AL137818, AJ271718,







AC006965, AC006501, AC009154, AL163613, AL159996, AC011489, AL136124,







AL162718, X71342, AC005008, AL110502, AL096773, AC009756, AC005996, AC005701,







AL158830, AB023052, AC008887, AC011440, AC005954, AL132653, AC004967,







AC005971, AC005253, AL121585, AC002418, AC005064, AC004149, AC035149,







AC011529, AP001685, Z95331, AC004068, AL139232, AC020663, AL133391, AC011442,







AC004549, AC008753, AL031289, AC004954, AP000513, AC011548, I40899, AP001752,







AC006077, AC007383, AC008474, AL109955, AL021332, AC004963, AF254822, Z85996,







AC005274, AC009248, AC005839, AJ289880, AC011452, AC006985, AL096853,







AC005914, AP001137, AC004816, AC008012, AC004042, AC004472, AC022173,







AP001712, AC004477, AL133448, U71218, AL132713, AC008372, AP001477, Z98752,







AF129756, AC006483, AC004765, AC006050, AP000512, M37469, AL353807, U63313,







AL020993, AF196969, AP001681, AL136303, X55448, AC005972, AC005235, AL445263,







AL121945, AL160397, AL391684, AC004867, AL157768, AP001710, AL391259,







AC009244, AC005184, AC007193, AC002544, AC005540, AC005520, AL159158,







AC008482, AC012442, AC073175, AL353706, AB020860, L44140, AC005057, Z85988,







AL133396, AC074178, I40904, Z85987, AC004448, AL139099, AL163285, AP000959,







AF168787, AL034548, AL445220, AF001549, AL157384, AC008179, AC004016,







AP000347, AL117352, Z64709, AL356575, AC006270, AB020859, AB000880, AF069291,







AL136179, AP000244, AL009029, AC004386, AL161743, AC079045, AC004882,







AL136984, U62293, and AL157791.


HTEDS39
133
745350
1–680
15–694
BF103655, AW408031, BE247532, AA029932, BE929465, AI718659, AI651912,







AA380908, BG232105, AW956578, BE049585, AA749217, AI984467, AI633684,







AA743043, AA748305, BF513581, BF895003, BF895254, AC007068, AF285089,







AF133299, AF285087, AF285088, and AC010186.






















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 (clonetech)
Human Lung




(clonetech)


AR057
Human Mammary
Human Mammary



(clontech)
(clontech)


AR058
Human Thymus
Human Thymus



(clonetech)
(clonetech)


AR059
Jurkat (unstimulated)
Jurkat




(unstimulated)


AR060
Kidney
Kidney


AR061
Liver
Liver


AR062
Liver (Clontech)
Liver (Clontech)


AR063
Lymphocytes chronic
Lymphocytes



lymphocytic leukaemia
chronic lymphocytic




leukaemia


AR064
Lymphocytes diffuse large
Lymphocytes



B cell lymphoma
diffuse large B cell




lymphoma


AR065
Lymphocytes follicular
Lymphocytes



lymphoma
follicular lymphoma


AR066
normal breast
normal breast


AR067
Normal Ovarian
Normal Ovarian



(4004901)
(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
Ovarian Cancer



(9702G001)
(9702G001)


AR073
Ovarian Cancer
Ovarian Cancer



(9707G029)
(9707G029)


AR074
Ovarian Cancer
Ovarian Cancer



(9804G011)
(9804G011)


AR075
Ovarian Cancer
Ovarian Cancer



(9806G019)
(9806G019)


AR076
Ovarian Cancer
Ovarian Cancer



(9807G017)
(9807G017)


AR077
Ovarian Cancer
Ovarian Cancer



(9809G001)
(9809G001)


AR078
ovarian cancer 15799
ovarian cancer




15799


AR079
Ovarian Cancer
Ovarian Cancer



17717AID
17717AID


AR080
Ovarian Cancer
Ovarian Cancer



4004664B1
4004664B1


AR081
Ovarian Cancer
Ovarian Cancer



4005315A1
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
Ovarian Cancer



9905C032RC
9905C032RC


AR088
Ovarian cancer 9907 C00
Ovarian cancer 9907



3rd
C00 3rd


AR089
Prostate
Prostate


AR090
Prostate (clonetech)
Prostate (clonetech)


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)
Small Intestine




(Clontech)


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
Tonsil geminal



centroblast
center centroblast


AR101
Tonsil germinal center B
Tonsil germinal



cell
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
002: Monocytes



(1 hr)
untreated (1 hr)


AR125
002: Monocytes untreated
002: Monocytes



(5 hrs)
untreated (5 hrs)


AR126
002: Control.1C
002: Control.1C


AR127
002: IL2.1C
002: 1L2.1C


AR130
003: Placebo-treated Rat
003: Placebo-



Lacrimal Gland
treated Rat Lacrimal




Gland


AR131
003: Placebo-treated Rat
003: Placebo-



Submandibular Gland
treated Rat




Submandibular




Gland


AR135
004: Monocytes untreated
004: Monocytes



(5 hrs)
untreated (5 hrs)


AR136
004: Monocytes untreated
004: Monocytes



1 hr
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
Adrenal Gland + Whole



Brain
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
Breast Cancer



(4004127A30)
(4004127A30)


AR194
Breast Cancer
Breast Cancer



(400443A21)
(400443A21)


AR195
Breast Cancer
Breast Cancer



(4004643A2)
(4004643A2)


AR196
Breast Cancer
Breast Cancer



(4004710A7)
(4004710A7)


AR197
Breast Cancer
Breast Cancer



(4004943A21)
(4004943A21)


AR198
Breast Cancer (400553A2)
Breast Cancer




(400553A2)


AR199
Breast Cancer
Breast Cancer



(9805C046R)
(9805C046R)


AR200
Breast Cancer
Breast Cancer



(9806C012R)
(9806C012R)


AR201
Breast Cancer (ODQ
Breast Cancer (ODQ



45913)
45913)


AR202
Breast Cancer
Breast Cancer



(ODQ45913)
(ODQ45913)


AR203
Breast Cancer
Breast Cancer



(ODQ4591B)
(ODQ4591B)


AR204
Colon Cancer (15663)
Colon Cancer




(15663)


AR205
Colon Cancer
Colon Cancer



(4005144A4)
(4005144A4)


AR206
Colon Cancer
Colon Cancer



(4005413A4)
(4005413A4)


AR207
Colon Cancer
Colon Cancer



(4005570B1)
(4005570B1)


AR208
Control RNA #1
Control RNA #1


AR209
Control RNA #2
Control RNA #2


AR210
Cultured Preadipocyte
Cultured



(blue)
Preadipocyte (blue)


AR211
Cultured Preadipocyte
Cultured



(Red)
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-
Liver Diabetic (99-



A281A)
09-A281A)


AR240
Lung
Lung


AR241
Lung (27270)
Lung (27270)


AR242
Lung (2727Q)
Lung (2727Q)


AR243
Lung Cancer
Lung Cancer



(4005116A1)
(4005116A1)


AR244
Lung Cancer
Lung Cancer



(4005121A5)
(4005121A5)


AR245
Lung Cancer
Lung Cancer



(4005121A5))
(4005121A5))


AR246
Lung Cancer
Lung Cancer



(4005340A4)
(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 (OCT)
Monocytes (OCT)


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-
Omentum Normal



B009)
(94-08-B009)


AR267
Omentum Normal (97-01-
Omentum Normal



A039A)
(97-01-A039A)


AR268
Omentum Normal (97-04-
Omentum Normal



A114C)
(97-04-A114C)


AR269
Omentum Normal (97-06-
Omentum Normal



A117C)
(97-06-A117C)


AR270
Omentum Normal (97-09-
Omentum Normal



B004C)
(97-09-B004C)


AR271
Ovarian Cancer
Ovarian Cancer



(17717AID)
(17717AID)


AR272
Ovarian Cancer
Ovarian Cancer



(9905C023RC)
(9905C023RC)


AR273
Ovarian Cancer
Ovarian Cancer



(9905C032RC)
(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-
Skeletal Muscle



A105)
(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-
Skeletal Muscle (91-



A105)
01-A105)


AR289
Skeletal Muscle (92-04-
Skeletal Muscle (92-



A059)
04-A059)


AR290
Skeletal Muscle (96-08-
Skeletal Muscle (96-



A171)
08-A171)


AR291
Skeletal Muscle (97-07-
Skeletal Muscle (97-



A190A)
07-A190A)


AR292
Skeletal Muscle Diabetic
Skeletal Muscle



(42352)
Diabetic (42352)


AR293
Skeletal Muscle Diabetic
Skeletal Muscle



(42366)
Diabetic (42366)


AR294
Skeletal Muscle Diabetic
Skeletal Muscle



(42395)
Diabetic (42395)


AR295
Skeletal Muscle Diabetic
Skeletal Muscle



(42483)
Diabetic (42483)


AR296
Skeletal Muscle Diabetic
Skeletal Muscle



(42491)
Diabetic (42491)


AR297
Skeletal Muscle Diabetic
Skeletal Muscle



42352
Diabetic 42352


AR298
Skeletal Musle (42461)
Skeletal Musle




(42461)


AR299
Small Intestine
Small Intestine


AR300
Stomach
Stomach


AR301
T-Cell + HDPBQ71.fc
T-Cell + HDPBQ71.fc



1449 16 hrs
1449




16 hrs


AR302
T-Cell + HDPBQ71.fc
T-Cell + HDPBQ71.fc



1449 6 hrs
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


H0007
Human Cerebellum
Human Cerebellum
Brain


Uni-ZAP XR


H0009
Human Fetal Brain




Uni-ZAP XR


H0012
Human Fetal Kidney
Human Fetal Kidney
Kidney


Uni-ZAP XR


H0013
Human 8 Week Whole
Human 8 Week Old
Embryo


Uni-ZAP XR



Embryo
Embryo


H0014
Human Gall Bladder
Human Gall Bladder
Gall


Uni-ZAP XR





Bladder


H0024
Human Fetal Lung III
Human Fetal Lung
Lung


Uni-ZAP XR


H0025
Human Adult Lymph
Human Adult
Lymph


Lambda ZAP



Node
Lymph Node
Node


II


H0028
Human Old Ovary
Human Old Ovary
Ovary


pBluescript


H0030
Human Placenta




Uni-ZAP XR


H0031
Human Placenta
Human Placenta
Placenta


Uni-ZAP XR


H0032
Human Prostate
Human Prostate
Prostate


Uni-ZAP XR


H0036
Human Adult Small
Human Adult Small
Small Int.


Uni-ZAP XR



Intestine
Intestine


H0038
Human Testes
Human Testes
Testis


Uni-ZAP XR


H0039
Human Pancreas Tumor
Human Pancreas
Pancreas

disease
Uni-ZAP XR




Tumor


H0040
Human Testes Tumor
Human Testes
Testis

disease
Uni-ZAP XR




Tumor


H0041
Human Fetal Bone
Human Fetal Bone
Bone


Uni-ZAP XR


H0042
Human Adult Pulmonary
Human Adult
Lung


Uni-ZAP XR




Pulmonary


H0046
Human Endometrial
Human Endometrial
Uterus

disease
Uni-ZAP XR



Tumor
Tumor


H0047
Human Fetal Liver
Human Fetal Liver
Liver


Uni-ZAP XR


H0050
Human Fetal Heart
Human Fetal Heart
Heart


Uni-ZAP XR


H0051
Human Hippocampus
Human
Brain


Uni-ZAP XR




Hippocampus


H0052
Human Cerebellum
Human Cerebellum
Brain


Uni-ZAP XR


H0056
Human Umbilical Vein,
Human Umbilical
Umbilical


Uni-ZAP XR



Endo. remake
Vein Endothelial
vein




Cells


H0057
Human Fetal Spleen




Uni-ZAP XR


H0059
Human Uterine Cancer
Human Uterine
Uterus

disease
Lambda ZAP




Cancer



II


H0060
Human Macrophage
Human Macrophage
Blood
Cell Line

pBluescript


H0063
Human Thymus
Human Thymus
Thymus


Uni-ZAP XR


H0068
Human Skin Tumor
Human Skin Tumor
Skin

disease
Uni-ZAP XR


H0069
Human Activated T-Cells
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR


H0075
Human Activated T-Cells
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR



(II)


H0078
Human Lung Cancer
Human Lung Cancer
Lung

disease
Lambda ZAP








II


H0081
Human Fetal Epithelium
Human Fetal Skin
Skin


Uni-ZAP XR



(Skin)


H0083
HUMAN JURKAT
Jurkat Cells



Uni-ZAP XR



MEMBRANE BOUND



POLYSOMES


H0085
Human Colon
Human Colon



Lambda ZAP








II


H0086
Human epithelioid
Epithelioid
Sk Muscle

disease
Uni-ZAP XR



sarcoma
Sarcoma, muscle


H0087
Human Thymus
Human Thymus



pBluescript


H0090
Human T-Cell Lymphoma
T-Cell Lymphoma
T-Cell

disease
Uni-ZAP XR


H0100
Human Whole Six Week
Human Whole Six
Embryo


Uni-ZAP XR



Old Embryo
Week Old Embryo


H0102
Human Whole 6 Week
Human Whole Six
Embryo


pBluescript



Old Embryo (II), subt
Week Old Embryo


H0108
Human Adult Lymph
Human Adult
Lymph


Uni-ZAP XR



Node, subtracted
Lymph Node
Node


H0109
Human Macrophage,
Macrophage
Blood
Cell Line

pBluescript



subtracted


H0123
Human Fetal Dura Mater
Human Fetal Dura
Brain


Uni-ZAP XR




Mater


H0124
Human
Human
Sk Muscle

disease
Uni-ZAP XR



Rhabdomyosarcoma
Rhabdomyosarcoma


H0125
Cem cells cyclohexamide
Cyclohexamide
Blood
Cell Line

Uni-ZAP XR



treated
Treated Cem, Jurkat,




Raji, and Supt


H0130
LNCAP untreated
LNCAP Cell Line
Prostate
Cell Line

Uni-ZAP XR


H0131
LNCAP + o.3 nM R1881
LNCAP Cell Line
Prostate
Cell Line

Uni-ZAP XR


H0134
Raji Cells, cyclohexamide
Cyclohexamide
Blood
Cell Line

Uni-ZAP XR



treated
Treated Cem, Jurkat,




Raji, and Supt


H0135
Human Synovial Sarcoma
Human Synovial
Synovium


Uni-ZAP XR




Sarcoma


H0136
Supt Cells, cyclohexamide
Cyclohexamide
Blood
Cell Line

Uni-ZAP XR



treated
Treated Cem, Jurkat,




Raji, and Supt


H0141
Activated T-Cells, 12 hrs.
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR


H0144
Nine Week Old Early
9 Wk Old Early
Embryo


Uni-ZAP XR



Stage Human
Stage Human


H0150
Human Epididymus
Epididymis
Testis


Uni-ZAP XR


H0154
Human Fibrosarcoma
Human Skin
Skin

disease
Uni-ZAP XR




Fibrosarcoma


H0156
Human Adrenal Gland
Human Adrenal
Adrenal

disease
Uni-ZAP XR



Tumor
Gland Tumor
Gland


H0158
Activated T-Cells, 4 hrs.,
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR



ligation 2


H0159
Activated T-Cells, 8 hrs.,
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR



ligation 2


H0163
Human Synovium
Human Synovium
Synovium


Uni-ZAP XR


H0166
Human Prostate Cancer,
Human Prostate
Prostate

disease
Uni-ZAP XR



Stage B2 fraction
Cancer, stage B2


H0167
Activated T-Cells, 24 hrs.
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR


H0169
Human Prostate Cancer,
Human Prostate
Prostate

disease
Uni-ZAP XR



Stage C fraction
Cancer, stage C


H0170
12 Week Old Early Stage
Twelve Week Old
Embryo


Uni-ZAP XR



Human
Early Stage Human


H0171
12 Week Old Early Stage
Twelve Week Old
Embryo


Uni-ZAP XR



Human, II
Early Stage Human


H0172
Human Fetal Brain,
Human Fetal Brain
Brain


Lambda ZAP



random primed




II


H0178
Human Fetal Brain
Human Fetal Brain
Brain


Uni-ZAP XR


H0179
Human Neutrophil
Human Neutrophil
Blood
Cell Line

Uni-ZAP XR


H0181
Human Primary Breast
Human Primary
Breast

disease
Uni-ZAP XR



Cancer
Breast Cancer


H0188
Human Normal Breast
Human Normal
Breast


Uni-ZAP XR




Breast


H0196
Human Cardiomyopathy,
Human
Heart


Uni-ZAP XR



subtracted
Cardiomyopathy


H0197
Human Fetal Liver,
Human Fetal Liver
Liver


Uni-ZAP XR



subtracted


H0204
Human Colon Cancer,
Human Colon
Colon


pBluescript



subtracted
Cancer


H0207
LNCAP, differential
LNCAP Cell Line
Prostate
Cell Line

pBluescript



expression


H0208
Early Stage Human Lung,
Human Fetal Lung
Lung


pBluescript



subtracted


H0212
Human Prostate,
Human Prostate
Prostate


pBluescript



subtracted


H0213
Human Pituitary,
Human Pituitary



Uni-ZAP XR



subtracted


H0221
Activated T-Cells, 4 hrs,
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR



differentially expressed


H0231
Human Colon, subtraction
Human Colon



pBluescript


H0234
human colon cancer,
Human Colon
Liver


pBluescript



metastatic to liver,
Cancer, metasticized



differentially expressed
to liver


H0247
Human Membrane Bound
Human Membrane
Blood
Cell Line

Uni-ZAP XR



Polysomes- Enzyme
Bound Polysomes



Subtraction


H0250
Human Activated
Human Monocytes



Uni-ZAP XR



Monocytes


H0251
Human Chondrosarcoma
Human
Cartilage

disease
Uni-ZAP XR




Chondrosarcoma


H0252
Human Osteosarcoma
Human
Bone

disease
Uni-ZAP XR




Osteosarcoma


H0253
Human adult testis, large
Human Adult Testis
Testis


Uni-ZAP XR



inserts


H0254
Breast Lymph node cDNA
Breast Lymph Node
Lymph


Uni-ZAP XR



library

Node


H0255
breast lymph node CDNA
Breast Lymph Node
Lymph


Lambda ZAP



library

Node


II


H0263
human colon cancer
Human Colon
Colon

disease
Lambda ZAP




Cancer



II


H0264
human tonsils
Human Tonsil
Tonsil


Uni-ZAP XR


H0265
Activated T-Cell
T-Cells
Blood
Cell Line

Uni-ZAP XR



(12 hs)/Thiouridine



labelledEco


H0266
Human Microvascular
HMEC
Vein
Cell Line

Lambda ZAP



Endothelial Cells, fract. A




II


H0267
Human Microvascular
HMEC
Vein
Cell Line

Lambda ZAP



Endothelial Cells, fract. B




II


H0268
Human Umbilical Vein
HUVE Cells
Umbilical
Cell Line

Lambda ZAP



Endothelial Cells, fract. A

vein


II


H0269
Human Umbilical Vein
HUVE Cells
Umbilical
Cell Line

Lambda ZAP



Endothelial Cells, fract. B

vein


II


H0270
HPAS (human pancreas,
Human Pancreas
Pancreas


Uni-ZAP XR



subtracted)


H0271
Human Neutrophil,
Human Neutrophil -
Blood
Cell Line

Uni-ZAP XR



Activated
Activated


H0272
HUMAN TONSILS,
Human Tonsil
Tonsil


Uni-ZAP XR



FRACTION 2


H0282
HBGB's differential
Human Primary
Breast


Uni-ZAP XR



consolidation
Breast Cancer


H0286
Human OB MG63 treated
Human
Bone
Cell Line

Uni-ZAP XR



(10 nM E2) fraction I
Osteoblastoma




MG63 cell line


H0288
Human OB HOS control
Human
Bone
Cell Line

Uni-ZAP XR



fraction I
Osteoblastoma HOS




cell line


H0292
Human OB HOS treated
Human
Bone
Cell Line

Uni-ZAP XR



(10 nM E2) fraction I
Osteoblastoma HOS




cell line


H0293
WI 38 cells




Uni-ZAP XR


H0295
Amniotic Cells - Primary
Amniotic Cells -
Placenta
Cell Line

Uni-ZAP XR



Culture
Primary Culture


H0305
CD34 positive cells (Cord
CD34 Positive Cells
Cord


ZAP Express



Blood)

Blood


H0306
CD34 depleted Buffy Coat
CD34 Depleted
Cord


ZAP Express



(Cord Blood)
Buffy Coat (Cord
Blood




Blood)


H0309
Human Chronic Synovitis
Synovium, Chronic
Synovium

disease
Uni-ZAP XR




Synovitis/




Osteoarthritis


H0310
human caudate nucleus
Brain
Brain


Uni-ZAP XR


H0318
HUMAN B CELL
Human B Cell
Lymph

disease
Uni-ZAP XR



LYMPHOMA
Lymphoma
Node


H0321
HUMAN SCHWANOMA
Schwanoma
Nerve

disease
Uni-ZAP XR


H0327
human corpus colosum
Human Corpus
Brain


Uni-ZAP XR




Callosum


H0328
human ovarian cancer
Ovarian Cancer
Ovary

disease
Uni-ZAP XR


H0331
Hepatocellular Tumor
Hepatocellular
Liver

disease
Lambda ZAP




Tumor



II


H0333
Hemangiopericytoma
Hemangiopericytoma
Blood

disease
Lambda ZAP





vessel


II


H0341
Bone Marrow Cell Line
Bone Marrow Cell
Bone
Cell Line

Uni-ZAP XR



(RS4; 11)
Line RS4; 11
Marrow


H0343
stomach cancer (human)
Stomach Cancer -


disease
Uni-ZAP XR




5383A (human)


H0346
Brain-medulloblastoma
Brain
Brain

disease
Uni-ZAP XR




(Medulloblastoma)-




9405C006R


H0351
Glioblastoma
Glioblastoma
Brain

disease
Uni-ZAP XR


H0352
wilm's tumor
Wilm's Tumor


disease
Uni-ZAP XR


H0354
Human Leukocytes
Human Leukocytes
Blood
Cell Line

pCMVSport 1


H0357
H. Normalized Fetal
Human Fetal Liver
Liver


Uni-ZAP XR



Liver, II


H0359
KMH2 cell line
KMH2



ZAP Express


H0369
H. Atrophic Endometrium
Atrophic



Uni-ZAP XR




Endometrium and




myometrium


H0370
H. Lymph node breast
Lymph node with


disease
Uni-ZAP XR



Cancer
Met. Breast Cancer


H0373
Human Heart
Human Adult Heart
Heart


pCMVSport 1


H0384
Brain, Kozak
Human Brain



pCMVSport 1


H0392
H. Meningima, M1
Human Meningima
brain


pSport1


H0393
Fetal Liver, subtraction II
Human Fetal Liver
Liver


pBluescript


H0394
A-14 cell line
Redd-Sternberg cell



ZAP Express


H0399
Human Kidney Cortex, re-
Human Kidney



Lambda ZAP



rescue
Cortex



II


H0402
CD34 depleted Buffy Coat
CD34 Depleted
Cord


ZAP Express



(Cord Blood), re-excision
Buffy Coat (Cord
Blood




Blood)


H0411
H Female Bladder, Adult
Human Female
Bladder


pSport1




Adult Bladder


H0412
Human umbilical vein
HUVE Cells
Umbilical
Cell Line

pSport1



endothelial cells, IL-4

vein



induced


H0413
Human Umbilical Vein
HUVE Cells
Umbilical
Cell Line

pSport1



Endothelial Cells,

vein



uninduced


H0414
Ovarian Tumor I, OV5232
Ovarian Tumor,
Ovary

disease
pSport1




OV5232


H0415
H. Ovarian Tumor, II,
Ovarian Tumor,
Ovary

disease
pCMVSport



OV5232
OV5232



2.0


H0416
Human Neutrophils,
Human Neutrophil -
Blood
Cell Line

pBluescript



Activated, re-excision
Activated


H0418
Human Pituitary,
Human Pituitary



pBluescript



subtracted VII


H0421
Human Bone Marrow, re-
Bone Marrow



pBluescript



excision


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


H0427
Human Adipose
Human Adipose, left



pSport1




hiplipoma


H0428
Human Ovary
Human Ovary
Ovary


pSport1




Tumor


H0432
H. Kidney Pyramid
Kidney pyramids
Kidney


pBluescript


H0435
Ovarian Tumor 10-3-95
Ovarian Tumor,
Ovary


pCMVSport




OV350721



2.0


H0436
Resting T-Cell Library, II
T-Cells
Blood
Cell Line

pSport1


H0438
H. Whole Brain #2, re-
Human Whole Brain



ZAP Express



excision
#2


H0441
H. Kidney Cortex,
Kidney cortex
Kidney


pBluescript



subtracted


H0444
Spleen metastic melanoma
Spleen, Metastic
Spleen

disease
pSport1




malignant




melanoma


H0445
Spleen, Chronic
Human Spleen, CLL
Spleen

disease
pSport1



lymphocytic leukemia


H0455
H. Striatum Depression,
Human Brain,
Brain


pBluescript



subt
Striatum Depression


H0457
Human Eosinophils
Human Eosinophils



pSport1


H0461
H. Kidney Medulla,
Kidney medulla
Kidney


pBluescript



subtracted


H0477
Human Tonsil, Lib 3
Human Tonsil
Tonsil


pSport1


H0478
Salivary Gland, Lib 2
Human Salivary
Salivary


pSport1




Gland
gland


H0483
Breast Cancer cell line,
Breast Cancer Cell



pSport1



MDA 36
line, MDA 36


H0484
Breast Cancer Cell line,
Breast Cancer Cell



pSport1



angiogenic
line, Angiogenic,




36T3


H0485
Hodgkin's Lymphoma I
Hodgkin's


disease
pCMVSport




Lymphoma I



2.0


H0486
Hodgkin's Lymphoma II
Hodgkin's


disease
pCMVSport




Lymphoma II



2.0


H0488
Human Tonsils, Lib 2
Human Tonsils



pCMVSport








2.0


H0489
Crohn's Disease
Ileum
Intestine

disease
pSport1


H0492
HL-60, RA 4h, Subtracted
HL-60 Cells, RA
Blood
Cell Line

Uni-ZAP XR




stimulated for 4H


H0494
Keratinocyte
Keratinocyte



pCMVSport








2.0


H0497
HEL cell line
HEL cell line

HEL

pSport1






92.1.7


H0505
Human Astrocyte
Human Astrocyte



pSport1


H0506
Ulcerative Colitis
Colon
Colon


pSport1


H0509
Liver, Hepatoma
Human Liver,
Liver

disease
pCMVSport




Hepatoma, patient 8



3.0


H0517
Nasal polyps
Nasal polyps



pCMVSport








2.0


H0518
pBMC stimulated w/ poly
pBMC stimulated



pCMVSport



I/C
with poly I/C



3.0


H0519
NTERA2, control
NTERA2,



pCMVSport




Teratocarcinoma



3.0




cell line


H0520
NTERA2 + retinoic acid,
NTERA2,



pSport1



14 days
Teratocarcinoma




cell line


H0521
Primary Dendritic Cells,
Primary Dendritic



pCMVSport



lib 1
cells



3.0


H0522
Primary Dendritic
Primary Dendritic



pCMVSport



cells, frac 2
cells



3.0


H0525
PCR, pBMC I/C treated
pBMC stimulated



PCRII




with poly I/C


H0528
Poly[I]/Poly[C] Normal
Poly[I]/Poly[C]



pCMVSport



Lung Fibroblasts
Normal Lung



3.0




Fibroblasts


H0529
Myoloid Progenitor Cell
TF-1 Cell Line;



pCMVSport



Line
Myoloid progenitor



3.0




cell line


H0538
Merkel Cells
Merkel cells
Lymph


pSport1





node


H0539
Pancreas Islet Cell Tumor
Pancreas Islet Cell
Pancreas

disease
pSport1




Tumor


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
Human endometrial



pCMVSport



stromal cells
stromal cells



3.0


H0545
Human endometrial
Human endometrial



pCMVSport



stromal cells-treated with
stromal cells-treated



3.0



progesterone
with proge


H0546
Human endometrial
Human endometrial



pCMVSport



stromal cells-treated with
stromal cells-treated



3.0



estradiol
with estra


H0547
NTERA2 teratocarcinoma
NTERA2,



pSport1



cell line + retinoic acid (14
Teratocarcinoma



days)
cell line


H0549
H. Epididiymus, caput &
Human



Uni-ZAP XR



corpus
Epididiymus, caput




and corpus


H0550
H. Epididiymus, cauda
Human



Uni-ZAP XR




Epididiymus, cauda


H0551
Human Thymus Stromal
Human Thymus



pCMVSport



Cells
Stromal Cells



3.0


H0553
Human Placenta
Human Placenta



pCMVSport








3.0


H0555
Rejected Kidney, lib 4
Human Rejected
Kidney

disease
pCMVSport




Kidney



3.0


H0556
Activated T-
T-Cells
Blood
Cell Line

Uni-ZAP XR



cell(12 h)/Thiouridine-re-



excision


H0559
HL-60, PMA 4H, re-
HL-60 Cells, PMA
Blood
Cell Line

Uni-ZAP XR



excision
stimulated 4H


H0560
KMH2
KMH2



pCMVSport








3.0


H0561
L428
L428



pCMVSport








3.0


H0563
Human Fetal Brain,
Human Fetal Brain



pCMVSport



normalized 50021F




2.0


H0565
Human Fetal Brain,
Human Fetal Brain



pCMVSport



normalized 100024F




2.0


H0569
Human Fetal Brain,
Human Fetal Brain



pCMVSport



normalized CO




2.0


H0571
Human Fetal Brain,
Human Fetal Brain



pCMVSport



normalized C500HE




2.0


H0572
Human Fetal Brain,
Human Fetal Brain



pCMVSport



normalized AC5002




2.0


H0574
Hepatocellular Tumor; re-
Hepatocellular
Liver

disease
Lambda ZAP



excision
Tumor



II


H0575
Human Adult
Human Adult
Lung


Uni-ZAP XR



Pulmonary; re-excision
Pulmonary


H0576
Resting T-Cell; re-
T-Cells
Blood
Cell Line

Lambda ZAP



excision




II


H0580
Dendritic cells, pooled
Pooled dendritic



pCMVSport




cells



3.0


H0581
Human Bone Marrow,
Human Bone
Bone


pCMVSport



treated
Marrow
Marrow


3.0


H0583
B Cell lymphoma
B Cell Lymphoma
B Cell

disease
pCMVSport








3.0


H0584
Activated T-cells, 24 hrs,
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR



re-excision


H0585
Activated T-Cells, 12 hrs,
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR



re-excision


H0586
Healing groin wound, 6.5
healing groin
groin

disease
pCMVSport



hours post incision
wound, 6.5 hours



3.0




post incision - 2/


H0587
Healing groin wound; 7.5
Groin-2/19/97
groin

disease
pCMVSport



hours post incision




3.0


H0589
CD34 positive cells (cord
CD34 Positive Cells
Cord


ZAP Express



blood), re-ex

Blood


H0590
Human adult small
Human Adult Small
Small Int.


Uni-ZAP XR



intestine, re-excision
Intestine


H0591
Human T-cell
T-Cell Lymphoma
T-Cell

disease
Uni-ZAP XR



lymphoma; re-excision


H0592
Healing groin wound -
HGS wound healing


disease
pCMVSport



zero hr post-incision
project; abdomen



3.0



(control)


H0593
Olfactory
Olfactory epithelium



pCMVSport



epithelium; nasalcavity
from roof of left



3.0




nasal cacit


H0594
Human Lung Cancer; re-
Human Lung Cancer
Lung

disease
Lambda ZAP



excision




II


H0595
Stomach cancer
Stomach Cancer -


disease
Uni-ZAP XR



(human); re-excision
5383A (human)


H0596
Human Colon Cancer; re-
Human Colon
Colon


Lambda ZAP



excision
Cancer



II


H0597
Human Colon; re-excision
Human Colon



Lambda ZAP








II


H0598
Human Stomach; re-
Human Stomach
Stomach


Uni-ZAP XR



excision


H0599
Human Adult Heart; re-
Human Adult Heart
Heart


Uni-ZAP XR



excision


H0604
Human Pituitary, re-
Human Pituitary



pBluescript



excision


H0606
Human Primary Breast
Human Primary
Breast

disease
Uni-ZAP XR



Cancer; re-excision
Breast Cancer


H0615
Human Ovarian Cancer
Ovarian Cancer
Ovary

disease
Uni-ZAP XR



Reexcision


H0616
Human Testes, Reexcision
Human Testes
Testis


Uni-ZAP XR


H0617
Human Primary Breast
Human Primary
Breast

disease
Uni-ZAP XR



Cancer Reexcision
Breast Cancer


H0618
Human Adult Testes,
Human Adult Testis
Testis


Uni-ZAP XR



Large Inserts, Reexcision


H0619
Fetal Heart
Human Fetal Heart
Heart


Uni-ZAP XR


H0620
Human Fetal Kidney;
Human Fetal Kidney
Kidney


Uni-ZAP XR



Reexcision


H0622
Human Pancreas Tumor;
Human Pancreas
Pancreas

disease
Uni-ZAP XR



Reexcision
Tumor


H0623
Human Umbilical Vein;
Human Umbilical
Umbilical


Uni-ZAP XR



Reexcision
Vein Endothelial
vein




Cells


H0624
12 Week Early Stage
Twelve Week Old
Embryo


Uni-ZAP XR



Human II; Reexcision
Early Stage Human


H0625
Ku 812F Basophils Line
Ku 812F Basophils



pSport1


H0626
Saos2 Cells; Untreated
Saos2 Cell Line;



pSport1




Untreated


H0628
Human Pre-Differentiated
Human Pre-



Uni-ZAP XR



Adipocytes
Differentiated




Adipocytes


H0631
Saos2, Dexamethosome
Saos2 Cell Line;



pSport1



Treated
Dexamethosome




Treated


H0632
Hepatocellular Tumor; re-
Hepatocellular
Liver


Lambda ZAP



excision
Tumor



II


H0633
Lung Carcinoma A549
TNFalpha activated


disease
pSport1



TNFalpha activated
A549 - Lung




Carcinoma


H0634
Human Testes Tumor, re-
Human Testes
Testis

disease
Uni-ZAP XR



excision
Tumor


H0635
Human Activated T-Cells,
Activated T-Cells
Blood
Cell Line

Uni-ZAP XR



re-excision


H0637
Dendritic Cells From
Dentritic cells from



pSport1



CD34 Cells
CD34 cells


H0638
CD40 activated monocyte
CD40 activated



pSport1



dendridic cells
monocyte dendridic




cells


H0641
LPS activated derived
LPS activated



pSport1



dendritic cells
monocyte derived




dendritic cells


H0644
Human Placenta (re-
Human Placenta
Placenta


Uni-ZAP XR



excision)


H0645
Fetal Heart, re-excision
Human Fetal Heart
Heart


Uni-ZAP XR


H0646
Lung, Cancer (4005313
Metastatic



pSport1



A3): Invasive Poorly
squamous cell lung



Differentiated Lung
carcinoma, poorly di



Adenocarcinoma,


H0647
Lung, Cancer (4005163
Invasive poorly


disease
pSport1



B7): Invasive, Poorly Diff.
differentiated lung



Adenocarcinoma,
adenocarcinoma



Metastatic


H0648
Ovary, Cancer: (4004562
Papillary Cstic


disease
pSport1



B6) Papillary Serous
neoplasm of low



Cystic Neoplasm, Low
malignant potentia



Malignant Pot


H0650
B-Cells
B-Cells



pCMVSport








3.0


H0651
Ovary, Normal:
Normal Ovary



pSport1



(9805C040R)


H0653
Stromal Cells
Stromal Cells



pSport1


H0656
B-cells (unstimulated)
B-cells



pSport1




(unstimulated)


H0657
B-cells (stimulated)
B-cells (stimulated)



pSport1


H0658
Ovary, Cancer
9809C332-Poorly
Ovary &

disease
pSport1



(9809C332): Poorly
differentiate
Fallopian



differentiated

Tubes



adenocarcinoma


H0659
Ovary, Cancer
Grade II Papillary
Ovary

disease
pSport1



(15395A1F): Grade II
Carcinoma, Ovary



Papillary Carcinoma


H0660
Ovary, Cancer:
Poorly differentiated


disease
pSport1



(15799A1F) Poorly
carcinoma, ovary



differentiated carcinoma


H0661
Breast, Cancer: (4004943
Breast cancer


disease
pSport1



A5)


H0662
Breast, Normal:
Normal Breast -
Breast


pSport1



(4005522B2)
#4005522(B2)


H0663
Breast, Cancer: (4005522
Breast Cancer -
Breast

disease
pSport1



A2)
#4005522(A2)


H0664
Breast, Cancer:
Breast Cancer
Breast

disease
pSport1



(9806C012R)


H0665
Stromal cells 3.88
Stromal cells 3.88



pSport1


H0667
Stromal cells(HBM3.18)
Stromal cell(HBM



pSport1




3.18)


H0668
stromal cell clone 2.5
stromal cell clone



pSport1




2.5


H0669
Breast, Cancer: (4005385
Breast Cancer
Breast


pSport1



A2)
(4005385A2)


H0670
Ovary, Cancer(4004650
Ovarian Cancer -



pSport1



A3): Well-Differentiated
4004650A3



Micropapillary Serous



Carcinoma


H0672
Ovary, Cancer: (4004576
Ovarian
Ovary


pSport1



A8)
Cancer(4004576A8)


H0673
Human Prostate Cancer,
Human Prostate
Prostate


Uni-ZAP XR



Stage B2; re-excision
Cancer, stage B2


H0674
Human Prostate Cancer,
Human Prostate
Prostate


Uni-ZAP XR



Stage C; re-excission
Cancer, stage C


H0676
Colon, Cancer:
Colon Cancer



pCMVSport



(9808C064R)-total RNA
9808C064R



3.0


H0682
Serous Papillary
serous papillary



pCMVSport



Adenocarcinoma
adenocarcinoma



3.0




(9606G304SPA3B)


H0683
Ovarian Serous Papillary
Serous papillary



pCMVSport



Adenocarcinoma
adenocarcinoma,



3.0




stage 3C (9804G01


H0684
Serous Papillary
Ovarian Cancer-
Ovaries


pCMVSport



Adenocarcinoma
9810G606



3.0


H0685
Adenocarcinoma of
Adenocarcinoma of



pCMVSport



Ovary, Human Cell Line,
Ovary, Human Cell



3.0



# OVCAR-3
Line, # OVCAR-


H0686
Adenocarcinoma of
Adenocarcinoma of



pCMVSport



Ovary, Human Cell Line
Ovary, Human Cell



3.0




Line, # SW-626


H0687
Human normal
Human normal
Ovary


pCMVSport



ovary(#9610G215)
ovary(#9610G215)



3.0


H0688
Human Ovarian
Human Ovarian



pCMVSport



Cancer(#9807G017)
cancer(#9807G017),



3.0




mRNA from Maura




Ru


H0690
Ovarian Cancer, #
Ovarian Cancer,



pCMVSport



9702G001
#9702G001



3.0


S0001
Brain frontal cortex
Brain frontal cortex
Brain


Lambda ZAP








II


S0002
Monocyte activated
Monocyte-activated
blood
Cell Line

Uni-ZAP XR


S0003
Human Osteoclastoma
Osteoclastoma
bone

disease
Uni-ZAP XR


S0007
Early Stage Human Brain
Human Fetal Brain



Uni-ZAP XR


S0010
Human Amygdala
Amygdala



Uni-ZAP XR


S0011
STROMAL -
Osteoclastoma
bone

disease
Uni-ZAP XR



OSTEOCLASTOMA


S0022
Human Osteoclastoma
Osteoclastoma



Uni-ZAP XR



Stromal Cells -
Stromal Cells



unamplified


S0026
Stromal cell TF274
stromal cell
Bone
Cell Line

Uni-ZAP XR





marrow


S0027
Smooth muscle, serum
Smooth muscle
Pulmanary
Cell Line

Uni-ZAP XR



treated

artery


S0028
Smooth muscle, control
Smooth muscle
Pulmanary
Cell Line

Uni-ZAP XR





artery


S0031
Spinal cord
Spinal cord
spinal cord


Uni-ZAP XR


S0032
Smooth muscle-ILb
Smooth muscle
Pulmanary
Cell Line

Uni-ZAP XR



induced

artery


S0036
Human Substantia Nigra
Human Substantia



Uni-ZAP XR




Nigra


S0037
Smooth muscle, IL1b
Smooth muscle
Pulmanary
Cell Line

Uni-ZAP XR



induced

artery


S0038
Human Whole Brain #2 -
Human Whole Brain



ZAP Express



Oligo dT > 1.5 Kb
#2


S0040
Adipocytes
Human Adipocytes



Uni-ZAP XR




from Osteoclastoma


S0044
Prostate BPH
prostate BPH
Prostate

disease
Uni-ZAP XR


S0045
Endothelial cells-control
Endothelial cell
endothelial
Cell Line

Uni-ZAP XR





cell-lung


S0046
Endothelial-induced
Endothelial cell
endothelial
Cell Line

Uni-ZAP XR





cell-lung


S0049
Human Brain, Striatum
Human Brain,



Uni-ZAP XR




Striatum


S0050
Human Frontal Cortex,
Human Frontal


disease
Uni-ZAP XR



Schizophrenia
Cortex,




Schizophrenia


S0051
Human
Human


disease
Uni-ZAP XR



Hypothalmus, Schizophrenia
Hypothalamus,




Schizophrenia


S0052
neutrophils control
human neutrophils
blood
Cell Line

Uni-ZAP XR


S0053
Neutrophils IL-1 and LPS
human neutrophil
blood
Cell Line

Uni-ZAP XR



induced
induced


S0114
Anergic T-cell
Anergic T-cell

Cell Line

Uni-ZAP XR


S0116
Bone marrow
Bone marrow
Bone


Uni-ZAP XR





marrow


S0126
Osteoblasts
Osteoblasts
Knee
Cell Line

Uni-ZAP XR


S0132
Epithelial-TNFa and INF
Airway Epithelial



Uni-ZAP XR



induced


S0134
Apoptotic T-cell
apoptotic cells

Cell Line

Uni-ZAP XR


S0140
eosinophil-IL5 induced
eosinophil
lung
Cell Line

Uni-ZAP XR


S0142
Macrophage-oxLDL
macrophage-
blood
Cell Line

Uni-ZAP XR




oxidized LDL




treated


S0144
Macrophage (GM-CSF
Macrophage (GM-



Uni-ZAP XR



treated)
CSF treated)


S0148
Normal Prostate
Prostate
prostate


Uni-ZAP XR


S0150
LNCAP prostate cell line
LNCAP Cell Line
Prostate
Cell Line

Uni-ZAP XR


S0152
PC3 Prostate cell line
PC3 prostate cell



Uni-ZAP XR




line


S0180
Bone Marrow Stroma,
Bone Marrow


disease
Uni-ZAP XR



TNF&LPS ind
Stroma, TNF & LPS




induced


S0182
Human B Cell 8866
Human B- Cell 8866



Uni-ZAP XR


S0192
Synovial Fibroblasts
Synovial Fibroblasts



pSport1



(control)


S0194
Synovial hypoxia
Synovial Fibroblasts



pSport1


S0196
Synovial IL-1/TNF
Synovial Fibroblasts



pSport1



stimulated


S0206
Smooth Muscle- HASTE
Smooth muscle
Pulmanary
Cell Line

pBluescript



normalized

artery


S0208
Messangial cell, frac 1
Messangial cell



pSport1


S0210
Messangial cell, frac 2
Messangial cell



pSport1


S0212
Bone Marrow Stromal
Bone Marrow



pSport1



Cell, untreated
Stromal




Cell, untreated


S0214
Human Osteoclastoma, re-
Osteoclastoma
bone

disease
Uni-ZAP XR



excision


S0218
Apoptotic T-cell, re-
apoptotic cells

Cell Line

Uni-ZAP XR



excision


S0222
H. Frontal
H. Brain, Frontal
Brain

disease
Uni-ZAP XR



cortex, epileptic; re-
Cortex, Epileptic



excision


S0242
Synovial Fibroblasts
Synovial Fibroblasts



pSport1



(I11/TNF), subt


S0250
Human Osteoblasts II
Human Osteoblasts
Femur

disease
pCMVSport








2.0


S0260
Spinal Cord, re-excision
Spinal cord
spinal cord


Uni-ZAP XR


S0276
Synovial hypoxia-RSF
Synovial fobroblasts
Synovial


pSport1



subtracted
(rheumatoid)
tissue


S0278
H Macrophage (GM-CSF
Macrophage (GM-



Uni-ZAP XR



treated), re-excision
CSF treated)


S0280
Human Adipose Tissue,
Human Adipose



Uni-ZAP XR



re-excision
Tissue


S0282
Brain Frontal Cortex, re-
Brain frontal cortex
Brain


Lambda ZAP



excision




II


S0292
Osteoarthritis (OA-4)
Human
Bone

disease
pSport1




Osteoarthritic




Cartilage


S0300
Frontal lobe, dementia; re-
Frontal Lobe
Brain


Uni-ZAP XR



excision
dementia/Alzheimer's


S0316
Human Normal
Human Normal



pSport1



Cartilage, Fraction I
Cartilage


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



pSport1



Fraction III
Cartilage


S0344
Macrophage-oxLDL; re-
macrophage-
blood
Cell Line

Uni-ZAP XR



excision
oxidized LDL




treated


S0346
Human Amygdala; re-
Amygdala



Uni-ZAP XR



excision


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


S0364
Human Quadriceps
Quadriceps muscle



pSport1


S0366
Human Soleus
Soleus Muscle



pSport1


S0370
Larynx carcinoma II
Larynx carcinoma


disease
pSport1


S0374
Normal colon
Normal colon



pSport1


S0376
Colon Tumor
Colon Tumor


disease
pSport1


S0378
Pancreas normal PCA4
Pancreas Normal



pSport1



No
PCA4 No


S0380
Pancreas Tumor PCA4 Tu
Pancreas Tumor


disease
pSport1




PCA4 Tu


S0386
Human Whole Brain, re-
Whole brain
Brain


ZAP Express



excision


S0388
Human
Human


disease
Uni-ZAP XR



Hypothalamus, schizophrenia,
Hypothalamus,



re-excision
Schizophrenia


S0390
Smooth muscle, control;
Smooth muscle
Pulmanary
Cell Line

Uni-ZAP XR



re-excision

artery


S0392
Salivary Gland
Salivary gland;



pSport1




normal


S0404
Rectum normal
Rectum, normal



pSport1


S0406
Rectum tumor
Rectum tumor



pSport1


S0408
Colon, normal
Colon, normal



pSport1


S0410
Colon, tumor
Colon, tumor



pSport1


S0412
Temporal cortex-
Temporal cortex,


disease
Other



Alzheizmer; subtracted
alzheimer


S0414
Hippocampus, Alzheimer
Hippocampus,



Other



Subtracted
Alzheimer




Subtracted


S0418
CHME Cell Line; treated 5 hrs
CHME Cell Line;



pCMVSport




treated



3.0


S0420
CHME Cell
CHME Cell line,



pSport1



Line, untreated
untreatetd


S0422
Mo7e Cell Line GM-CSF
Mo7e Cell Line



pCMVSport



treated (1 ng/ml)
GM-CSF treated



3.0




(1 ng/ml)


S0424
TF-1 Cell Line GM-CSF
TF-1 Cell Line



pSport1



Treated
GM-CSF Treated


S0426
Monocyte activated; re-
Monocyte-activated
blood
Cell Line

Uni-ZAP XR



excision


S0428
Neutrophils control; re-
human neutrophils
blood
Cell Line

Uni-ZAP XR



excision


S0432
Sinus piniformis Tumor
Sinus piniformis



pSport1




Tumor


S0434
Stomach Normal
Stomach Normal


disease
pSport1


S0436
Stomach Tumor
Stomach Tumor


disease
pSport1


S0438
Liver Normal Met5No
Liver Normal



pSport1




Met5No


S0440
Liver Tumor Met 5 Tu
Liver Tumor



pSport1


S0442
Colon Normal
Colon Normal



pSport1


S0444
Colon Tumor
Colon Tumor


disease
pSport1


S0448
Larynx Normal
Larynx Normal



pSport1


S0452
Thymus
Thymus



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


S3012
Smooth Muscle Serum
Smooth muscle
Pulmanary
Cell Line

pBluescript



Treated, Norm

artery


S3014
Smooth muscle, serum
Smooth muscle
Pulmanary
Cell Line

pBluescript



induced, re-exc

artery


S6014
H. hypothalamus, frac A
Hypothalamus
Brain


ZAP Express


S6016
H. Frontal Cortex,
H. Brain, Frontal
Brain

disease
Uni-ZAP XR



Epileptic
Cortex, Epileptic


S6024
Alzheimers, spongy
Alzheimer's/Spongy
Brain

disease
Uni-ZAP XR



change
change


S6028
Human Manic Depression
Human Manic
Brain

disease
Uni-ZAP XR



Tissue
depression tissue


T0002
Activated T-cells
Activated T-Cell,
Blood
Cell Line

pBluescript




PBL fraction



SK-


T0010
Human Infant Brain
Human Infant Brain



Other


T0040
HSC172 cells
SA172 Cells



pBluescript








SK-


T0041
Jurkat T-cell G1 phase
Jurkat T-cell



pBluescript








SK-


T0042
Jurkat T-Cell, S phase
Jurkat T-Cell Line



pBluescript








SK-


T0048
Human Aortic
Human Aortic



pBluescript



Endothelium
Endothilium



SK-


T0049
Aorta endothelial cells + TNF-a
Aorta endothelial



pBluescript




cells



SK-


T0067
Human Thyroid
Human Thyroid



pBluescript








SK-


T0082
Human Adult Retina
Human Adult Retina



pBluescript








SK-


T0109
Human (HCC) cell line




pBluescript



liver (mouse) metastasis,




SK-



remake


T0110
Human colon carcinoma




pBluescript



(HCC) cell line, remake




SK-


L0002
Atrium cDNA library



Human heart


L0021
Human adult (K. Okubo)


L0022
Human adult lung 3′′



directed MboI cDNA


L0040
Human colon mucosa


L0055
Human promyelocyte


L0096
Subtracted human retina


L0097
Subtracted human retinal



pigment epithelium (RPE)


L0105
Human aorta polyA + (TFujiwara)
aorta


L0142
Human placenta cDNA
placenta



(TFujiwara)


L0157
Human fetal brain

brain



(TFujiwara)


L0163
Human heart cDNA

heart



(YNakamura)


L0352
Normalized infant brain,




BA, M13-



Bento Soares




derived


L0361
Stratagene ovary

ovary


Bluescript SK



(#937217)


L0362
Stratagene ovarian cancer




Bluescript



(#937219)




SK-


L0363
NCI_CGAP_GC2
germ cell tumor



Bluescript








SK-


L0364
NCI_CGAP_GC5
germ cell tumor



Bluescript








SK-


L0366
Stratagene schizo brain
schizophrenic brain



Bluescript



S11
S-11 frontal lobe



SK-


L0368
NCI_CGAP_SS1
synovial sarcoma



Bluescript








SK-


L0369
NCI_CGAP_AA1
adrenal adenoma
adrenal


Bluescript





gland


SK-


L0371
NCI_CGAP_Br3
breast tumor
breast


Bluescript








SK-


L0372
NCI_CGAP_Co12
colon tumor
colon


Bluescript








SK-


L0373
NCI_CGAP_Co11
tumor
colon


Bluescript








SK-


L0374
NCI_CGAP_Co2
tumor
colon


Bluescript








SK-


L0375
NCI_CGAP_Kid6
kidney tumor
kidney


Bluescript








SK-


L0378
NCI_CGAP_Lu1
lung tumor
lung


Bluescript








SK-


L0381
NCI_CGAP_HN4
squamous cell
pharynx


Bluescript




carcinoma



SK-


L0382
NCI_CGAP_Pr25
epithelium (cell line)
prostate


Bluescript








SK-


L0387
NCI_CGAP_GCB0
germinal center B-
tonsil


Bluescript




cells



SK-


L0389
NCI_CGAP_HN5
normal gingiva (cell



Bluescript




line from primary



SK-




keratinocyt


L0411
1-NIB




Lafmid BA


L0438
normalized infant brain
total brain
brain


lafmid BA



cDNA


L0439
Soares infant brain 1NIB

whole


Lafmid BA





brain


L0455
Human retina cDNA
retina
eye


lambda gt10



randomly primed



sublibrary


L0456
Human retina cDNA
retina
eye


lambda gt10



Tsp509I-cleaved



sublibrary


L0471
Human fetal heart,




Lambda ZAP



Lambda ZAP Express




Express


L0480
Stratagene cat#937212




Lambda ZAP,



(1992)




pBluescript








SK(−)


L0483
Human pancreatic islet




Lambda








ZAPII


L0485
STRATAGENE Human
skeletal muscle
leg muscle


Lambda



skeletal muscle cDNA




ZAPII



library, cat. #936215.


L0500
NCI_CGAP_Brn20
oligodendroglioma
brain


pAMP1


L0502
NCI_CGAP_Br15
adenocarcinoma
breast


pAMP1


L0509
NCI_CGAP_Lu26
invasive
lung


pAMP1




adenocarcinoma


L0515
NCI_CGAP_Ov32
papillary serous
ovary


pAMP1




carcinoma


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


L0526
NCI_CGAP_Pr12
metastatic prostate



pAMP10




bone lesion


L0527
NCI_CGAP_Ov2
ovary



pAMP10


L0528
NCI_CGAP_Pr5
prostate



pAMP10


L0545
NCI_CGAP_Pr4.1
prostatic
prostate


pAMP10




intraepithelial




neoplasia —high




grade


L0547
NCI_CGAP_Pr16
tumor
prostate


pAMP10


L0553
NCI_CGAP_Co22
colonic
colon


pAMP10




adenocarcinoma


L0563
Human Bone Marrow
bone marrow



pBluescript



Stromal Fibroblast


L0564
Jia bone marrow stroma
bone marrow stroma



pBluescript


L0565
Normal Human
Bone
Hip


pBluescript



Trabecular Bone Cells


L0581
Stratagene liver (#937224)

liver


pBluescript








SK


L0586
HTCDL1




pBluescript








SK(−)


L0588
Stratagene endothelial cell




pBluescript



937223




SK-


L0589
Stratagene fetal retina




pBluescript



937202




SK-


L0591
Stratagene HeLa cell s3




pBluescript



937216




SK-


L0592
Stratagene hNT neuron




pBluescript



(#937233)




SK-


L0593
Stratagene




pBluescript



neuroepithelium




SK-



(#937231)


L0594
Stratagene




pBluescript



neuroepithelium




SK-



NT2RAMI 937234


L0595
Stratagene NT2 neuronal
neuroepithelial cells
brain


pBluescript



precursor 937230




SK-


L0596
Stratagene colon

colon


pBluescript



(#937204)




SK-


L0598
Morton Fetal Cochlea
cochlea
ear


pBluescript








SK-


L0599
Stratagene lung (#937210)

lung


pBluescript








SK-


L0600
Weizmann Olfactory
olfactory epithelium
nose


pBluescript



Epithelium




SK-


L0601
Stratagene pancreas

pancreas


pBluescript



(#937208)




SK-


L0603
Stratagene placenta

placenta


pBluescript



(#937225)




SK-


L0604
Stratagene muscle 937209
muscle
skeletal


pBluescript





muscle


SK-


L0605
Stratagene fetal spleen
fetal spleen
spleen


pBluescript



(#937205)




SK-


L0608
Stratagene lung carcinoma
lung carcinoma
lung
NCI-H69

pBluescript



937218




SK-


L0623
HM3
pectoral muscle



pcDNAII




(after mastectomy)



(Invitrogen)


L0630
NCI_CGAP_CNS1
substantia nigra
brain


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


L0637
NCI_CGAP_Brn53
three pooled
brain


pCMV-




meningiomas



SPORT6


L0638
NCI_CGAP_Brn35
tumor, 5 pooled (see
brain


pCMV-




description)



SPORT6


L0639
NCI_CGAP_Brn52
tumor, 5 pooled (see
brain


pCMV-




description)



SPORT6


L0640
NCI_CGAP_Br18
four pooled high-
breast


pCMV-




grade tumors,



SPORT6




including two prima


L0641
NCI_CGAP_Co17
juvenile granulosa
colon


pCMV-




tumor



SPORT6


L0643
NCI_CGAP_Co19
moderately
colon


pCMV-




differentiated



SPORT6




adenocarcinoma


L0645
NCI_CGAP_Co21
moderately
colon


pCMV-




differentiated



SPORT6




adenocarcinoma


L0646
NCI_CGAP_Co14
moderately-
colon


pCMV-




differentiated



SPORT6




adenocarcinoma


L0648
NCI_CGAP_Eso2
squamous cell
esophagus


pCMV-




carcinoma



SPORT6


L0649
NCI_CGAP_GU1
2 pooled high-grade
genitourinary


pCMV-




transitional cell
tract


SPORT6




tumors


L0650
NCI_CGAP_Kid13
2 pooled Wilms'
kidney


pCMV-




tumors, one primary



SPORT6




and one metast


L0651
NCI_CGAP_Kid8
renal cell tumor
kidney


pCMV-








SPORT6


L0653
NCI_CGAP_Lu28
two pooled
lung


pCMV-




squamous cell



SPORT6




carcinomas


L0655
NCI_CGAP_Lym12
lymphoma,
lymph


pCMV-




follicular mixed
node


SPORT6




small and large cell


L0656
NCI_CGAP_Ov38
normal epithelium
ovary


pCMV-








SPORT6


L0657
NCI_CGAP_Ov23
tumor, 5 pooled (see
ovary


pCMV-




description)



SPORT6


L0658
NCI_CGAP_Ov35
tumor, 5 pooled (see
ovary


pCMV-




description)



SPORT6


L0659
NCI_CGAP_Pan1
adenocarcinoma
pancreas


pCMV-








SPORT6


L0661
NCI_CGAP_Mel15
malignant
skin


pCMV-




melanoma,



SPORT6




metastatic to lymph




node


L0662
NCI_CGAP_Gas4
poorly differentiated
stomach


pCMV-




adenocarcinoma



SPORT6




with signet r


L0663
NCI_CGAP_Ut2
moderately-
uterus


pCMV-




differentiated



SPORT6




endometrial




adenocarcino


L0664
NCI_CGAP_Ut3
poorly-differentiated
uterus


pCMV-




endometrial



SPORT6




adenocarcinoma,


L0665
NCI_CGAP_Ut4
serous papillary
uterus


pCMV-




carcinoma, high



SPORT6




grade, 2 pooled t


L0666
NCI_CGAP_Ut1
well-differentiated
uterus


pCMV-




endometrial



SPORT6




adenocarcinoma, 7


L0667
NCI_CGAP_CML1
myeloid cells, 18
whole


pCMV-




pooled CML cases,
blood


SPORT6




BCR/ABL rearra


L0716
PMA-induced HL60 cell


PMA-

pSPORT 1



subtraction library


induced






HL60






human






leukemic






cell line


L0717
Gessler Wilms tumor




pSPORT1


L0731
Soares_pregnant_uterus—

uterus


pT7T3-Pac



NbHPU


L0738
Human colorectal cancer




pT7T3D


L0740
Soares melanocyte
melanocyte



pT7T3D



2NbHM




(Pharmacia)








with a








modified








polylinker


L0741
Soares adult brain

brain


pT7T3D



N2b4HB55Y




(Pharmacia)








with a








modified








polylinker


L0742
Soares adult brain

brain


pT7T3D



N2b5HB55Y




(Pharmacia)








with a








modified








polylinker


L0743
Soares breast 2NbHBst

breast


pT7T3D








(Pharmacia)








with a








modified








polylinker


L0744
Soares breast 3NbHBst

breast


pT7T3D








(Pharmacia)








with a








modified








polylinker


L0745
Soares retina N2b4HR
retina
eye


pT7T3D








(Pharmacia)








with a








modified








polylinker


L0746
Soares retina N2b5HR
retina
eye


pT7T3D








(Pharmacia)








with a








modified








polylinker


L0747
Soares_fetal_heart_NbHH

heart


pT7T3D



19W




(Pharmacia)








with a








modified








polylinker


L0748
Soares fetal liver spleen

Liver and


pT7T3D



1NFLS

Spleen


(Pharmacia)








with a








modified








polylinker


L0749
Soares_fetal_liver_spleen—

Liver and


pT7T3D



1NFLS_S1

Spleen


(Pharmacia)








with a








modified








polylinker


L0750
Soares_fetal_lung_NbHL1

lung


pT7T3D



9W




(Pharmacia)








with a








modified








polylinker


L0751
Soares ovary tumor
ovarian tumor
ovary


pT7T3D



NbHOT




(Pharmacia)








with a








modified








polylinker


L0752
Soares_parathyroid_tumor—
parathyroid tumor
parathyroid


pT7T3D



NbHPA

gland


(Pharmacia)








with a








modified








polylinker


L0753
Soares_pineal_gland_N3H

pineal


pT7T3D



PG

gland


(Pharmacia)








with a








modified








polylinker


L0754
Soares placenta Nb2HP

placenta


pT7T3D








(Pharmacia)








with a








modified








polylinker


L0755
Soares_placenta_8to9weeks—

placenta


pT7T3D



2NbHP8to9W




(Pharmacia)








with a








modified








polylinker


L0756
Soares_multiple_sclerosis—
multiple sclerosis



pT7T3D



2NbHMSP
lesions



(Pharmacia)








with a








modified








polylinker








V_TYPE


L0757
Soares_senescent_fibroblasts—
senescent fibroblast



pT7T3D



NbHSF




(Pharmacia)








with a








modified








polylinker








V_TYPE


L0758
Soares_testis_NHT




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0759
Soares_total_fetus_Nb2H




pT7T3D-Pac



F8_9w




(Pharmacia)








with a








modified








polylinker


L0761
NCI_CGAP_CLL1
B-cell, chronic



pT7T3D-Pac




lymphotic leukemia



(Pharmacia)








with a








modified








polylinker


L0763
NCI_CGAP_Br2
breast



pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0764
NCI_CGAP_Co3
colon



pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0766
NCI_CGAP_GCB1
germinal centre B



pT7T3D-Pac




cell



(Pharmacia)








with a








modified








polylinker


L0767
NCI_CGAP_GC3
pooled germ cell



pT7T3D-Pac




tumors



(Pharmacia)








with a








modified








polylinker


L0768
NCI_CGAP_GC4
pooled germ cell



pT7T3D-Pac




tumors



(Pharmacia)








with a








modified








polylinker


L0769
NCI_CGAP_Brn25
anaplastic
brain


pT7T3D-Pac




oligodendroglioma



(Pharmacia)








with a








modified








polylinker


L0770
NCI_CGAP_Brn23
glioblastoma
brain


pT7T3D-Pac




(pooled)



(Pharmacia)








with a








modified








polylinker


L0771
NCI_CGAP_Co8
adenocarcinoma
colon


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0772
NCI_CGAP_Co10
colon tumor RER+
colon


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0773
NCI_CGAP_Co9
colon tumor RER+
colon


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0774
NCI_CGAP_Kid3

kidney


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0775
NCI_CGAP_Kid5
2 pooled tumors
kidney


pT7T3D-Pac




(clear cell type)



(Pharmacia)








with a








modified








polylinker


L0776
NCI_CGAP_Lu5
carcinoid
lung


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0777
Soares_NhHMPu_S1
Pooled human
mixed (see


pT7T3D-Pac




melanocyte, fetal
below)


(Pharmacia)




heart, and pregnant



with a








modified








polylinker


L0779
Soares_NFL_T_GBC_S1

pooled


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0780
Soares_NSF_F8_9W_OT—

pooled


pT7T3D-Pac



PA_P_S1




(Pharmacia)








with a








modified








polylinker


L0783
NCI_CGAP_Pr22
normal prostate
prostate


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0784
NCI_CGAP_Lei2
leiomyosarcoma
soft tissue


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0786
Soares_NbHFB

whole


pT7T3D-Pac





brain


(Pharmacia)








with a








modified








polylinker


L0787
NCI_CGAP_Sub1




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0788
NCI_CGAP_Sub2




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0789
NCI_CGAP_Sub3




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0790
NCI_CGAP_Sub4




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0791
NCI_CGAP_Sub5




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0792
NCI_CGAP_Sub6




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0793
NCI_CGAP_Sub7




pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0794
NCI_CGAP_GC6
pooled germ cell



pT7T3D-Pac




tumors



(Pharmacia)








with a








modified








polylinker


L0796
NCI_CGAP_Brn50
medulloblastoma
brain


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0800
NCI_CGAP_Co16
colon tumor, RER+
colon


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0803
NCI_CGAP_Kid11

kidney


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0804
NCI_CGAP_Kid12
2 pooled tumors
kidney


pT7T3D-Pac




(clear cell type)



(Pharmacia)








with a








modified








polylinker


L0805
NCI_CGAP_Lu24
carcinoid
lung


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0806
NCI_CGAP_Lu19
squamous cell
lung


pT7T3D-Pac




carcinoma, poorly



(Pharmacia)




differentiated (4



with a








modified








polylinker


L0807
NCI_CGAP_Ov18
fibrotheoma
ovary


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L0809
NCI_CGAP_Pr28

prostate


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L4747
NCI_CGAP_Brn41
oligodendroglioma
brain


pT7T3D-Pac








(Pharmacia)








with a








modified








polylinker


L5565
NCI_CGAP_Brn66
glioblastoma with
brain


pCMV-




probably TP53



SPORT6




mutation and witho


L5566
NCI_CGAP_Brn70
anaplastic
brain


pCMV-




oligodendroglioma



SPORT6.ccdb


L5569
NCI_CGAP_HN17
normal epithelium
nasopharynx


pAMP10


L5575
NCI_CGAP_Brn65
glioblastoma
brain


pCMV-




without EGFR



SPORT6




amplification

















TABLE 5





OMIM



Reference
Description







104311
Alzheimer disease-3


107250
Anterior segment mesenchymal dysgenesis


109150
Machado-Joseph disease


112262
Fibrodysplasia ossificans progressiva, 135100


118485
Polycystic ovary syndrome with hyperandrogenemia


120215
Ehlers-Danlos syndrome, type I, 130000


120215
Ehlers-Danlos syndrome, type II, 130010


136550
Macular dystrophy, North Carolina type


138981
Pulmonary alveolar proteinosis, 265120


151670
Hepatic lipase deficiency


164731
Ovarian carcinoma, 167000


172400
Hemolytic anemia due to glucosephosphate isomerase



deficiency


172400
Hydrops fetalis, one form


180901
Malignant hyperthermia susceptibility 1, 145600


180901
Central core disease, 117000


181600
Sclerotylosis


182600
Spastic paraplegia-3A


182870
Spherocytosis-1


182870
Elliptocytosis-3


182870
Anemia, neonatal hemolytic, fatal and near-fatal


188826
Sorsby fundus dystrophy, 136900


189800
Preeclampsia/eclampsia


190040
Meningioma, SIS-related


190040
Dermatofibrosarcoma protuberans


190040
Giant-cell fibroblastoma


190198
Leukemia, T-cell acute lymphoblastic


203310
Ocular albinism, autosomal recessive


208400
Aspartylglucosaminuria


221770
Polycystic lipomembranous osteodysplasia with sclerosing



leukencephalopathy


225500
Ellis-van Creveld syndrome


231675
Glutaricaciduria, type IIC


231680
Glutaricaciduria, type IIA


245200
Krabbe disease


248600
Maple syrup urine disease, type Ia


266300
[Hair color, red]


269920
Salla disease


272800
Tay-Sachs disease


272800
[Hex A pseudodeficiency]


272800
GM2-gangliosidosis, juvenile, adult


276700
Tyrosinemia, type I


600225
Phenylketonuria, atypical, due to GCH1 deficiency, 233910


600225
Dystonia, DOPA-responsive, 128230


600374
Bardet-Biedl syndrome 4


600593
Craniosynostosis, Adelaide type


600850
Schizophrenia disorder-4


600918
Cystinuria, type III


601208
Insulin-dependent diabetes mellitus-11


601669
Hirschsprung disease, one form


601780
Ceroid-lipofuscinosis, neuronal-6, variant late infantile


602363
Ellis-van Creveld-like syndrome


602716
Nephrosis-1, congenital, Finnish type, 256300


602772
Retinitis pitmentosa-24









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 secreted protein.
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 ATCC deposit Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention.
Signal Sequences
The present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide 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. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence which 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.
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 5 residues (i.e., + or −5 residues) of the predicted cleavage point. 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 desribed below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
Polynucleotide and Polypeptide Variants
The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone.
The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by a deposited clone.
“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.
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% or 99% identical to, for example, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence contained in a deposited cDNA clone or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in a deposited clone, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:Y, the polypeptide sequence encoded by the cDNA contained in a deposited clone, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).
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 shown in Table 1A, 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 presence 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 in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identiy 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 lenght 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 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, an amino acid sequences shown in Table 1A (SEQ ID NO:Y) or to the amino acid sequence encoded by cDNA contained in a deposited clone can be determined conventionally using known computer programs. A preferred method for determing 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 in 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 sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
The variants 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, variants in which 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 secreted protein without substantial loss of biological function. The authors of 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].” (See, Abstract.) 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 substantial biological activity. 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. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., 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. (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) substitution with one or more of 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), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, 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. (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 a polypeptide which comprises the amino acid sequence of the present invention 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. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains 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 number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1–5,5–10, 5–25, 5–50, 10–50 or 50–150, conservative amino acid substitutions are preferable.
Polynucleotide and Polypeptide Fragments
The present invention is also directed to polynucleotide fragments of the polynucleotides of the invention.
In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:X or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. The nucleotide 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 a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. In this context “about” includes the particularly recited value, 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., 50, 150, 500, 600, 2000 nucleotides) are preferred.
Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting 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, 651–700, 701–750, 751–800, 800–850, 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, or 2001 to the end of SEQ ID NO:X, 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. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone. 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, 102–120, 121–140, 141–160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and 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 polypeptides are also encompassed by the invention.
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.
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 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.
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.
For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide of the invention for binding to an antibody of the polypeptide of the invention, 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 for a polypeptide of the invention 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, E., et al., 1995, Microbiol. Rev. 59:94–123. In another embodiment, physiological correlates of binding of a polypeptide of the invention to its substrates (signal transduction) can be assayed.
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 invention and fragments, variants derivatives and analogs thereof to elicit related biological activity related to that of the polypeptide of the invention (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.
Epitopes and Antibodies
The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions 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), 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 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, 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)).
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).
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 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 (“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.
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.
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 SEQ ID NO:Y, 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), 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, by size in contiguous amino acid residues, or listed in the Tables and Figures. 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, 10−2M, 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, 10−8M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11M, 10−11 M, 5×10−12M, 10−12 M, 5×10−13M, 10−13 M, 5×10−14M, 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. Preferrably, 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, but not limited to, 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 use 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 covalently and non-covalently 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 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, phosphylation, 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 (e.g., Example 16). 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 ATCC. 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.
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 Brinkinan 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., AJR134:26–34 (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 4,816,397, 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 regions 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; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, 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. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
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 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.
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.
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, W138, 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 which 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); 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)).
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. (EP 394,827; 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. (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. (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, thioepa 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, e.g., 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. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is 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, 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 at 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 at 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 at 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.
Therapeutic Uses
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 treat, inhibit or prevent 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. 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. 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 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, 10−8 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, 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 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 aspect, 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; Biolistic, Dupont), 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 that contains 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 Tlymphocytes, Blymphocytes, 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 controlling the presence or absence of the 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 an antibody of the invention. In a preferred aspect, 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 sialastic 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, i.e., 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; Biolistic, Dupont), 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, or monitor diseases, disorders, and/or 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 disorder, 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 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.
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 aspect 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 (MI).
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 which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which 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 which 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 calorimetric substrate (Sigma, 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.
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, the polypeptides of the present invention 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.
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.
Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of 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), 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).) Polynucleotides comprising or alternatively consisting of nucleic acids which encode these fusion proteins are also encompassed by the invention.
Similarly, EP-A-O 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).)
Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide 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).)
Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
Vectors, Host Cells, and Protein Production
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by 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 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 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 (ATCC 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.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223–3, pKK233–3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. 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.
Introduction of the construct 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.
A polypeptide of this 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, and preferably the secreted form, 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 the polypeptide of the present 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 protein of the invention by virtue of the strong AOXI 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 PA0815, 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 the 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, resulting in the formation of a new transcription unit (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 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 sequence of the invention 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 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.
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, 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, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), 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. Sulffiydryl 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 a 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 which 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 monmethoxy 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-trifluoreothane 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-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in 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 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 the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides 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 polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing 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 the sequence listing, or contained in the polypeptide encoded by a deposited clone). 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 of the invention.
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 an 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, oseteoprotegerin (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 seuqence. In a further embodiment, associations 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 hyrophobic 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).
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 polynucleotide of the present invention can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15–25 bp) from the sequences shown in SEQ ID NO:X. Primers can 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 the 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).
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).) 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 the polynucleotide 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 polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.
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.
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 present invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the present 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 disorder, 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 present 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 polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide 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 disorder or being determined by averaging levels from a population of individuals not having a 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 which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and other 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 preferrably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides 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 present invention attached may be used to identify polymorphisms between the polynucleotide sequences, 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, including 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 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 P. E. Nielsen, M. Egholm, R. H. Berg and 0. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, 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 (T.sub.m) 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 present invention is useful for 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 diseases, disorders, and/or conditions 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. , Wiemik, 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 would not be limited to treatment of proliferative diseases, disorders, and/or conditions of hematopoietic cells and tissues, 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 can be used to control gene expression through triple helix formation or 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. 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 or prevent disease.
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.
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 specific to particular tissue prepared from the sequences of the present invention. 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.
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.
A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., 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 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, 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.
In addition to assaying secreted protein levels 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), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. 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).)
Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (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 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, prevent, and/or diagnose disease. 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 supressor), 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, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and 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 following biological activities.
Gene Therapy Methods
Another aspect of the present invention is to gene therapy methods for treatingor 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 a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that 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 invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207–216 (1993); Ferrantini et al., Cancer Research, 53:107–1112 (1993); Ferrantini et al., J. Immunology 153: 4604–4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221–229 (1995); Ogura et al., Cancer Research 50: 5102–5106 (1990); Santodonato, et al., Human Gene Therapy 7:1–10 (1996); Santodonato, et al., Gene Therapy 4:1246–1255 (1997); and Zhang, 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 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, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin 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 of the invention 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 Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; 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 polynucleotide sequence of the invention. 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 polynucleotides of the 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 of the invention 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, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
In certain embodiments, the polynucleotide constructs of the invention 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, 84:7413–7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077–6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem., 265:10189–10192 (1990), 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 Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413–7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
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., 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), dioleoylphoshatidyl 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, 101:512–527 (1983), 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, 394:483 (1975); Wilson et al., Cell, 17:77 (1979)); ether injection (Deamer et al., 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 et al., Proc. Natl. Acad. Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980); Szoka et al., 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 (which are herein incorporated by reference) 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 polypeptides of the 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, T19–14×, 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 polypeptides of the 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 polypeptides of the invention.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the 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 (Schwartzet 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 et al., Science, 252:431–434 (1991); Rosenfeld et al., Cell, 68:143–155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).
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 which 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, 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 containing polynucleotides of the invention 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 of the invention. 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 the desired gene product.
Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) 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). 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 polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding other angiongenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), 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.
Preferably, the polynucleotide encoding a polypeptide of the invention contains 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, biolistic 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.
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
Biological Activities
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.
Polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with the following systems.
Immune Activity
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing 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 1A, column 8 (Tissue Distribution Library Code).
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies. 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 treated, prevented, diagnosed, and/or prognosing 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 treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, or antagonists or agonists thereof.
Other immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed 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 treated, prevented, diagnosed and/or prognosed 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 treating, preventing, diagnosing and/or prognosing 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 treated, prevented, diagnosed and/or prognosed 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, 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), and insulin-resistant diabetes mellitus.
Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, 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 treated, prevented, diagnosed and/or prognosed 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 treated, prevented, diagnosed and/or prognosed 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 treated, prevented, diagnosed and/or prognosed using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed 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 treated, prevented, diagnosed and/or prognosed 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 treating, preventing, prognosing, and/or diagnosing 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 treated, prevented, diagnosed and/or prognosed 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 treat, prevent, diagnose and/or prognose 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 diagnosis, prognosis, prevention, and/or treatment 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; 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, 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, dernatitis, 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 diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease. 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 other embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat 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, 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 Immunodificiency.
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 decribed 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 diagnose, prognose, treat, and/or prevent 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 prevented, diagnosed, prognosed, and/or treated 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 diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed, or treated 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 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 in an organ system listed above.
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 prevent, diagnose, prognose, and/or treat 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 occulsion 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 extrcorporeal devices (e.g., intravascular canulas, 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 prevent, diagnose, prognose, and/or treat 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 1A, column 8 (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 prevention, detection, diagnosis and/or treatment 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 prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example eosinophilia.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to prevent, treat, or diagnose 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 treating, preventing, and/or diagnosing anemias. Anemias that may be treated prevented or diagnosed by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary siderob; astic 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 treating, preventing, and/or diagnosing 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 treating, preventing, and/or diagnosing anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfadrugs. Additionally, rhe polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing 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 treating, preventing, and/or diagnosing 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 diagnosing, prognosing, preventing, and/or treating 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 diagnosing, prognosing, preventing, and/or treating 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 Bemard-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 diagnosing, prognosing, preventing, and/or treating 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 diagnosing, prognosing, preventing, and/or treating 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 diagnosing, prognosing, preventing, and/or treating leukopenia. In other specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating 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 diagnosing, prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia. Neutropenias that may be diagnosed, prognosed, prevented, and/or treated 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 B12 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 diagnosing, prognosing, preventing, and/or treating 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 Syndome, severe combined immunodeficiency, ataxia telangiectsia).
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating 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 diagnosing, prognosing, preventing, and/or treating 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 diagnosing, prognosing, preventing, and/or treating 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 leukenia), 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 diagnosing, prognosing, preventing, and/or treating 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 treating, preventing, and/or diagnosing myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday 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 preventing, diagnosing, and/or treating 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 diagnose, prognose, prevent, and/or treat 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 diagnosed, prognosed, prevented, and/or treated 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, a typical 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 diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, a typical 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 diagnosed, prognosed, prevented, and/or treated 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, ophthalmomandibulomelic 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 diagnosed, prognosed, prevented, and/or treated 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 prognose 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 1A, column 8 (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 diagnosed, prognosed, prevented, and/or treated 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 diagnosed, prognosed, prevented, and/or treated 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 diagnosed, prognosed, prevented, and/or treated 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 diagnosed, prognosed, prevented, and/or treated 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 diagnosed, prognosed, prevented, and/or treated 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 poynucleotides 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, lipofectin, 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 diagnostic, 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 fragements thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragements thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−6M, 10−6M, 5×107M, 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, 5×10−14 M, 10−14M, 5×10−15M, and 10−5M.
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 adjuviants, 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 elsewere 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 thereapeutic affects 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 antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodes of the invention may be associated with 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 treat, prevent, diagnose, and/or prognose disorders of the renal system. Renal disorders which can be diagnosed, prognosed, prevented, and/or treated 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 diagnosed, prognosed, prevented, and/or treated 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 diagnose, prognose, prevent, and/or treat 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 diagnose, prognose, prevent, and/or treat 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, biolistic 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 treat, prevent, diagnose, and/or prognose cardiovascular 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 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.
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, biolistic 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 treat, prevent, diagnose, and/or prognose 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), a typical 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 pneumoconioses), 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 which 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.
Within 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 muco-adhesive polymer which 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, sclerodema, trachoma, and vascular adhesions.
Moreover, disorders and/or states, which can be treated, prevented, diagnosed, and/or prognosed with the 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, vascluogenesis, 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; Angostatic 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 treated, prevented, diagnosed, and/or prognosed 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 treated, prevented, diagnosed, and/or prognesed 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 doudenal 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 brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, 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 displasia, 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 tetraholoride 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 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, Wemicke 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 diagnose and/or treat or prevent 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, Wemicke'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 Wemicke Aphasia, Dyslexia such as Acquired Dyslexia, language development disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wemicke 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, ophthalmoplegia such as diplopia, Duane's Syndrome, Homer's Syndrome, Chronic progressive external ophthalmoplegia 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, Homer'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, ophthalmoplegia such as Duane's Syndrome, Horner's Syndrome, Chronic Progressive External Ophthalmoplegia 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 treat, prevent, diagnose, and/or prognose 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 diagnose, prognose, prevent, and/or treat 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 1A, column 8 (Tissue Distribution Library Code).
Reproductive System Disorders
The polynucleotides or polypeptides, or agonists or antagonists of the invention may be used for the diagnosis, treatment, or prevention 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 diagnosis, treatment, and/or prevention 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 diagnosis, treatment, and/or prevention of diseases and/or 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 diagnosis, treatment, and/or prevention 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 diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicomuate uterus, septate uterus, simple unicornuate uterus, unicomuate 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, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod 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 diagnosis, treatment, and/or prevention 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 diagnosed, treated, and/or prevented 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, Bimaviridae, 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, Picomaviridae, 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, bronchiollitis, 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 virenia, 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 dificile, 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, 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, noscomial 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, diptheria, botulism, and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed 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 treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose 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 treat, prevent, diagnose, and/or prognose gastrointestinal 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 stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, 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.
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, daunorubisin, 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.
Polypeptides of the 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 polypeptide of the invention 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: contacting a polypeptide of the invention with a plurality of molecules; and identifying a molecule that binds the polypeptide of the invention.
The step of contacting the polypeptide of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the polypeptide of the invention on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptide of the invention. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized polypeptide of the invention. The molecules having a selective affinity for the polypeptide of the invention can then be purified by affinity selection. The nature of the solid support, process for attachment of the polypeptide of the invention 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 polypeptide 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 polypeptide of the invention and the individual clone. Prior to contacting the polypeptide of the invention 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 a polypeptide of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the polypeptide 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 polypeptide of the invention, or alterntatively, unbound polypeptides, from a mixture of the polypeptide 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 polypeptide of the invention or the plurality of polypeptides is 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 to a polypeptide 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, pyrrolinones, 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 a polypeptide of the invention can be carried out by contacting the library members with a polypeptide of the invention immobilized on a solid phase and harvesting those library members that bind to the polypeptide 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 a polypeptide of the invention.
Where the polypeptide of the invention 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 polypeptide of the invention 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 polypeptide of the invention binding polypeptide has in the range of 15–100 amino acids, or 20–50 amino acids.
The selected polypeptide of the invention binding polypeptide can be obtained by chemical synthesis or recombinant expression.
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 nucleotide sequences contained a deposited clone. 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, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Anitsense 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, 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, 10 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 mature 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 of the invention. 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 a polypeptide of the 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 (Bemoist 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 interest. 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 of the invention, 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 sequence of the invention 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., Nature, 372:333–335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′non-translated, non-coding regions of a polynucleotide sequence of the invention 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, 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., Proc. Natl. Acad. Sci. U.S.A. 86:6553–6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648–652 (1987); 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., BioTechniques, 6:958–976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539–549 (1988)). 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, xantine, 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-N-6-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., Nucl. Acids Res., 15:6625–6641 (1987)). The oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131–6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327–330 (1987)).
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. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448–7451 (1988)), etc.
While antisense nucleotides complementary to the coding region sequence of the invention 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 corresponding to the polynucleotides of the invention, 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 each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5 end of the mRNA corresponding to the polynucleotides of the invention; 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 the polynucleotides of the invention 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, prevent, and/or diagnose the diseases described herein.
Thus, the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions 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.
invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention
Other Activities
The polypeptide 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. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
The polypeptide 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.
The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the 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.
The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
The polypeptide of the 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, the polypeptides 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.
The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.
The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
The polypeptide or polynucleotides and/or agonist or antagonists 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, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to treat weight disorders, including but not limited to, obesity, cachexia, wasting disease, anorexia, and bulimia.
Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), 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. Polypeptide or polynucleotides and/or agonist or antagonists 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.
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 wherein X is 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 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.
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.
A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:X 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.
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.
Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said isolated nucleic acid molecule 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 a human cDNA clone identified by a cDNA Clone Identifier in Table 1A, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table 1A for said cDNA Clone Identifier.
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 in the nucleotide sequence of the cDNA of a human cDNA clone identified by a cDNA Clone Identifier in Table 1A, which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table 1A. Further preferred is the above nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said human cDNA clone. In addition, an isolated nucleic acid molecule of the invention may comprise a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence of the cDNA in said human cDNA clone. 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 of the cDNA in said human cDNA clone. 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 the cDNA in said human cDNA clone.
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 wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A; which method comprises: (a) 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 (b) determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence. The step of comparing sequences in the above method may further comprise 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, the step of comparing sequences in the above method may be 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 wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. This method described above may further 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 gene encoding a secreted protein identified in Table 1A, which 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 wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. This method described above may further 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 wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
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 amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A. 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 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.
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.
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.
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 complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. Further preferred is the above isolated polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a secreted portion of the secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.
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 the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.
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 the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.
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: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.
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: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A; which method comprises: (a) 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 (b) 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. The step in the above method of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group may further comprise 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: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. Further, the step of comparing sequences in the above method may be 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: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. This method may further comprise 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 gene encoding a secreted protein identified in Table 1A, 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: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.
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: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. Further preferred is the above isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host. Similarly preferred is the above isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.
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 of the invention 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 secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of: 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 the ATCC Deposit Number shown for said cDNA clone in Table 1A. 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 secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.
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.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
EXAMPLES
Example 1
Isolation of a Selected cDNA Clone from the Deposited Sample
Each cDNA clone in a cited ATCC deposit 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 table immediately below 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 Zap,” the corresponding deposited clone is in pBluescript.”











Vector Used to Construct Library
Corresponding Deposited Plasmid







Lambda Zap
pBluescript (pBS)


Uni. Zap XR
pBluescript (pBS)


Zap Express
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 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), pBluescript (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 Stratagene 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 Stratagene. 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 SacI 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 ori generates sense strand DNA and in the other, antisense.
Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, 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 Technologies. (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 Technologies. (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 ATCC Deposit Number cited in Table 1A 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 ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table 1A. Typically, each ATCC deposit sample cited in Table 1A comprises a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; but such a deposit sample may include plasmids for more or less than 50 cDNA clones, up to about 500 cDNA clones.
Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNAs cited for that clone in Table 1A. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to 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 (Stratagene)) 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 SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded by the 5′ NT and the 3′ NT of the clone defined in Table 1A) 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 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5–5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM 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 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 cDNA sequence corresponding to SEQ ID NO:X., according to the method described in Example 1. (See also, Sambrook.)
Example 3
Tissue Distribution of Polypeptide
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 SPIN-100TM column (Clontech 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) (Clontech) are examined with the labeled probe using ExpressHybTM hybridization solution (Clontech) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at −70 degree C. overnight, and the films developed according to standard procedures.
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 degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree 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 is 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-bistidine 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 lacI 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 ug/ml) and Kan (25 ug/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 lacI 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 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3–4 hours at 4 degree 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 immidazole. Immidazole 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 degree C. or frozen at −80 degree C.
In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC 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-Delgamo sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequence and operator sequences are made synthetically.
DNA can be inserted into the pHEa 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 degree C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4–10 degree 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 ihe solution through a microfluidizer (Microfuidics, 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 degree 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 degree 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 um 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 ug 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 and the naturally associated leader sequence identified in Table 1A, is amplified using the PCR protocol described in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, 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 (“Geneclean,” 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 (“Geneclean” BIO 101 Inc., La Jolla, Calif.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB 101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene 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 ug of a plasmid containing the polynucleotide is co-transfected with 1.0 ug 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 ug of BaculoGoldTM virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul 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 (ATCC 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 degrees 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 degrees 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 Technologies 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 Technologies 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 ul 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 degree 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 11 medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of 35S-methionine and 5 uCi 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, HII 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 (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 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, 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 (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC 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 the naturally occurring signal sequence is used to produce the secreted protein, the vector 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., WO 96/34891.)
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” 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 HB 101 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 is used for transfection. Five μg of the expression plasmid pC6 a pC4 is cotransfected with 0.5 ug of the plasmid pSVneo using lipofectin (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 metothrexate 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 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100–200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase BPLC 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 EP A 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 (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 secreted protein, 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., WO 96/34891.)
Human IgG Fc region:








Human IgG Fc region:









(SEQ ID NO:1)









GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC



 


CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA


 


ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG


 


TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG


 


GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA


 


CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT


 


GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA


 


ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC


 


ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG


 


TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG


 


GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC


 


CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG


 


ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT


 


GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG


 


TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT






Example 10
Production of an Antibody from a Polypeptide
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 is administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the secreted protein 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.
In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler 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, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/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 ATCC. 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.
Alternatively, additional antibodies capable of binding to the polypeptide 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 which 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 protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-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, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. 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).)
Example 11
Production of Secreted Protein for High-Throughput Screening Assays
The following protocol produces a supernatant containing a polypeptide to be tested. This supernatant can then be used in the Screening Assays described herein.
First, dilute Poly-D-Lysine (644 587 Boehringer-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 ul 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 2×10 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 ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2 ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8 or 9, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50 ul of the Lipofectamine/Optimem 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 ul Optimem 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 a 12-channel pipetter with tips on every other channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degrees C. for 6 hours.
While cells are incubating, prepare appropriate media, either 1% BSA in DMEM with 1× penstrep, or 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—H2O; 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-H2O; 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-H2O; 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-Phenylalainine; 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-Tryrosine-2Na-2H2O; 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; and 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 uM 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; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal) with 2 mm glutamine and 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul 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 degrees 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 ul multichannel pipetter, aliquot 600 ul in one iml 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 13–20.
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 directly (e.g., as a secreted protein) or by the polypeptide 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 12
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. StatS 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, 1L-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-Xxx-Trp-Ser (SEQ ID NO:2)).
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.













GAS











JAKs

(elements)













Ligand
tyk2
Jak1
Jak2
Jak3
STATS
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 (Pleiotrophic)
+
+
+
?
1, 3
GAS(IRF1 >








Lys6 > IFP)


Il-11(Pleiotrophic)
?
+
?
?
1, 3


OnM(Pleiotrophic)
?
+
+
?
1, 3


LIF(Pleiotrophic)
?
+
+
?
1, 3


CNTF(Pleiotrophic)
−/+
+
+
?
1, 3


G-CSF(Pleiotrophic)
?
+
?
?
1, 3


IL-12(Pleiotrophic)
+
−
+
+
1, 3


g-C family


IL-2 (lymphocytes)
−
+
−
+
1, 3, 5
GAS


IL-4 (lymph/
−
+
−
+
6
GAS(IRF1 =


myeloid)





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 13–14, 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:3)










5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTT



 


CCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′






The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:4)
PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:








(SEQ ID NO:5)









5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGA



 


AATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTC


 


CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA


 


TTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGG


 


CCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGA


 


GGCCTAGGCTTTTGCAAAAAGCTT: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 Clontech 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 (Clontech), 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 13–14.
Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing NFK-B and EGR promoter sequences are described in Examples 15 and 16. 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, II-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 13
High-Throughput Screening Assay for T-cell Activity
The following protocol is used to assess T-cell activity by identifying factors, and determining whether supemate 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 12. 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 (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC 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 Technologies)(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 genticin 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 ul 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-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul 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-MEM to a final concentration of 107 cells/ml. Then add 1 ml of 1×107 cells in OPTI-MEM to T25 flask and incubate at 37 degrees 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 Genticin, and 1% Pen-Strep. These cells are treated with supernatants containing polypeptides of the invention and/or induced polypeptides of the invention as produced by the protocol described in Example 11.
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 ul of cells into each well (therefore adding 100,000 cells per well).
After all the plates have been seeded, 50 ul 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 ul 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 degrees C. until SEAP assays are performed according to Example 17. The plates containing the remaining treated cells are placed at 4 degrees 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 14
High-Throughput Screening Assay Identifying Myeloid Activity
The following protocol is used to assess myeloid activity by determining whether polypeptides of the invention proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. 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 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth & Differentiation, 5:259–265) is used. First, harvest 2×10e7 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 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na2HPO4.7H2O, 1 mM MgCl2, and 675 uM 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 degrees C. for 36 hr.
The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/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 ug/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 ul cells per well in the 96-well plate (or 1×105 cells/well).
Add 50 ul of the supernatant prepared by the protocol described in Example 11. Incubate at 37 degrees 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 17.
Example 15
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.
Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma 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 can be assessed.
The EGRISEAP 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:









(SEQ ID NO:6)










5′GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′



 









(SEQ ID NO:7)










5′GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′







Using the GAS:SEAP/Neo vector produced in Example 12, 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 ug/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 Lipofectamine protocol described in Example 11. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/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 ug/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 ul of the cell suspension to each well of 96-well plate (equivalent to 1×105 cells/well). Add 50 ul supernatant produced by Example 11, 37° 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/ul 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 17.
Example 16
High-Throughput Screenin 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 11. Activators or inhibitors of NF-KB would be useful in treating 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:8), 18 bp of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site:








(SEQ ID NO:9)









5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGG



ACTTTCCATCCTGCCATCTCAATTAG:3′






The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a Hind III site:









5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′
(SEQ ID NO:4)







PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence:








(SEQ ID NO:10)









5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTT



 


CCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCG


 


CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGG


 


CTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG


 


AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC


 


AAAAAGCTT:3′






Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) 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 (Clontech), 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 13. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 13. As a positive control, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10, and H111, with a 5–10 fold activation typically observed.
Example 17
Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 13–16, 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 ul of 2.5× dilution buffer into Optiplates containing 35 ul 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 ul 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 luminometer, 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 8
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 meassure 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 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul 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 ul of 12 ug/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 ul 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 ul 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 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul 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 ul. Increased emission at 530 nm indicates an extracellular signaling event which has resulted in an increase in the intracellular Ca++ concentration.
Example 19
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, Ick, 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, the identification of novel human secreted proteins capable of activating tyrosine kinase signal transduction pathways are of interest. Therefore, the following protocol is designed to identify those novel human secreted proteins 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 Loprodyne 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 Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel 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 alamarBlue 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 Loprodyne 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 Loprodyne 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 ul of the supernatant produced in Example 11, 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 mM Na3VO4, 2 mM Na4P207 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 degrees 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 degrees 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 Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 ul of SuM Biotinylated Peptide, then 10 ul ATP/Mg2+(5 mM ATP/50 mM MgCl2), then 10 ul of 5× Assay Buffer (40 mM imidazole hydrochloride, pH 7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl2, 5 mM MnCl2, 0.5 mg/ml BSA), then Sul of Sodium Vanadate (1 mM), and then 5 ul of water. Mix the components gently and preincubate the reaction mix at 30 degrees C. for 2 min. Initial the reaction by adding 10 ul of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degrees C. for 20 min. This allows the streptavadin 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 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37 degrees C. for one hour. Wash the well as above.
Next add 100 ul of peroxidase substrate solution (Boehringer 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 20
High-Throughput Screening Assay Identifying Phosphorylation Activity
As a potential alternative and/or compliment to the assay of protein tyrosine kinase activity described in Example 19, 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 degrees C. until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne 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 ul of the supernatants obtained in Example 11 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.
Example 21
Method of Determining Alterations in a Gene Corresponding to a Polynucleotide
RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be 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. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60–120 seconds at 52–58 degrees C.; and 60–120 seconds at 70 degrees 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 borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.
PCR products is 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 (Boehringer 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 22
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 ug/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 unbounded polypeptide.
Next, 50 ul 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 unbounded conjugate.
Add 75 ul 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 23
Formulation
The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) 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 ug/kg/hour to about 50 ug/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 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. 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 mirocapsules), 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, Neutrokine-alpha and/or Neutrokine-alphaSV 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 Neutrokine-alpha and/or Neutrokine-alphaSV 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 Neutrokine-alpha and/or Neutrokine-alphaSV 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 Neutrokine-alpha and/or Neutrokine-alphaSV compositions are poly(lactide-co-glycolides). Polymer properties such as molecular weight, hydrophobicity, and lactide/glycolide ratio may be modified to obtain the desired drug Neutrokine-alpha and/or Neutrokine-alphaSV release profile (See, e.g., Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732–741 (2000), which is hereby incorporated by refernce 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 limted to, N-methyl-2-pyrrolidone, 2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, 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 alchohol, 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 Neutrokine-alpha and/or Neutrokine-alphaSV 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, and the like. 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 Neutrokine-alpha and/or Neutrokine-alphaSV 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); E P 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 (Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable prepartions 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/diptheria, 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 not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, 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 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/Abbott; COVIRACILTM (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro; Triangle/Abbott); 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/Abbott); 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 β-L-FD4C and β-L-FddC (WO 98/17281).
Additional NNRTIs include COACTINONTM (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINETM (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); GW-420867×(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; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIRTM (PNU-140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); 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 recpetor 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 compunds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOXTM (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; Roche).
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; Chiron), 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 targetted 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, PENTAMDINETM, 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 LEUCOVORNTM 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, 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 (Entremed, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), 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, (Celgene, 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) (OXiGENE, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (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 (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (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 compositons of the invention include, but are not lmited to, AG-3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aetema, 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 compositons of the invention include, but are not lmited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, 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 compositons of the invention include, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787!ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositons of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, 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; M1H), 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 horomone 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 (Roche, RO-32-3555), Leflunomide (also known as AravaTM from Hoechst Marion Roussel), KineretTM (an IL-1 Receptor antagonist also known as Anakinra from Amgen, 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 radisotopes. 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-1 alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, 1L-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), 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-6821 10; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PIGF-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, LEUKETM, PROKINETM), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGENTM), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGENTM, PROCRITM), 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, 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, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, 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, methyclothiazide, 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, 127, 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 congugated estrogens such as ESTRACETM (estradiol), ESTINYLTM (ethinyl estradiol), PREMARINTM, ESTRATABTM, ORTHO-ESTTM, OGENTM and estropipate (estrone), ESTROVISTMTM (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), LEVLENMTM, NORDETTEMTM, 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), TESTEXυ (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 eh (hydrocortisone) acetate), LOCOIDTM (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATEM (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), FLUOR-OPTM and FMLTM (fluorometholone), CORDRANTM (flurandrenolide), HALOGTM (halcinonide), HMS LIZUFIFILMTM (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).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to 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), and troglitazone; 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 biguamide 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), PROMETRWM® 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, phenytoin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantidine, 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, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide mononitrate, 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.
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 24
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 an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. 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 polypeptide 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 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 23.
Example 25
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, preferably a secreted form, 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 formulation of the antisense polynucleotide is provided in Example 23.
Example 26
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 HB 101, 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 27
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 Na2 HPO4, 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 pUC 18 (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 pUC 18 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 28
Method of Treatment Using Gene Therapy—In Vivo
Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases 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 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, 5705151, 5580859; 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 carrer.
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, lipofectin 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 therapies 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 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% Avertin. 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 um 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 use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.
Example 29
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 diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.
Example 30
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., 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, eg., 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 which, 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 diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.
Example 31
Production of an Antibody
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 polypeptide(s) of the invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of polypeptide(s) of the 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 polypeptide(s) of the 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 polypeptide(s) of the 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/kg/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 (SP20), available from the ATCC. 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(s) of the invention.
Alternatively, additional antibodies capable of binding polypeptide(s) of the 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 which 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(s) of the invention protein-specific antibody can be blocked by polypeptide(s) of the invention. Such antibodies comprise anti-idiotypic antibodies to the polypeptide(s) of the invention protein-specific antibody and are used to immunize an animal to induce formation of further polypeptide(s) of the invention protein-specific antibodies.
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., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)
Isolation of Antibody Fragments Directed polypeptide(s) of the 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 polypeptide(s) of the 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 PCT publication 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 innoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see PCT publication 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 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication 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 ttg 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 i.OM Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TGI 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., PCT publication 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 32
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, ILIO, 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 CD 154, 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—Purified polypeptides of the invention, or truncated forms thereof, is 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 polypeptides 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, 100U/ml penicillin, 10 ug/ml streptomycin, and 10−5 dilution of SAC) in a total volume of 150 ul. 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 a polypeptide 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 polypeptides of the invention identify the results of the activity of the polypeptides 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 polypeptide is used to indicate whether the polypeptide 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 polypeptide-treated mice.
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 of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.
Example 33
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 microliters per well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B 33.1) overnight at 4° C. (1 microgram/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 TNF Delta and/or TNF Epsilon protein (total volume 200 microliters). 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 microliters of supernatant is removed and stored −20° C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 microliters of medium containing 0.5 microcuries 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 controls for the effects of TNF Delta and/or TNF Epsilon proteins.
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 Ficoll (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 microliters. 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 ul 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 (*), IFN□, TNF□, IL-10 and TR2. In addition to the control supernatants, recombinant human IL-2 (R & D Systems, Minneapolois, Minn.) at a final concentration of 10 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 uCi 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, IFN□, TNF; 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, which 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 ug/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 100 ul and incubated overnight at 4C. Plates are washed twice with PBS before use. PBMC are isolated by Ficoll (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 ul. 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 ul 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, IFN□, TNF□, 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 uCi 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 ug/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 500 ul and incubated overnight at 4C. Plates are washed twice with PBS before use. PBMC are isolated by Ficoll (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 ul. The supernatants (293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 300 ul are added to 600 ul 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, IFN□, 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 ul) are collected 2 days and 5 days after the beginning of the assay. ELISAs to test for IFN□ 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 1L-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 (nayve) 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 (Sigma, Saint Louis, Mo.) for three days. The cells are then washed in PBS and cultured in 10% FCSIRPMI 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% FCSIRPMI) forl6 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 ul. 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 ul 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, IFN□, TNF□, 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 uCi 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 test activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.
Example 34
Effect of Polypeptides 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 polypeptides 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).
Effect on the production of cvtokines. 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 polypeptides 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. Increase 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 polypeptides 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 degreesC. 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. Polypeptides, 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 Histopaque gradient (Sigma). 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 process (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 incubaed 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 monocytesimacrophages 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 the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide 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-w plate at 2-1×105 cell/well. Increasing concentrations of polypeptides 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 a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polypeptides, polynucleotides (e.g., gene therapy), agonists, z z and/or antagonists of the invention.
Example 35
Biological Effects of Polypeptides of the Invention
Astrocyte and Neuronal Assays
Recombinant polypeptides 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 a polypeptide 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 a polypeptide 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 Blue (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 CytoFluor 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 polypeptides 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 polypeptides 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 polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue 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 polypeptides 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, polypeptides 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 a polypeptide 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 dopminergic 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 a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.
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
The Effect of Polypeptides 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. A polypeptide having the amino acid sequence of SEQ ID NO:Y, 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 polypeptide of the invention may proliferate 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 (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 37
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-sulfopheny 1)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). 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 38
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 39
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 um (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) HUIVEC 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 40
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 KI 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 41
Effect of Polypepides 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-esteradiol (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 42
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 qual (Coturnix cotumix) are incubated at 37.8° C. and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old qual 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. Thermanox 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 43
Angiogenesis Assay Using a Matripel 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 (Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C. and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid “plug” of Matrigel is removed and examined for the presence of new blood vessels. Matrigel is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.
When thawed at 4 degree C. the Matrigel material is a liquid. The Matrigel 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 Matrigel 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 Matrigel 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). Matrigel 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 44
Rescue of Ischeinia 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 arteries 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 (Takeshitaet 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 45
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 46
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 47
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 48
Ischenic 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 49
Rat Corneal Wound Healing Model
This animal model shows the effect of a polypeptide of the invention on neovascularization. The experimental protocol includes:
Making a 1–1.5 mm long incision from the center of cornea into the stromal layer. Inserting a spatula below the lip of the incision facing the outer corner of the eye. Making a pocket (its base is 1–1.5 mm form the edge of the eye). Positioning a pellet, containing 50 ng–5 ug of a polypeptide of the invention, within the pocket.
Treatment with a polypeptide 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 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 50
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
Diabetic db+/db+Mouse Model
To demonstrate that a polypeptide 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 C57BLUKsJ (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 Avertin (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.
A polypeptide 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 a polypeptide 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 can serve as a positive tissue control and human brain tissue can be used as a negative tissue control. Each specimen includes 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); Wahlet 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 impaired 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 a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide 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/kgirat 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 polypeptide 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.
Four 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 a polypeptide 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 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 51
Lymphadema Animal Model
The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of a polypeptide 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 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 and 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 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 then 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), both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into 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 −80EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.
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 52
Suppression of TNF alpha-induced adhesion molecule expression by a Polypeptide 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 a polypeptide 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 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul 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. Do not allow the wells to dry. Add 10 μl of diluted primary antibody 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 X3 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 X3 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>10−1.50.5 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 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 53
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 a 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 pt 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 l of prepared cytokines, 50 μl SID (supernatants at 1:2 dilution=501) 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 t. 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 μlI 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 OmniFilter assemblies consisting of one OmniFilter plate and one OmniFilter Tray. 60 μl Microscint 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 is 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 diagnosis and treatment 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 54
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 responsible for stimulating stem cell self-renewal has 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 are tested with appropriate negative controls in the presence and absence of SCF(5.0 ng/ml), where test factor supernates 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 gene product is found to be a stimulator of hematopoietic progenitors, polynucleotides and polypeptides corresponding to the gene may be 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 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 treatment and diagnosis 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 55
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 @ 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 37C until day 2.
On day 2, serial dilutions and templates of the polypeptide of interest are designed which should 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). Then add 1/3 vol media containing controls or supernatants and incubate at 37C/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 4C until Day 6 (for IL6 ELISA). To the remaining 100 μl in the cell culture plate, aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10%1). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530 nm and emission at 590 nm using the CytoFluor. This yields the growth stimulation/inhibition data.
On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50–100 ul/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.
Wash plates with wash buffer and blot 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. Wash plates with wash buffer. Blot 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 gene product of interest 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 gene/gene product of interest. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, polypeptides of the gene product and polynucleotides of the gene may be used in wound healing and dermal regeneration, as well as the promotion of vasculargenesis, 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 of the gene product and polynucleotides of the gene may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular (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 of the gene product and polynucleotides of the gene 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 56
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 Phosphotase (1:5,000 dilution, refered 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>10−5. 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 57
Alamar Blue 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 Blue 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 370 C incubator for three days. After three days 10 ml of stock alamar blue (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 CytoFluor fluorescence reader. Direct output is recorded in relative fluorescence units.
Alamar blue 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 58
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 Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor is 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) is 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 JC 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.
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.
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. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties. Additionally, the specificatons and sequence listings of U.S. Provisional Patent Application Serial No. 60/239,899, filed Oct. 13, 2000; U.S. patent application Ser. No. 09/227,357, filed Jan. 8, 1999; International Patent Application Serial Nos: PCT/US98/13684, filed Jul. 7, 1998; U.S. Provisional Patent Applications Serial Nos. 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, and 60/051,928, filed Jul. 8, 1997; U.S. Provisional Patent Applications Serial Nos. 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, and 60/055,954, filed Aug. 18, 1997; and U.S. Provisional Patent Applications Serial Nos. 60/058,785, 60/058,664, 60/058,660, and 60/058,661, filed Sep. 12, 1997 are hereby incorporated by reference in their entirety.






















TABLE 6





Res
Position
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV






























Met
1
.
.
B
.
.
.
.
0.09
0.54
.
*
.
−0.40
0.49


Gly
2
.
.
B
.
.
.
.
0.59
0.51
.
.
.
−0.40
0.67


Phe
3
.
.
B
.
.
.
.
0.98
0.09
.
.
.
0.05
1.02


Pro
4
.
.
B
.
.
.
.
1.16
0.06
.
.
.
0.05
1.78


Gln
5
.
.
.
.
T
.
.
1.20
−0.13
.
.
F
1.45
2.79


Arg
6
.
.
B
.
.
.
.
0.99
−0.13
.
.
F
1.30
3.19


Gln
7
.
.
B
.
.
T
.
1.03
−0.23
.
.
F
1.75
1.70


Pro
8
.
.
.
.
.
T
C
1.39
−0.27
.
.
F
2.20
1.32


Gly
9
.
.
.
.
T
T
.
0.79
−0.24
.
.
F
2.50
0.66


Leu
10
.
.
.
.
.
T
C
−0.02
0.44
.
.
F
1.15
0.32


Ser
11
.
.
B
.
.
.
.
−0.94
0.73
.
.
F
0.50
0.17


Gly
12
.
A
B
.
.
.
.
−1.76
0.99
.
.
.
−0.10
0.14


Leu
13
.
A
B
.
.
.
.
−2.40
1.24
.
.
.
−0.35
0.14


Leu
14
.
A
B
.
.
.
.
−2.34
1.20
.
.
.
−0.60
0.08


Leu
15
.
A
B
.
.
.
.
−2.12
1.73
.
.
.
−0.60
0.08


Leu
16
.
A
B
.
.
.
.
−2.63
1.80
.
.
.
−0.60
0.10


Val
17
.
A
B
.
.
.
.
−2.88
1.80
.
.
.
−0.60
0.10


Trp
18
.
A
B
.
.
.
.
−2.36
1.61
.
.
.
−0.60
0.12


Ala
19
.
A
B
.
.
.
.
−1.76
1.84
.
.
.
−0.60
0.16


Leu
20
.
A
B
.
.
.
.
−1.76
1.59
.
.
.
−0.60
0.33


Ala
21
.
A
.
.
T
.
.
−1.16
1.63
.
.
.
−0.20
0.26


Trp
22
.
A
.
.
.
.
C
−0.97
1.14
.
.
.
−0.40
0.40


Pro
23
.
A
.
.
.
.
C
−1.28
1.21
.
.
.
−0.40
0.26


Leu
24
.
.
.
.
.
.
C
−0.99
1.14
.
.
.
−0.20
0.25


Pro
25
.
.
.
.
T
.
.
−0.99
1.03
.
.
.
0.00
0.32


Cys
26
.
A
.
.
T
.
.
−0.40
0.80
.
*
.
−0.20
0.17


Met
27
.
A
B
.
.
.
.
−0.92
0.37
.
.
.
−0.30
0.36


Ser
28
.
A
B
.
.
.
.
−1.60
0.37
.
.
.
−0.30
0.19


Leu
29
.
A
B
.
.
.
.
−1.00
0.63
.
.
.
−0.60
0.25


Glu
30
.
A
B
.
.
.
.
−1.03
0.49
.
.
.
−0.60
0.39


Leu
31
.
A
B
.
.
.
.
−0.68
0.63
.
.
.
−0.60
0.46


Ile
32
.
A
B
.
.
.
.
−0.29
0.73
.
.
.
−0.60
0.80


Pro
33
.
A
.
.
T
.
.
0.01
0.47
*
*
.
−0.20
0.72


Tyr
34
.
.
.
B
T
.
.
−0.07
0.87
*
*
.
−0.05
1.50


Thr
35
.
.
B
B
.
.
.
−0.38
0.87
*
*
F
−0.30
1.50


Pro
36
.
.
B
B
.
.
.
−0.16
0.67
.
*
F
−0.30
1.40


Gln
37
.
.
B
B
.
.
.
0.44
0.74
.
*
F
−0.45
0.91


Ile
38
.
.
B
B
.
.
.
0.66
0.90
.
.
.
−0.60
0.66


Thr
39
.
.
B
B
.
.
.
0.09
0.41
.
*
.
−0.60
0.71


Ala
40
.
.
B
B
.
.
.
0.40
0.67
.
*
.
−0.60
0.34


Trp
41
.
.
B
B
.
.
.
0.27
0.27
.
*
.
−0.30
0.84


Asp
42
.
.
B
B
.
.
.
0.31
0.01
.
*
.
−0.30
0.57


Leu
43
.
A
.
.
.
.
C
0.34
−0.47
.
*
.
0.65
1.14


Glu
44
.
A
.
.
T
.
.
0.34
−0.33
.
*
F
0.85
0.80


Gly
45
.
A
.
B
T
.
.
0.34
−0.76
.
*
F
1.15
0.69


Lys
46
.
A
.
B
T
.
.
0.32
−0.26
.
*
F
0.85
0.85


Val
47
.
A
B
B
.
.
.
0.01
−0.46
.
*
F
0.45
0.71


Thr
48
.
A
B
B
.
.
.
0.12
0.03
.
*
F
0.00
1.03


Ala
49
.
.
B
B
.
.
.
−0.18
0.39
.
*
F
−0.15
0.45


Thr
50
.
.
B
B
.
.
.
−0.64
0.77
.
*
F
−0.45
0.81


Thr
51
.
.
B
B
.
.
.
−0.69
0.81
.
*
.
−0.60
0.46


Phe
52
.
.
B
B
.
.
.
0.17
0.33
.
.
.
−0.03
0.79


Ser
53
.
.
B
B
.
.
.
0.27
0.23
.
.
.
0.24
0.95


Leu
54
.
.
.
.
T
.
.
0.97
0.17
.
.
F
1.41
1.02


Glu
55
.
.
.
.
T
.
.
0.61
−0.31
*
.
F
2.28
2.30


Gln
56
.
.
.
.
.
T
C
0.07
−0.53
*
.
F
2.70
0.92


Pro
57
.
.
.
.
T
T
.
−0.04
−0.27
*
*
F
2.33
0.83


Arg
58
.
.
.
.
T
T
.
0.26
−0.27
*
*
F
2.06
0.39


Cys
59
.
.
B
.
.
T
.
0.72
−0.27
*
*
.
1.24
0.38


Val
60
.
.
B
.
.
.
.
−0.09
−0.24
*
*
.
0.77
0.24


Leu
61
.
.
B
.
.
.
.
−0.68
0.01
*
*
.
−0.10
0.10


Asp
62
.
.
B
.
.
.
.
−0.81
0.51
*
*
.
−0.40
0.19


Gly
63
.
.
B
.
.
.
.
−1.78
0.37
*
*
.
−0.10
0.26


Leu
64
.
.
B
B
.
.
.
−1.70
0.37
*
.
.
−0.30
0.23


Ala
65
.
.
B
B
.
.
.
−1.14
0.19
*
.
.
−0.30
0.14


Gly
66
.
.
B
B
.
.
.
−0.64
0.57
*
.
.
−0.60
0.19


Val
67
.
.
B
B
.
.
.
−1.53
0.63
*
.
.
−0.60
0.33


Ala
68
.
.
B
B
.
.
.
−1.48
0.63
.
.
.
−0.60
0.23


Ser
69
.
.
B
B
.
.
.
−1.48
1.04
.
.
.
−0.60
0.25


Thr
70
.
.
B
B
.
.
.
−1.74
1.30
.
.
.
−0.60
0.27


Ile
71
.
.
B
B
.
.
.
−2.26
1.30
.
.
.
−0.60
0.20


Trp
72
.
.
B
B
.
.
.
−1.99
1.44
.
.
.
−0.60
0.11


Leu
73
.
.
B
B
.
.
.
−2.10
1.56
.
.
.
−0.60
0.08


Val
74
.
.
B
B
.
.
.
−2.10
1.86
.
.
.
−0.60
0.10


Val
75
.
.
B
B
.
.
.
−1.79
1.56
.
.
.
−0.60
0.12


Ala
76
.
.
B
B
.
.
.
−1.49
1.04
.
.
.
−0.60
0.24


Phe
77
.
.
B
.
.
T
.
−1.50
0.86
*
.
.
0.14
0.32


Ser
78
.
.
.
.
.
T
C
−0.58
0.60
*
.
.
0.68
0.59


Asn
79
.
.
.
.
.
T
C
0.28
−0.04
*
.
F
2.22
1.13


Ala
80
.
.
.
.
.
T
C
0.43
−0.54
*
.
F
2.86
2.19


Ser
81
.
.
.
.
T
T
.
1.02
−0.54
*
.
F
3.40
1.41


Arg
82
.
.
.
.
T
T
.
1.72
−0.53
*
.
F
3.06
1.52


Asp
83
.
.
.
.
T
T
.
1.81
−0.53
*
.
F
2.72
2.42


Phe
84
.
.
.
.
T
T
.
1.81
−0.60
*
.
F
2.38
2.80


Gln
85
.
.
.
.
.
.
C
2.09
−0.59
*
*
F
1.94
2.47


Asn
86
.
.
.
.
.
T
C
2.50
−0.10
*
*
F
1.80
2.14


Pro
87
.
.
.
.
.
T
C
1.80
−0.10
*
*
F
2.10
4.83


Gln
88
.
.
.
.
.
T
C
1.80
−0.39
.
*
F
2.40
2.82


Thr
89
.
.
.
.
.
T
C
1.61
−0.79
.
*
F
3.00
3.04


Arg
90
.
A
B
.
.
.
.
1.40
−0.50
.
*
F
1.80
1.38


Ala
91
.
A
B
.
.
.
.
0.81
−0.50
.
*
F
1.50
1.23


Glu
92
.
A
B
.
.
.
.
0.32
−0.40
.
*
.
0.90
0.86


Ile
93
.
A
B
.
.
.
.
0.11
−0.10
*
*
.
0.60
0.38


Pro
94
.
A
B
.
.
.
.
0.53
0.33
*
*
.
−0.30
0.58


Ala
95
.
A
B
.
.
.
.
−0.39
−0.17
*
*
.
0.30
0.66


Phe
96
.
A
B
.
.
.
.
−0.61
0.51
*
*
.
−0.60
0.77


Pro
97
.
A
B
.
.
.
.
−0.92
0.51
*
*
.
−0.60
0.41


Arg
98
.
A
B
.
.
.
.
−0.03
0.57
*
*
.
−0.60
0.59


Leu
99
.
A
B
.
.
.
.
−0.17
0.07
*
*
F
0.00
1.18


Leu
100
.
A
.
.
.
.
C
0.39
−0.29
*
*
F
0.65
0.76


Thr
101
.
A
.
.
.
.
C
0.84
−0.21
*
*
F
0.65
0.52


Glu
102
.
.
.
.
T
.
.
0.46
0.54
*
*
F
0.15
1.00


Gly
103
.
.
.
.
T
.
.
0.03
0.47
*
*
.
0.15
1.20


His
104
.
.
B
.
.
.
.
0.03
0.27
.
.
.
0.05
1.20


Tyr
105
.
.
B
.
.
.
.
0.63
0.47
.
.
.
−0.40
0.57


Met
106
.
.
B
.
.
.
.
0.13
0.90
.
.
.
−0.40
0.89


Thr
107
.
.
B
.
.
.
.
−0.17
1.16
.
*
.
−0.40
0.54


Leu
108
.
.
B
.
.
.
.
−0.63
1.04
.
*
.
−0.40
0.46


Pro
109
.
.
B
.
.
.
.
−0.60
0.97
.
*
.
−0.40
0.38


Leu
110
.
A
B
.
.
.
.
−0.36
0.36
.
*
.
−0.30
0.44


Ser
111
.
A
B
.
.
.
.
−0.57
0.27
.
*
.
−0.30
0.93


Leu
112
.
A
B
.
.
.
.
−0.47
0.27
*
*
.
−0.30
0.50


Asp
113
.
A
.
.
T
.
.
−0.32
0.27
*
*
F
0.25
0.93


Gln
114
.
A
B
.
.
.
.
−0.11
0.16
.
.
.
−0.30
0.37


Leu
115
.
.
B
.
.
T
.
0.70
0.17
.
.
.
0.10
0.78


Pro
116
.
.
B
.
.
T
.
0.79
−0.51
.
.
.
1.00
0.78


Cys
117
.
.
B
.
.
T
.
1.01
−0.09
.
.
.
0.70
0.70


Gln
118
.
.
B
.
.
T
.
0.67
0.01
.
.
F
0.56
0.86


Asp
119
.
.
B
.
.
T
.
0.32
−0.24
.
.
F
1.47
0.55


Pro
120
.
.
.
.
T
T
.
0.79
−0.24
*
*
F
2.33
1.01


Ala
121
.
.
.
.
T
T
.
1.11
−0.39
*
.
F
2.49
0.58


Gly
122
.
.
.
.
T
T
.
1.78
−0.79
*
.
F
3.10
0.68


Gly
123
.
.
.
.
T
.
.
0.92
−0.79
*
.
F
2.59
0.73


Gly
124
.
.
.
.
T
T
.
0.71
−0.57
*
.
F
2.48
0.54


Arg
125
.
.
B
.
.
T
.
0.11
−0.64
*
.
F
1.77
0.84


Asp
126
.
.
B
.
.
T
.
−0.11
−0.39
*
*
F
1.16
0.70


Val
127
.
.
B
.
.
T
.
0.34
−0.13
.
*
F
0.85
0.59


Pro
128
.
.
B
.
.
.
.
−0.17
−0.56
.
*
.
0.80
0.59


Leu
129
.
.
B
B
.
.
.
−0.17
0.09
.
*
.
−0.30
0.26


Leu
130
.
.
B
B
.
.
.
−0.28
0.51
*
*
.
−0.33
0.35


Arg
131
.
.
B
B
.
.
.
−0.28
0.27
*
.
.
0.24
0.36


Val
132
.
.
B
B
.
.
.
0.37
−0.16
*
.
.
1.11
0.73


Gly
133
.
.
.
B
T
.
.
0.23
−0.41
*
*
F
2.08
1.37


Asn
134
.
.
.
.
T
.
.
0.38
−0.67
*
*
F
2.70
0.69


Asp
135
.
.
.
.
.
T
C
0.38
−0.10
*
*
F
2.13
0.50


Pro
136
.
.
.
.
T
T
.
−0.32
−0.06
*
*
F
2.06
0.42


Gly
137
.
.
.
.
T
T
.
0.53
0.01
*
.
.
1.04
0.26


Cys
138
.
.
B
.
.
T
.
0.07
−0.39
*
.
.
0.97
0.26


Leu
139
.
A
B
.
.
.
.
−0.74
0.30
*
.
.
−0.30
0.14


Ala
140
.
A
B
.
.
.
.
−0.74
0.56
*
.
.
−0.60
0.12


Asp
141
.
A
B
.
.
.
.
−0.74
0.53
*
.
.
−0.60
0.38


Leu
142
.
A
B
.
.
.
.
−0.61
0.39
*
.
.
−0.30
0.70


Leu
143
.
A
B
.
.
.
.
−0.19
0.13
*
.
.
−0.15
1.08


Gln
144
.
A
B
.
.
.
.
−0.04
0.39
*
.
F
0.00
1.01


Pro
145
.
.
B
.
.
T
.
0.54
0.96
.
.
F
−0.05
0.66


Pro
146
.
.
.
.
T
T
.
0.24
0.67
.
.
F
0.50
1.28


Tyr
147
.
.
.
.
T
T
.
0.84
0.37
.
.
.
0.50
0.99


Cys
148
.
.
.
.
T
T
.
0.84
0.40
.
.
.
0.20
0.99


Asn
149
.
.
B
.
.
.
.
0.63
0.66
.
.
F
−0.25
0.53


Ser
150
.
.
B
.
.
T
.
0.54
0.66
.
.
F
−0.05
0.52


Pro
151
.
.
B
.
.
T
.
0.54
0.29
.
.
F
0.40
1.31


Leu
152
.
.
.
.
.
T
C
0.44
0.14
.
.
F
0.60
1.26


Pro
153
.
.
.
.
.
T
C
0.90
0.17
.
.
F
0.45
0.93


Ser
154
.
.
.
.
.
T
C
0.66
0.21
.
*
F
0.61
0.93


Pro
155
.
.
.
.
.
T
C
1.07
0.54
.
*
F
0.62
1.76


Gly
156
.
.
.
.
.
T
C
0.42
−0.14
.
*
F
1.68
2.23


Pro
157
.
.
B
.
.
T
.
1.28
0.07
.
*
F
1.04
1.24


Tyr
158
.
.
B
.
.
.
.
0.79
−0.31
.
*
F
1.60
1.60


Arg
159
.
A
B
.
.
.
.
0.28
0.04
.
*
.
0.49
1.40


Val
160
.
A
B
.
.
.
.
−0.32
0.30
.
*
.
0.18
0.75


Lys
161
.
A
B
.
.
.
.
−0.58
0.56
.
*
.
−0.28
0.39


Phe
162
.
A
B
.
.
.
.
−0.37
0.41
*
*
.
−0.44
0.20


Leu
163
.
A
B
.
.
.
.
−0.71
0.41
.
*
.
−0.60
0.45


Leu
164
.
A
B
.
.
.
.
−0.71
0.27
.
*
.
0.04
0.23


Met
165
.
A
B
.
.
.
.
−0.20
0.27
*
*
.
0.38
0.51


Asp
166
.
.
.
.
T
T
.
−0.54
−0.09
*
*
.
2.12
0.61


Ala
167
.
.
.
.
T
T
.
−0.06
−0.39
*
*
F
2.61
0.99


Arg
168
.
.
.
.
T
T
.
0.76
−0.64
*
*
F
3.40
1.55


Gly
169
.
.
.
.
.
T
C
0.98
−0.86
.
*
F
2.86
1.61


Ser
170
.
.
.
.
.
T
C
1.58
−0.36
.
*
F
2.22
1.61


Pro
171
.
.
.
.
.
T
C
1.27
−0.86
*
*
F
2.18
1.42


Gln
172
.
.
.
.
.
T
C
1.97
−0.37
*
*
F
1.54
2.08


Ala
173
.
.
B
.
.
T
.
1.57
−0.80
*
*
F
1.64
3.04


Glu
174
.
.
B
.
.
.
.
1.61
−0.27
*
*
F
1.48
2.06


Thr
175
.
.
B
.
.
.
.
1.91
−0.31
*
*
F
1.82
1.60


Arg
176
.
.
.
.
T
.
.
1.91
−0.71
*
*
F
2.86
2.64


Trp
177
.
.
.
.
T
T
.
1.02
−0.79
*
*
F
3.40
2.36


Ser
178
.
.
.
.
.
T
C
1.02
−0.10
*
*
F
2.56
1.15


Asp
179
.
.
.
.
.
T
C
0.21
−0.09
*
*
F
2.07
0.59


Pro
180
.
.
.
.
.
T
C
0.49
0.60
*
*
.
0.68
0.46


Ile
181
.
.
B
.
.
.
.
0.38
0.19
.
*
.
0.24
0.47


Ala
182
.
.
B
.
.
.
.
0.32
0.20
.
.
.
0.18
0.49


Leu
183
.
.
B
.
.
.
.
0.67
0.63
.
.
.
0.16
0.31


His
184
.
.
B
.
.
T
.
0.37
0.20
.
.
.
0.94
0.89


Gln
185
.
.
.
.
T
T
.
0.37
−0.10
.
.
F
2.52
1.18


Gly
186
.
.
.
.
T
T
.
0.67
−0.17
.
.
F
2.80
2.22


Lys
187
.
.
.
.
.
T
C
0.96
−0.36
*
*
F
2.32
1.64


Ser
188
.
.
.
.
.
T
C
0.88
−0.47
.
*
F
2.04
1.27


Pro
189
.
.
.
.
.
T
C
0.91
−0.19
.
*
F
1.61
0.90


Ala
190
.
.
B
.
.
T
.
0.60
−0.61
.
*
F
1.43
0.75


Ser
191
.
.
B
.
.
T
.
0.66
−0.13
.
*
F
0.85
0.81


Ile
192
.
.
B
.
.
.
.
0.40
0.40
.
*
F
−0.25
0.55


Asp
193
.
.
B
.
.
.
.
0.36
0.40
*
*
F
−0.25
0.84


Thr
194
.
.
B
.
.
.
.
0.68
0.33
.
.
F
0.05
0.62


Trp
195
.
.
B
.
.
T
.
1.38
−0.06
.
.
F
1.34
1.74


Pro
196
.
.
.
.
.
T
C
1.38
−0.74
.
.
F
2.18
2.04


Gly
197
.
.
.
.
T
T
.
1.92
−0.36
.
.
F
2.42
1.90


Arg
198
.
.
.
.
T
T
.
1.58
−0.41
.
.
F
2.76
1.78


Arg
199
.
.
.
.
T
T
.
1.29
−0.90
*
.
F
3.40
1.14


Ser
200
.
.
.
.
T
T
.
0.69
−0.71
*
.
F
3.06
1.14


Gly
201
.
.
.
.
T
T
.
0.04
−0.46
*
.
F
2.27
0.41


Gly
202
.
.
B
.
.
T
.
−0.50
0.19
*
.
F
0.93
0.15


Met
203
.
.
B
B
.
.
.
−0.92
0.87
.
.
.
−0.26
0.08


Ile
204
.
.
B
B
.
.
.
−1.33
0.97
*
*
.
−0.60
0.12


Val
205
.
.
B
B
.
.
.
−1.92
0.93
.
.
.
−0.60
0.16


Ile
206
.
.
B
B
.
.
.
−2.39
1.19
.
.
.
−0.60
0.11


Thr
207
.
.
B
B
.
.
.
−2.34
1.26
*
.
.
−0.60
0.13


Ser
208
.
.
B
B
.
.
.
−2.04
0.96
*
.
.
−0.60
0.24


Ile
209
.
.
B
B
.
.
.
−1.97
0.70
*
.
.
−0.60
0.46


Leu
210
.
A
B
B
.
.
.
−1.70
0.70
*
.
.
−0.60
0.26


Ser
211
.
A
B
.
.
.
.
−1.11
0.71
*
.
.
−0.60
0.20


Ser
212
.
A
.
.
.
.
C
−1.19
0.71
*
.
.
−0.40
0.38


Leu
213
.
A
B
.
.
.
.
−1.70
0.46
*
.
.
−0.60
0.59


Ala
214
.
A
B
.
.
.
.
−1.62
0.46
*
.
.
−0.60
0.36


Ser
215
.
A
B
.
.
.
.
−1.62
0.76
*
.
.
−0.60
0.22


Xxx
216
.
A
B
.
.
.
.
−1.91
1.06
*
.
.
−0.60
0.22


Leu
217
.
A
B
.
.
.
.
−2.31
0.87
.
.
.
−0.60
0.22


Leu
218
.
A
B
.
.
.
.
−2.31
1.16
.
.
.
−0.60
0.14


Leu
219
.
A
B
.
.
.
.
−2.31
1.46
.
.
.
−0.60
0.09


Ala
220
.
A
B
.
.
.
.
−2.60
1.53
.
.
.
−0.60
0.11


Phe
221
.
A
B
.
.
.
.
−2.51
1.34
.
.
.
−0.60
0.14


Leu
222
.
A
B
.
.
.
.
−2.01
1.04
.
.
.
−0.60
0.23


Ala
223
.
A
B
.
.
.
.
−1.59
0.84
*
.
.
−0.60
0.32


Ala
224
.
A
B
.
.
.
.
−0.67
0.77
*
*
.
−0.32
0.48


Ser
225
.
.
B
.
.
T
.
−0.78
−0.01
.
*
.
1.41
1.13


Thr
226
.
.
.
.
.
T
C
−0.38
0.09
*
*
F
1.29
0.97


Xxx
227
.
.
.
.
T
T
.
0.13
−0.03
*
.
F
2.52
1.29


Arg
228
.
.
.
.
T
T
.
−0.09
−0.14
*
*
F
2.80
1.29


Phe
229
.
.
.
.
T
.
.
0.21
0.16
*
.
F
1.57
0.74


Ser
230
.
.
.
.
.
T
C
0.22
0.59
*
*
.
0.84
0.61


Ser
231
.
.
.
.
.
T
C
0.32
1.07
*
*
.
0.56
0.33


Leu
232
.
.
.
.
.
T
C
0.71
1.50
.
*
.
0.28
0.58


Trp
233
.
.
.
.
.
T
C
0.60
0.71
.
*
.
0.00
0.75


Trp
234
.
A
.
.
.
.
C
0.71
0.33
.
.
.
−0.10
0.97


Pro
235
.
A
.
.
.
.
C
0.80
0.44
.
.
F
−0.10
1.19


Glu
236
.
A
.
.
.
.
C
1.10
0.19
.
.
F
0.20
1.75


Glu
237
.
A
.
.
.
.
C
1.91
−0.73
*
.
F
1.10
2.88


Ala
238
.
A
.
.
.
.
C
1.39
−1.24
*
*
F
1.10
3.22


Pro
239
.
A
.
.
.
.
C
1.79
−0.99
*
*
F
1.10
1.53


Glu
240
.
A
.
.
T
.
.
1.11
−0.99
*
*
F
1.30
1.73


Gln
241
A
.
.
B
.
.
.
0.77
−0.30
*
*
F
0.60
1.20


Leu
242
A
.
.
B
.
.
.
0.47
−0.37
.
*
.
0.30
0.77


Arg
243
.
.
B
B
.
.
.
0.36
−0.41
*
*
.
0.30
0.60


Ile
244
.
.
B
B
.
.
.
−0.03
0.37
*
*
.
−0.30
0.30


Gly
245
.
.
B
B
.
.
.
−0.38
0.59
*
*
.
−0.60
0.36


Ser
246
.
.
B
B
.
.
.
−0.33
0.33
*
*
.
−0.30
0.18


Phe
247
.
.
B
B
.
.
.
0.59
0.33
*
*
.
−0.05
0.52


Met
248
.
.
.
B
.
.
C
0.23
−0.36
*
.
.
1.15
1.02


Gly
249
.
.
.
.
T
T
.
0.52
−0.03
*
.
.
2.00
1.19


Lys
250
.
.
.
.
T
T
.
0.56
0.20
*
.
.
1.65
1.36


Arg
251
.
.
.
.
T
T
.
0.82
−0.10
.
.
.
2.50
1.99


Tyr
252
.
.
B
.
.
T
.
1.49
−0.21
*
.
.
1.85
2.73


Met
253
.
.
B
B
.
.
.
1.20
−0.14
*
.
.
1.20
1.86


Thr
254
.
.
B
B
.
.
.
1.33
0.54
*
.
.
−0.10
0.67


His
255
.
.
B
B
.
.
.
1.08
0.97
*
.
.
−0.35
0.66


His
256
.
.
.
B
.
.
C
0.67
0.64
*
.
.
−0.01
1.03


Ile
257
.
.
.
B
.
.
C
0.91
0.41
.
.
.
0.08
0.95


Pro
258
.
.
.
.
.
T
C
0.92
−0.07
.
.
F
1.92
1.21


Pro
259
.
.
.
.
.
T
C
0.64
−0.07
.
.
F
2.01
0.90


Ser
260
.
.
.
.
.
T
C
0.37
−0.07
.
.
F
2.40
1.30


Glu
261
.
.
B
.
.
T
.
−0.41
−0.27
.
.
F
1.96
1.21


Ala
262
.
.
.
.
T
.
.
0.27
−0.01
.
.
.
1.62
0.65


Ala
263
.
.
B
.
.
.
.
−0.38
−0.01
.
.
.
0.98
0.75


Thr
264
.
.
B
.
.
.
.
−0.56
0.24
.
.
.
0.14
0.32


Leu
265
.
.
B
.
.
.
.
−0.92
0.67
.
*
.
−0.40
0.40


Pro
266
.
.
B
.
.
.
.
−0.92
0.74
.
*
.
−0.40
0.21


Val
267
.
.
B
.
.
.
.
−0.54
0.24
.
*
.
−0.10
0.26


Xxx
268
.
.
B
.
.
.
.
−0.30
0.19
.
*
.
−0.10
0.48


Cys
269
.
.
B
.
.
.
.
−0.80
−0.07
.
*
.
0.67
0.31


Glu
270
.
.
B
.
.
T
.
0.01
0.19
.
*
F
0.59
0.34


Pro
271
.
.
B
.
.
T
.
0.01
−0.46
.
*
F
1.36
0.45


Gly
272
.
.
.
.
T
T
.
0.06
−0.46
.
*
F
2.08
1.29


Leu
273
.
.
B
.
.
T
.
0.23
−0.34
*
.
F
1.70
0.62


Asp
274
.
.
B
.
.
.
.
0.60
0.09
*
.
F
0.73
0.62


Pro
275
.
.
B
.
.
.
.
−0.21
0.04
*
.
F
0.56
0.83


Leu
276
.
.
B
.
.
T
.
−0.30
0.30
*
.
F
0.59
0.83


Pro
277
.
.
B
.
.
T
.
−0.17
0.00
*
.
F
0.42
0.67


Ser
278
.
.
.
.
T
T
.
0.26
0.43
*
.
F
0.35
0.67


Leu
279
.
.
B
.
.
T
.
−0.13
0.43
*
.
F
0.10
1.04


Ser
280
.
.
B
.
.
.
.
−0.31
0.17
.
.
.
−0.10
0.86


Pro
281
.
.
B
.
.
.
.
0.11
0.17
.
.
.
−0.10
0.82









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. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.

## Claims

1. An isolated protein comprising amino acid residues 2 to 111 of SEQ ID NO:164.

2. The An isolated protein comprising amino acid residues 1 to 111 of SEQ ID NO:164.

3. The protein of claim 2 which further comprises a heterologous polypeptide sequence.

4. A composition comprising the protein of claim 2 and a pharmaceutically acceptable carrier.

5. An isolated protein produced by the method comprising:
(a) expressing the protein of claim 2 by a cell; and
(b) recovering said protein.

6. An isolated protein comprising the amino acid sequence of the complete polypeptide encoded by the HHTLF25 cDNA contained in ATCC Deposit No. 209125, excepting the N-terminal methionine.

7. An isolated protein comprising the amino acid sequence of the complete polypeptide encoded by the HHTLF25 cDNA contained in ATCC Deposit No. 209125.

8. The protein of claim 7 which further comprises a heterologous polypeptide sequence.

9. A composition comprising the protein of claim 7 and a pharmaceutically acceptable carrier.

10. An isolated protein produced by the method comprising:
(a) expressing the protein of claim 7 by a cell; and
(b) recovering said protein.