# Patent

## Title
Compositions and methods for the therapy and diagnosis of influenza

## Abstract

The present invention provides compositions, vaccines, and methods for diagnosing, treating, and preventing influenza infection using a combination of antibodies raised against the influenza hemagglutinin and the matrix 2 ectodomain polypeptides.

## Background
RELATED APPLICATIONS
This application claims the benefit of provisional application U.S. Ser. No. 61/442,733, filed Feb. 14, 2011, the contents of which are herein incorporated by reference in their entirety.


INCORPORATION OF SEQUENCE LISTING
The contents of the text file named “37418-518001US_ST25.txt,” which was created on Jan. 6, 2012 and is 910 KB in size, are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates generally to prevention, diagnosis, therapy and monitoring of influenza infection. The invention is more specifically related to compositions containing a combination of human antibodies raised against either the influenza hemagglutinin or matrix 2 protein. Such compositions are useful in pharmaceutical compositions for the prevention and treatment of influenza, and for the diagnosis and monitoring of influenza infection.
BACKGROUND OF THE INVENTION
Influenza virus infects 5-20% of the population and results in 30,000-50,000 deaths each year in the U.S. Disease caused by influenza A viral infections is typified by its cyclical nature. Antigenic drift and shift allow for different A strains to emerge every year. Added to that, the threat of highly pathogenic strains entering into the general population has stressed the need for novel therapies for flu infections.
SUMMARY OF THE INVENTION
The invention provides diagnostic, prophylactic, and therapeutic compositions including a human antibody raised against the Influenza hemagglutinin protein and a human monoclonal antibody raised against the Influenza M2 protein. Moreover, the invention provides diagnostic, prophylactic, and therapeutic compositions including an isolated human antibody raised against an epitope of the Influenza hemagglutinin protein and an isolated human monoclonal antibody raised against an epitope of the Influenza M2 protein. Furthermore, these compositions are pharmaceutical compositions that include a pharmaceutical carrier. These compositions address a long-felt need in the art for pharmaceutical compositions that both strongly neutralizes Influenza virus infection and recognizes constant regions within proteins common to all Influenza strains.
Specifically, the invention provides a composition including: (a) an isolated human antibody that specifically binds to an epitope of the hemagglutinin (HA) glycoprotein of an influenza virus; and (b) an isolated human monoclonal antibody that specifically binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus. In certain embodiments of this composition, the isolated human monoclonal antibody that specifically binds an epitope of the M2e polypeptide is TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, or 3242_P05. Moreover, the isolated human antibody that specifically binds an epitope of the HA glycoprotein is optionally TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN 558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), TCN-504 (3251_K17), SC06-141, SC06-255, SC06-257, SC06-260, SC06-261, SC06-262, SC06-268, SC06-272, SC06-296, SC06-301, SC06-307, SC06-310, SC06-314, SC06-323, SC06-325, SC06-327, SC06-328, SC06-329, SC06-331, SC06-332, SC06-334, SC06-336, SC06-339, SC06-342, SC06-343, SC06-344, CR6141, CR6255, CR6257, CR6260, CR6261, CR6262, CR6268, CR6272, CR6296, CR6301, CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329, CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, CR6344, 2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98.
The epitope of the HA glycoprotein is optionally GVTNKVNSIIDK (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO: 283), GVTNKENSIIDK (SEQ ID NO: 202), GVTNKVNRIIDK (SEQ ID NO: 201), GITNKVNSVIEK (SEQ ID NO: 281), GITNKENSVIEK (SEQ ID NO: 257), GITNKVNSIIDK (SEQ ID NO: 225), and KITSKVNNIVDK (SEQ ID NO: 216). The influenza hemaglutinin (HA) glycoprotein includes an HA1 and HA2 subunit. Exemplary epitopes of the HA glycoprotein include the HA1 subunit, HA2 subunit, or both the HA1 and HA2 subunits. Alternatively, or in addition, the epitope of the M2e polypeptide is a discontinuous epitope. For example, the epitope of the M2e polypeptide includes the amino acid at positions 2, 5, and 6 of MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1) or the amino acid at positions 2, 5, and 6 of SLLTEV (SEQ ID NO: 42).
The invention further provides a composition including: (a) an isolated human anti-HA antibody, or an antigen-binding fragment thereof, including a heavy chain variable region (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain each contain three complementarity determining regions 1 to 3 (CDR1-3), and wherein each CDR includes the following amino acid sequences: VH CDR1: SEQ ID NOs: 247, 571, 586, 597, 603, 609, 615, 627, 633, 637, 643, 649, 658, 664, 670, 303, 251, 242, or 222; VH CDR2: SEQ ID NOs: 248, 572, 587, 592, 598, 604, 610, 616, 628, 634, 638, 644, 650, 655, 659, 665, 671, 306, 249, 307, or 221; VH CDR3: SEQ ID NOs: 568, 573, 588, 593, 599, 605, 611, 617, 629, 635, 639, 645, 651, 656, 660, 666, 672, 725, 246, 290, or 220; VL CDR1: SEQ ID NOs: 569, 574, 577, 580, 583, 589, 594, 612, 618, 621, 624, 640, 646, 652, 661, 667, 285, 289, 245, 224, or 219; VL CDR2: SEQ ID NOs: 570, 575, 578, 581, 584, 590, 595, 601, 607, 613, 619, 622, 625, 631, 653, 662, 668, 305, 223, or 231;VL CDR3: SEQ ID NOs: 289, 576, 579, 582, 585, 591, 596, 602, 608, 614, 620, 623, 626, 632, 636, 642, 648, 654, 657, 663, 669, 308, 250, 227, or 280; and (b) an isolated anti-matrix 2 ectodomain (M2e) antibody, or antigen-binding fragment thereof, including a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain each contain three complementarity determining regions 1 to 3 (CDR1-3), and wherein each CDR includes the following amino acid sequences: VH CDR1: SEQ ID NOs: 72, 103, 179, 187, 203, 211, 228, 252, 260, 268, 284, 293, or 301; VH CDR2: SEQ ID NOs: 74, 105, 180, 188, 204, 212, 229, 237, 253, 261, 269, 285, or 294; VH CDR3 SEQ ID NOs: 76, 107, 181, 189, 197, 205, 213, 230, 238, 254, 262, 270, 286, or 295; VL CDR1: SEQ ID NOs: 59, 92, 184, 192, 208, 192, 233, 241, 265, or 273; VL CDR2: SEQ ID NOs: 61, 94, 185, 193, 209, 217, 226, 234, 258, 274, or 282; and VL CDR3: SEQ ID NOs: 63, 96, 186, 194, 210, 218, 243, 259, 267, 275, 291, or 300.
Alternatively, or in addition, the invention provides a composition including: (a) an isolated human anti-HA antibody, or an antigen-binding fragment thereof, including a heavy chain variable region (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain each contain three complementarity determining regions 1 to 3 (CDR1-3), and wherein each CDR includes the following amino acid sequences: VH CDR1: SEQ ID NOs: 247, 571, 586, 597, 603, 609, 615, 627, 633, 637, 643, 649, 658, 664, 670, 303, 251, 242, or 222; VH CDR2: SEQ ID NOs: 248, 572, 587, 592, 598, 604, 610, 616, 628, 634, 638, 644, 650, 655, 659, 665, 671, 306, 249, 307, or 221; VH CDR3: SEQ ID NOs: 568, 573, 588, 593, 599, 605, 611, 617, 629, 635, 639, 645, 651, 656, 660, 666, 672, 725, 246, 290, or 220; VL CDR1: SEQ ID NOs: 569, 574, 577, 580, 583, 589, 594, 612, 618, 621, 624, 640, 646, 652, 661, 667, 285, 289, 245, 224, or 219; VL CDR2: SEQ ID NOs: 570, 575, 578, 581, 584, 590, 595, 601, 607, 613, 619, 622, 625, 631, 653, 662, 668, 305, 223, or 231; VL CDR3: SEQ ID NOs: 289, 576, 579, 582, 585, 591, 596, 602, 608, 614, 620, 623, 626, 632, 636, 642, 648, 654, 657, 663, 669, 308, 250, 227, or 280; and (b) an isolated anti-matrix 2 ectodomain (M2e) antibody, or antigen-binding fragment thereof, including a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain each contain three complementarity determining regions 1 to 3 (CDR1-3), and wherein each CDR includes the following amino acid sequences: VH CDR1: SEQ ID NOs: 109, 112, 182, 190, 206, 214, 239, 255, 263, 271, 287, 296, or 304; VH CDR2: SEQ ID NOs: 110, 113, 183, 191, 207, 215, 232, 240, 256, 264, 272, 288, or 297; VH CDR3 SEQ ID NOs: 76, 107, 181, 189, 197, 205, 213, 230, 238, 254, 262, 270, 286, or 295; VL CDR1: SEQ ID NOs: 59, 92, 184, 192, 208, 192, 233, 241, 265, or 273; VL CDR2: SEQ ID NOs: 61, 94, 185, 193, 209, 217, 226, 234, 258, 274, or 282; and VL CDR3: SEQ ID NOs: 63, 96, 186, 194, 210, 218, 243, 259, 267, 275, 291, or 300.
The invention provides a composition including: (a) an isolated human anti-HA antibody, or an antigen-binding fragment thereof, including a heavy chain variable region (VH) domain, wherein the VH domain includes the following amino acid sequences: SEQ ID NOs 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 199, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483, 489, 495, 501, 507, 513, 519, 525, 531, 537, 543, 550, 556, or 562, and a light chain variable (VL) domain, wherein the VL domain includes the following amino acid sequences: SEQ ID NOs 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492, 498, 504, 510, 516, 522, 528, 534, 540, 547, 553, 559, or 565; and (b) an isolated anti-matrix 2 ectodomain (M2e) antibody, or antigen-binding fragment thereof, including a heavy chain variable (VH) domain, wherein the VH domain includes the following amino acid sequences: SEQ ID NOs 44, 277, 276, 50, 236, 235, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, or 176, and a light chain variable (VL) domain, wherein the VL domain includes the following amino acid sequences: SEQ ID NOs 46, 292, 52, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, or 178.
Furthermore, the invention provides a multivalent vaccine composition including any of the compositions described herein containing an isolated human anti-HA antibody, or an antigen-binding fragment thereof and an isolated anti-matrix 2 ectodomain (M2e) antibody, or antigen-binding fragment thereof. Alternatively, the multivalent vaccine includes antibodies that bind to the epitopes to which the antibodies of the invention bind. Exemplary antibodies of the invention include, but are not limited to, TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, 3242_P05, TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN 558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), TCN-504 (3251_K17), SC06-141, SC06-255, SC06-257, SC06-260, SC06-261, SC06-262, SC06-268, SC06-272, SC06-296, SC06-301, SC06-307, SC06-310, SC06-314, SC06-323, SC06-325, SC06-327, SC06-328, SC06-329, SC06-331, SC06-332, SC06-334, SC06-336, SC06-339, SC06-342, SC06-343, SC06-344, CR6141, CR6255, CR6257, CR6260, CR6261, CR6262, CR6268, CR6272, CR6296, CR6301, CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329, CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, CR6344, D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98. For example, the multivalent vaccine may include one or more of the following epitopes: GVTNKVNSIIDK (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO: 283), GVTNKENSIIDK (SEQ ID NO: 202), GVTNKVNRIIDK (SEQ ID NO: 201), GITNKVNSVIEK (SEQ ID NO: 281), GITNKENSVIEK (SEQ ID NO: 257), GITNKVNSIIDK (SEQ ID NO: 225), KITSKVNNIVDK (SEQ ID NO: 216), MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1), and MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1) provided in its native conformation.
The multivalent vaccine also includes a composition including: (a) a human antibody that specifically binds to an epitope of the hemagglutinin (HA) glycoprotein of an influenza virus; and (b) a human monoclonal antibody that specifically binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus.
The invention provides a pharmaceutical composition including any one of the compositions described herein. Moreover, the pharmaceutical composition includes a pharmaceutical carrier.
The invention provides a method for stimulating an immune response in a subject, including administering to the subject the pharmaceutical composition described herein. The pharmaceutical composition may administered prior to or after exposure of the subject to an Influenza virus.
The invention also provides a method for the treatment of an influenza virus infection in a subject in need thereof, including administering to the subject the pharmaceutical composition described herein. The subjection may have been exposed to an influenza virus. Alternatively, or in addition, the subject has not been diagnosed with an influenza infection. The pharmaceutical composition may administered prior to or after exposure of the subject to an Influenza virus. Preferably, the pharmaceutical composition is administered at a dose sufficient to promote viral clearance or eliminate influenza infected cells.
The invention further provides a method for the prevention of an influenza virus infection in a subject in need thereof, including administering to the subject a vaccine composition described herein, prior to exposure of the subject to an influenza virus. In certain embodiments of this method, the subject is at risk of contracting an influenza infection. The pharmaceutical composition may administered prior to or after exposure of the subject to an Influenza virus. Preferably, the pharmaceutical composition is administered at a dose sufficient to promote viral clearance or eliminate influenza infected cells.
The treatment and prevention methods provided by the invention further include administering an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor. Exemplary anti-viral drugs include, but are not limited to, a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor, or an M2 ion channel inhibitor. In certain aspects of these methods, the M2 ion channel inhibitor is amantadine or rimantadine. In other aspects of these methods, the neuraminidase inhibitor is zanamivir or oseltamivir phosphate. The antiviral drug may administered prior to or after exposure of the subject to an Influenza virus.
The treatment and prevention methods provided by the invention further include administering a second anti-Influenza A antibody. The second antibody is optionally an antibody described herein. The second antibody may administered prior to or after exposure of the subject to an Influenza virus.
The invention provides a method for determining the presence of an Influenza virus infection in a subject, including the steps of: (a) contacting a biological sample obtained from the subject with any one of the antibodies or pharmaceutical compositions described herein; (b) detecting an amount of the antibody that binds to the biological sample; and (c) comparing the amount of antibody that binds to the biological sample to a control value, and therefrom determining the presence of the Influenza virus in the subject. Optionally, the control value is determined by contacting a control sample obtained from the subject with any one of the antibodies or pharmaceutical compositions described herein and detecting an amount of the antibody that binds to the control sample.
The invention also provides a diagnostic kit including any one of the antibodies, compositions, or pharmaceutical compositions described herein.
The invention further provides a prophylactic kit including a vaccine composition described herein. Preferably, the vaccine is a multivalent vaccine. The term “multivalent vaccine” describes a single vaccine that elicits an immune response either to more than one infectious agent, e.g. the influenza HA glycoprotein and the influenza M2e polypeptide, or to several different epitopes of a molecule, e.g. HA epitopes shown in SEQ ID NOs 198, 283, 202, 201, 281, 257, 225, and 216. Alternatively, or in addition, the term multivalent vaccine is meant to describe the administration of a combination of human antibodies raised against more than one infectious agent, e.g. the influenza HA glycoprotein and the influenza M2e polypeptide.
Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the binding of three antibodies of the present invention and control hu14C2 antibody to 293-HEK cells transfected with an M2 expression construct or control vector, in the presence or absence of free M2 peptide.
FIGS. 2A and B are graphs showing human monoclonal antibody binding to influenza A/Puerto Rico/8/32.
FIG. 3A is a chart showing amino acid sequences of extracellular domains of M2 variants (SEQ ID NOS 1-3, 679 & 5-40, respectively, in order of appearance).
FIGS. 3B and C are bar charts showing binding of human monoclonal anti-influenza antibody binding to M2 variants shown in FIG. 3A.
FIGS. 4A and B are bar charts showing binding of human monoclonal anti-influenza antibody binding to M2 peptides subjected to alanine scanning mutagenesis.
FIG. 5 is a series of bar charts showing binding of MAbs 8i10 and 23K12 to M2 protein representing influenza strain A/HK/483/1997 sequence that was stably expressed in the CHO cell line DG44.
FIG. 6A is a chart showing cross reactivity binding of anti-M2 antibodies to variant M2 peptides (SEQ ID NOS 680-704, respectively, in order of appearance).
FIG. 6B is a chart showing binding activity of M2 antibodies to truncated M2 peptides (SEQ ID NOS 680, 705-724 & 19, respectively, in order of appearance).
FIG. 7 is a graph showing survival of influenza infected mice treated with human anti-influenza monoclonal antibodies.
FIG. 8 is an illustration showing the anti-M2 antibodies bind a highly conserved region in the N-Terminus of M2e (SEQ ID NO: 19).
FIG. 9 is a graph showing anti-M2 rHMAb clones from crude supernatant bound to influenza on ELISA, whereas the control anti-M2e mAb 14C2 did not readily bind virus.
FIG. 10 is a series of photographs showing anti-M2 rHMAbs bound to cells infected with influenza. MDCK cells were or were not infected with influenza A/PR/8/32 and Ab binding from crude supernatant was tested 24 hours later. Data were gathered from the FMAT plate scanner.
FIG. 11 is a graph showing anti-M2 rHMAb clones from crude supernatant bound to cells transfected with the influenza subtypes H3N2, HK483, and VN1203 M2 proteins. Plasmids encoding full length M2 cDNAs corresponding to influenza strains H3N2, HK483, and VN1203, as well as a mock plasmid control, were transiently transfected into 293 cells. The 14C2, 8i10, 23K12, and 21B15 mABs were tested for binding to the transfectants, and were detected with an AF647-conjugated anti-human IgG secondary antibody. Shown are the mean fluorescence intensities of the specific mAB bound after FACS analysis.
FIGS. 12A-B are amino acid sequences of the variable regions of anti-M2e mAbs. Framework regions 1-4 (FR 1-4) and complementarity determining regions 1-3 (CDR 1-3) for VH and Vk are shown. FR, CDR, and gene names are defined using the nomenclature in the IMGT database (IMGT®, the International ImMunoGeneTics Information system® http://www.imgt.org). Grey boxes denote identity with the germline sequence which is shown in light blue boxes, hyphens denote gaps, and white boxes are amino acid replacement mutations from the germline.
FIG. 13 is a graph depicting the results of a competition binding analysis of a panel of anti-M2e mAbs with TCN-032 Fab. The indicated anti-M2e mAbs were used to bind to the stable CHO transfectant expressing M2 of A/Hong Kong/483/97 that had previously been treated with or without 10 μg/mL TCN-032 Fab fragment. The anti-M2e mAb bound to the cell surface was detected with goat anti-huIgG FcAlexafluor488 FACS and analyzed by flow cytometry. The results are derived from one experiment.
FIG. 14A is a graph depicting the ability of anti-M2e mAbs TCN-032 and TCN-031 to bind virus particles and virus-infected cells but not M2e-derived synthetic peptide. Purified influenza virus (A/Puerto Rico/8/34) was coated at 10 μg/ml on ELISA wells and binding of anti-M2e mAbs TCN-031, TCN-032, ch14C2, and the HCMV mAbs 2N9 was evaluated using HRP-labeled goat anti-human Fc. Results shown are representative of 3 experiments.
FIG. 14B is a graph depicting the ability of anti-M2e mAbs TCN-032 and TCN-031 to bind virus particles and virus-infected cells but not M2e-derived synthetic peptide. 23 mer synthetic peptide of M2 derived from A/Fort Worth/1/50 was coated at 1 μg/ml on ELISA wells and binding of mAbs TCN-031, TCN-032, ch14C2, and 2N9 were evaluated as in panel a. Results shown are representative of 3 experiments.
FIG. 14C is a graph depicting the ability of anti-M2e mAbs TCN-032 and TCN-031 to bind virus particles and virus-infected cells but not M2e-derived synthetic peptide. MDCK cells were infected with A/Puerto Rico/8/34 (PR8) and subsequently stained with mAbs TCN-031, TCN-032, ch14C2 and the HCMV mAb 5J12. Binding of antibodies was detected using Alexafluor 647-conjugated goat anti-Human IgG H&L antibody and quantified by flow cytometry. Results shown are representative of 3 experiments.
FIG. 14D is a series of photographs depicting HEK 293 cells stably transfected with the M2 ectodomain of A/Fort Worth/1/50 (D20) were stained with transient transfection supernatant containing mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FMAT for binding to M2 in the presence or absence of 5 ug/ml M2e peptide. Mock transfected cells are 293 cells stably transfected with vector alone. Results shown are representative of one experiment.
FIGS. 15A-D are graphs depicting the Therapeutic efficacy of anti-M2 mAbs TCN-031 and TCN-032 in mice. Mice (n=10) were infected by intranasal inoculation with 5×LD50 A/Vietnam/1203/04 (H5N1) (panels A-B) or (n=5) with 5×LD50 A/Puerto Rico 8/34 (H1N1) (panels C-D), followed by 3 intraperitoneal (ip) injections with mAbs at 24, 72, and 120 hours post-infection (a total of 3 mAb injections per mouse) and weighed daily for 14 days. Percentage survival is shown in a and c, whereas percent weight change of mice is shown in B and D. The results shown for the treatment study of mice infected with A/Vietnam/1203/04 (H5N1) are representative of 2 experiments.
FIG. 16 is a series of graphs depicting the viral titers in lung, liver, and brain of mice treated with anti-M2e mAbs TCN-031 and TCN-032 after challenge with H5N1 A/Vietnam/1203/04. BALB/C mice (n=19) were treated i.p. injection of a 400 μg/200 μL dose of TCN-031, TCN-032, control human mAb 2N9, control chimeric mAb ch14C2, PBS, or left untreated. Tissue viral titers were determined from 3 mice per group at 3 and 6 days post-infection in the lungs (as an indicator of local replication) and in liver and brain (as an indicator of the systemic spread which is characteristic of H5N1 infection).
FIG. 17 is a graph depicting the ability of TCN-031 and TCN-032 can potentiate cytolysis by NK cells. MDCK cells were infected with A/Solomon Island/3/2006 (H1N1) virus, and were treated with mAbs TCN-031, TCN-032, or the subclass-matched negative control mAb 2N9. The cells were then challenged with purified human NK cells, and the lactate dehydrogenase released as a result of cell lysis was measured through light absorbance. The results are representative of two separate experiments with two different normal human donors.
FIG. 18 is a graph depicting complement-dependent cytolysis (CDC) of M2-expressing cells bound with anti-M2 mAb. The stable transfectant expressing M2 of A/Hong Kong/483/97 and a mock control were treated with the indicated mAbs and subsequently challenged with human complement. Lysed cells were visualized by Propidium Iodide staining followed by FACS analysis. The data are representative of two experiments.
FIGS. 19A-C are graphs depicting binding of anti-M2e mAbs TCN-031 and TCN-032 to M2 mutants indicates the epitope is located in the highly conserved N-terminal of M2e. Mutants with alanine substituted at each position of the M2 ectodomain of A/Fort Worth/1/50 (D20) (A) or forty wild-type M2 mutants including A/Vietnam/1203/04 (VN) and A/Hong Kong/483/97 (HK) (B) were transiently transfected into 293 cells. The identity of each wild-type M2 mutant is listed in Table 6. Transfected cells were stained with mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FACS for binding to M2 at 24 hours post-transfection. mAbs TCN-031 and TCN-032 do not bind variants with amino acid substitutions at positions 1, 4, or 5 of M2e. (C) The deduced epitope for TCN-031 and TCN-032 occurs in a highly conserved region of M2e and is distinct from that found for ch14C2. Results shown for (A) and (B) are representative of 3 experiments.
FIG. 20 is a graph depicting mAbs TCN-031 and TCN-032 recognize the same region on M2e. The CHO transfectant stably expressing M2 for A/Hong Kong/483/97 as stained with 10 μg/mL TCN-031, TCN-032, or 2N9, followed by detection with Alexafluor647-labeled TCN-031 (TCN-031AF647) or TCN-032(TCN-032AF647) and analysis by flow cytometry. The results are representative of three experiments.
FIG. 21 is a graph depicting anti-M2e mAbs TCN-031 and TCN-032 bind cells that have been infected with H1N1 A/California/4/09. MDCK cells were infected with Influenza A strain H1N1 A/Memphis/14/96, H1N1 A/California/4/09, or mock infected. Twenty four hours post-infection cells were stained with mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FACS for binding to M2. Results shown are for one experiment.



DETAILED DESCRIPTION
Influenza viruses consist of three types, A, B and C. Influenza A viruses infect a wide variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In animals most influenza A viruses cause mild localized infections of the respiratory and intestinal tract. However, highly pathogenic influenza A strains such as H5N1 exist that cause systemic infections in poultry in which mortality may reach 100%. Animals infected with influenza A often act as a reservoir for the influenza viruses and certain subtypes have been shown to cross the species barrier to humans.
Influenza A viruses can be classified into subtypes based on allelic variations in antigenic regions of two genes that encode surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) which are required for viral attachment and cellular release. Other major viral proteins include the nucleoprotein, the nucleocapsid structural protein, membrane proteins (M1 and M2), polymerases (PA, PB and PB2) and non-structural proteins (NS1 and NS2). Currently, sixteen subtypes of HA (H1-H16) and nine NA (N1-N9) antigenic variants are known in influenza A virus. Previously, only three subtypes have been known to circulate in humans (H1N1, H1N2, and H3N2).
However, in recent years, the pathogenic H5N1 subtype of avian influenza A has been reported to cross the species barrier and infect humans as documented in Hong Kong in 1997 and 2003, leading to the death of several patients. In humans, the avian influenza virus infects cells of the respiratory tract as well as the intestinal tract, liver, spleen, kidneys and other organs. Symptoms of avian influenza infection include fever, respiratory difficulties including shortness of breath and cough, lymphopenia, diarrhea and difficulties regulating blood sugar levels. In contrast to seasonal influenza, the group most at risk is healthy adults, which make up the bulk of the population. Due to the high pathogenicity of certain avian influenza A subtypes, particularly H5N1, and their demonstrated ability to cross over to infect humans, there is a significant economic and public health risk associated with these viral strains, including a real epidemic and pandemic threat. The scale of the threat is illustrated by the 1918 influenza pandemic which killed over 50 million people.
Currently, no effective vaccines for H5N1 infection are available, so passive immunotherapy with immunoglobulins may be an alternative strategy. Use of passive immunization during the 1918 pandemic reportedly halved the death rate. In view of their therapeutic benefit in humans, there is thus a need for antibodies, preferably human antibodies, capable of neutralizing influenza infection, including H5N1.
The invention provides compositions including human antibodies raised against two influenza proteins, hemagglutinin (HA) and matrix 2 ectodomain (M2e), and shows that these compositions can be used in medicine, in particular for diagnosis, prevention and treatment of influenza infections, including H5N1.
HuM2e Antibodies
The present invention provides fully human monoclonal antibodies specifically directed against M2e. Optionally, the antibody is isolated form a B-cell from a human donor. Exemplary monoclonal antibodies include TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, and 3242_P05.described herein. Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, and 3242_P05. The antibodies respectively referred to herein are huM2e antibodies. The huM2e antibody has one or more of the following characteristics: a) binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus; b) binds to influenza A infected cells; or c) binds to influenza A virus.
The epitope that huM2e antibody binds to is a non-linear epitope of a M2 polypeptide. Preferably, the epitope includes the amino terminal region of the M2e polypeptide. More preferably the epitope wholly or partially includes the amino acid sequence SLLTEV (SEQ ID NO: 42). Most preferably, the epitope includes the amino acid at position 2, 5 and 6 of the M2e polypeptide when numbered in accordance with SEQ ID NO: 1. The amino acid at position 2 is a serine; at position 5 is a threonine; and at position 6 is a glutamic acid.
A huM2e antibody contains a heavy chain variable having the amino acid sequence of SEQ ID NOs: 44, 277, 276, 50, 236, 235, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, or 176 and a light chain variable having the amino acid sequence of SEQ ID NOs: 46, 52, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, or 178. Preferably, the three heavy chain CDRs include an amino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NOs: 72, 74, 76, 103, 105, 107, 179, 180, 181, 187, 188, 189, 197, 203, 204, 205, 21, 212, 213, 228, 229, 230, 237, 238, 252, 253, 254, 260, 261, 262, 268, 269, 270, 284, 285, 286, 293, 294, 295, and 301 (as determined by the Kabat method) or SEQ ID NOs: 109, 110, 76, 112, 113, 107, 182, 183, 181, 190, 191, 189, 197, 206, 207, 205, 214, 215, 213, 232, 230, 239, 240, 238, 255, 256, 254, 263, 264, 262, 271, 272, 270, 287, 288, 286, 296, 297, 295, and 304 (as determined by the Chothia method) and a light chain with three CDRs that include an amino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NOs: 59, 60, 61, 92, 94, 96, 184, 185, 186, 192, 193, 194, 208, 209, 210, 217, 218, 226, 223, 234, 241, 243, 258, 259, 265, 267, 273, 274, 275, 282, 291, and 300 (as determined by the Kabat method) or SEQ ID NOs: 59, 60, 61, 92, 94, 96, 184, 185, 186, 192, 193, 194, 208, 209, 210, 217, 218, 226, 223, 234, 241, 243, 258, 259, 265, 267, 273, 274, 275, 282, 291, and 300 (as determined by the Chothia method). The antibody binds M2e.
The heavy chain of a M2e antibody is derived from a germ line V (variable) gene such as, for example, the IgHV4 or the IgHV3 germline gene.
The M2e antibodies of the invention include a variable heavy chain (VH) region encoded by a human IgHV4 or the IgHV3 germline gene sequence. A IgHV4 germline gene sequence are shown, e.g., in Accession numbers L10088, M29812, M95114, X56360 and M95117. IgHV3 germline gene sequence are shown, e.g., in Accession numbers X92218, X70208, Z27504, M99679 and AB019437. The M2e antibodies of the invention include a VH region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgHV4 or the IgHV3 germline gene sequence. Preferably, the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgHV4 or the IgHV3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the IgHV4 or the IgHV3 germline gene sequence. The VH region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VH region encoded by the IgHV4 or the IgHV3 VH germline gene sequence. Preferably, the amino acid sequence of VH region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgHV4 or the IgHV3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by the IgHV4 or the IgHV3 germline gene sequence.
The M2e antibodies of the invention also include a variable light chain (VL) region encoded by a human IgKV1 germline gene sequence. A human IgKV1 VL germline gene sequence is shown, e.g., Accession numbers X59315, X59312, X59318, J00248, and Y14865. Alternatively, the M2e antibodies include a VL region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgKV1 germline gene sequence. Preferably, the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgKV1 germline gene sequence, and more preferably, at least 98%, 99% homologous to the IgKV1 germline gene sequence. The VL region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VL region encoded the IgKV1 germline gene sequence. Preferably, the amino acid sequence of VL region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgKV1 germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by e the IgKV1 germline gene sequence.
In another aspect the invention provides a composition including an huM2e antibody according to the invention. In various aspects the composition further includes an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor. The anti-viral drug is for example a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor. The M2 ion channel inhibitor is for example amantadine or rimantadine. The neuraminidase inhibitor for example zanamivir, or oseltamivir phosphate. In a further aspect the composition further includes a second anti-influenza A antibody.
In a further aspect the huM2e antibodies according to the invention are operably-linked to a therapeutic agent or a detectable label.
Additionally, the invention provides methods for stimulating an immune response, treating, preventing or alleviating a symptom of an influenza viral infection by administering an huM2e antibody to a subject
Optionally, the subject is further administered with a second agent such as, but not limited to, an influenza virus antibody, an anti-viral drug such as a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or a viral attachment inhibitor. The M2 ion channel inhibitor is, for example, amantadine or rimantadine. The neuraminidase inhibitor is, for example, zanamivir or oseltamivir phosphate. The subject is suffering from or is predisposed to developing an influenza virus infection, such as, for example, an autoimmune disease or an inflammatory disorder.
In another aspect, the invention provides methods of administering the huM2e antibody of the invention to a subject prior to, and/or after exposure to an influenza virus. For example, the huM2e antibody of the invention is used to treat or prevent rejection influenza infection. The huM2e antibody is administered at a dose sufficient to promote viral clearance or eliminate influenza A infected cells.
Also included in the invention is a method for determining the presence of an influenza virus infection in a patient, by contacting a biological sample obtained from the patient with a humM2e antibody; detecting an amount of the antibody that binds to the biological sample; and comparing the amount of antibody that binds to the biological sample to a control value.
The invention further provides a diagnostic kit comprising a huM2e antibody.
Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims.
The present invention provides fully human monoclonal antibodies specific against the extracellular domain of the matrix 2 (M2) polypeptide. The antibodies are respectively referred to herein as huM2e antibodies.
M2 is a 96 amino acid transmembrane protein present as a homotetramer on the surface of influenza virus and virally infected cells. M2 contains a 23 amino acid ectodomain (M2e) that is highly conserved across influenza A strains. Few amino acid changes have occurred since the 1918 pandemic strain thus M2e is an attractive target for influenza therapies. In prior studies, monoclonal antibodies specific to the M2 ectodomain (M2e) were derived upon immunizations with a peptide corresponding to the linear sequence of M2e. In contrast, the present invention provides a novel process whereby full-length M2 is expressed in cell lines, which allows for the identification of human antibodies that bound this cell-expressed M2e. The huM2e antibodies have been shown to bind conformational determinants on the M2-transfected cells, as well as native M2, either on influenza infected cells, or on the virus itself. The huM2e antibodies did not bind the linear M2e peptide, but they do bind several natural M2 variants, also expressed upon cDNA transfection into cell lines. Thus, this invention has allowed for the identification and production of human monoclonal antibodies that exhibit novel specificity for a very broad range of influenza A virus strains. These antibodies may be used diagnostically to identify influenza A infection and therapeutically to treat influenza A infection.
The huM2e antibodies of the invention have one or more of the following characteristics: the huM2e antibody binds a) to an epitope in the extracellular domain of the matrix 2 (M2) polypeptide of an influenza virus; b) binds to influenza A infected cells; and/or c) binds to influenza A virus (i.e., virons). The huM2e antibodies of the invention eliminate influenza infected cells through immune effector mechanisms, such as ADCC, and promote direct viral clearance by binding to influenza virons. The huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide. Preferably, the huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide wherein the N-terminal methionine residue is absent. Exemplary M2e sequences include those sequences listed on Table 1 below











TABLE 1





Type
Name
Subtype
M2E Sequence
SEQ ID NO







A
BREVIG MISSION.1.1918
H1N1
MSLLTEVETPTRNEWGCRCNDSSD
SEQ ID NO: 1


 


A
FORT MONMOUTH.1.1947
H1N1
MSLLTEVETPTKNEWECRCNDSSD
SEQ ID NO: 2


 


A
.SINGAPORE.02.2005
H3N2
MSLLTEVETPIRNEWECRCNDSSD
SEQ ID NO: 3


 


A
WISCONSIN.10.98
H1N1
MSLLTEVETPIRNGWECKCNDSSD
SEQ ID NO: 4


 


A
WISCONSIN.301.1976
H1N1
MSLLTEVETPIRSEWGCRCNDSSD
SEQ ID NO: 5


 


A
PANAMA.1.66
H2N2
MSFLPEVETPIRNEWGCRCNDSSD
SEQ ID NO: 6


 


A
NEW YORK.321.1999
H3N2
MSLLTEVETPIRNEWGCRCNDSSN
SEQ ID NO: 7


 


A
CARACAS.1.71
H3N2
MSLLTEVETPIRKEWGCRCNDSSD
SEQ ID NO: 8


 


A
TAIWAN.3.71
H3N2
MSFLTEVETPIRNEWGCRCNDSSD
SEQ ID NO: 9


 


A
WUHAN.359.95
H3N2
MSLPTEVETPIRSEWGCRCNDSSD
SEQ ID NO: 10


 


A
HONG KONG.1144.99
H3N2
MSLLPEVETPIRNEWGCRCNDSSD
SEQ ID NO: 11


 


A
HONG KONG.1180.99
H3N2
MSLLPEVETPIRNGWGCRCNDSSD
SEQ ID NO: 12


 


A
HONG KONG.1774.99
H3N2
MSLLTEVETPTRNGWECRCSGSSD
SEQ ID NO: 13


 


A
NEW YORK.217.02
H1N2
MSLLTEVETPIRNEWEYRCNDSSD
SEQ ID NO: 14


 


A
NEW YORK.300.2003
H1N2
MSLLTEVETPIRNEWEYRCSDSSD
SEQ ID NO: 15


 


A
SWINE.SPAIN.54008.2004
H3N2
MSLLTEVETPTRNGWECRYSDSSD
SEQ ID NO: 16


 


A
GUANGZHOU.333.99
H9N2
MSFLTEVETLTRNGWECRCSDSSD
SEQ ID NO: 17


 


A
HONG KONG.1073.99
H9N2
MSLLTEVETLTRNGWECKCRDSSD
SEQ ID NO: 18


 


A
HONG KONG.1.68
H3N2
MSLLTEVETPIRNEWGCRCNDSSD
SEQ ID NO: 19


 


A
SWINE.HONG
H3N2
MSLLTEVETPIRSEWGCRCNDSGD
SEQ ID NO: 20



KONG.126.1982





 


A
NEW YORK.703.1995
H3N2
MSLLTEVETPIRNEWECRCNGSSD
SEQ ID NO: 21


 


A
SWINE.QUEBEC.192.81
H1N1
MSLPTEVETPIRNEWGCRCNDSSD
SEQ ID NO: 22


 


A
PUERTO RICO.8.34
H1N1
MSLLTEVETPIRNEWGCRCNGSSD
SEQ ID NO: 23


 


A
HONG KONG.485.97
H5N1
MSLLTEVDTLTRNGWGCRCSDSSD
SEQ ID NO: 24


 


A
HONG KONG.542.97
H5N1
MSLLTEVETLTKNGWGCRCSDSSD
SEQ ID NO: 25


 


A
SILKY
H9N2
MSLLTEVETPTRNGWECKCSDSSD
SEQ ID NO: 26



CHICKEN.SHANTOU.1826.2004





 


A
CHICKEN.TAIWAN.0305.04
H6N1
MSLLTEVETHTRNGWECKCSDSSD
SEQ ID NO: 27


 


A
QUAIL.ARKANSAS.16309-7.94
H7N3NSA
MSLLTEVKTPTRNGWECKCSDSSD
SEQ ID NO: 28


 


A
HONG KONG.486.97
H5N1
MSLLTEVETLTRNGWGCRCSDSSD
SEQ ID NO: 29


 


A
CHICKEN.PENNSYLVANIA.13552-
H7N2NSB
MSLLTEVETPTRDGWECKCSDSSD
SEQ ID NO: 30



1.98





 


A
CHICKEN.HEILONGJIANG.48.01
H9N2
MSLLTEVETPTRNGWGCRCSDSSD
SEQ ID NO: 31


 


A
SWINE.KOREA.S5.2005
H1N2
MSLLTEVETPTRNGWECKCNDSSD
SEQ ID NO: 32


 


A
HONG KONG.1073.99
H9N2
MSLLTEVETLTRNGWECKCSDSSD
SEQ ID NO: 33


 


A
WISCONSIN.3523.88
H1N1
MSLLTEVETPIRNEWGCKCNDSSD
SEQ ID NO: 34


 


A
X-31 VACCINE STRAIN
H3N2
MSFLTEVETPIRNEWGCRCNGSSD
SEQ ID NO: 35


 


A
CHICKEN.ROSTOCK.8.1934
H7N1
MSLLTEVETPTRNGWECRCNDSSD
SEQ ID NO: 36


 


A
ENVIRONMENT.NEW
H7N2
MSLLTEVETPIRKGWECNCSDSSD
SEQ ID NO: 37



YORK.16326-1.2005





 


A
INDONESIA.560H.2006
H5N1
MSLLTEVETPTRNEWECRCSDSSD
SEQ ID NO: 38


 


A
CHICKEN.HONG
H9N2
MSLLTGVETHTRNGWGCKCSDSSD
SEQ ID NO: 39



KONG.SF1.03





 


A
CHICKEN.HONGKONG.YU427.03
H9N2
MSLLPEVETHTRNGWGCRCSDSSD
SEQ ID NO: 40









In one embodiment, the huM2e antibodies of the invention bind to a M2e that wholly or partially includes the amino acid residues from position 2 to position 7 of M2e when numbered in accordance with SEQ ID NO: 1. For example, the huM2e antibodies of the invention bind wholly or partially to the amino acid sequence SLLTEVET (SEQ ID NO: 41) Most preferably, the huM2e antibodies of the invention bind wholly or partially to the amino acid sequence SLLTEV (SEQ ID NO: 42). Preferably, the huM2e antibodies of the invention bind to non-linear epitope of the M2e protein. For example, the huM2e antibodies bind to an epitope comprising position 2, 5, and 6 of the M2e polypeptide when numbered in accordance to SEQ ID NO: 1 where the amino acid at a) position 2 is a serine; b) position 5 is a threonine; and c) position 6 is a glutamic acid. Exemplary huM2e monoclonal antibodies that bind to this epitope are the TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, and 3242_P05 antibodies described herein.
The TCN-032 (8I10) antibody includes a heavy chain variable region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in SEQ ID NO: 43, a short heavy chain variable region (SEQ ID NO: 277) encoded by the nucleic acid sequence shown below in SEQ ID NO: 278, a long heavy chain variable region (SEQ ID NO: 276) encoded by the nucleic acid sequence shown below in SEQ ID NO: 196, and a light chain variable region (SEQ ID NO: 46) encoded by the nucleic acid sequence shown in SEQ ID NO: 45.
The amino acids encompassing the CDRs as defined by Chothia, C. et al. (1989, Nature, 342: 877-883) are underlined and those defined by Kabat E. A. et al.(1991, Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Heath and Human Services.) are highlighted in bold in the sequences below.
The heavy chain CDRs of the TCN-032 (8I10) antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the TCN-032 (8I10) antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).
The heavy chain CDRs of the TCN-032 (8I10) antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 109), FIYYGGNTK (SEQ ID NO: 110) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the TCN-032 (8I10) antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).







TCN-032 (8I10) VH nucleotide sequence:


(SEQ ID NO: 43)


CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT


 


GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT


 


ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT


 


CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT


 


CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT


 


AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC


 


CGTCTCG


 


TCN-032 (8I10) VH amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 44)


Q V Q L Q E S G P G L V K P S E T L S L T


 


C T V S G S S I S N Y Y W S W I R Q S P G


 


K G L E W I G F I Y Y G G N T K Y N P S L


 


K S R V T I S Q D T S K S Q V S L T M S S


 


V T A A E S A V Y F C A R A S C S G G Y C


 


I L D Y W G Q G T L V T V S


 


TCN-032 (8I10) VH short nucleotide sequence:


(SEQ ID NO: 278)


CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT


 


GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT


 


ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT


 


CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT


 


CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT


 


AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC


 


CGT


 


TCN-032 (8I10) VH short amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 277)


Q V Q L Q E S G P G L V K P S E T L S L T


 


C T V S G S S I S N Y Y W S W I R Q S P G


 


K G L E W I G F I Y Y G G N T K Y N P S L


 


K S R V T I S Q D T S K S Q V S L T M S S


 


V T A A E S A V Y F C A R A S C S G G Y C


 


I L D Y W G Q G T L V T


 


TCN-032 (8I10) VH long nucleotide sequence:


(SEQ ID NO: 196)


CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT


 


GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT


 


ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT


 


CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT


 


CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT


 


AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC


 


CGTCTCGAGC


 


TCN-032 (8I10) VH long amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 276)


Q V Q L Q E S G P G L V K P S E T L S L T


 


C T V S G S S I S N Y Y W S W I R Q S P G


 


K G L E W I G F I Y Y G G N T K Y N P S L


 


K S R V T I S Q D T S K S Q V S L T M S S


 


V T A A E S A V Y F C A R A S C S G G Y C


 


I L D Y W G Q G T L V T V S S


 


TCN-032 (8I10) VL nucleotide sequence:


(SEQ ID NO: 45)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAA


 


ATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCT


 


GCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTG


 


CAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGA


 


GGGACCAGGGTGGAGATCAAAC


 


TCN-032 (8I10) VL amino acid sequence:


Kabat Bold, Chothia underlined


(SEQ ID NO: 46)


D I Q M T Q S P S S L S A S V G D R V T I


 


T C R A S Q N I Y K Y L N W Y Q Q R P G K


 


A P K G L I S A A S G L Q S G V P S R F S


 


G S G S G T D F T L T I T S L Q P E D F A


 


T Y Y C Q Q S Y S P P L T F G G G T R V E


 


I K






The 21B15 antibody includes a heavy chain variable region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in SEQ ID NO: 47, a short heavy chain variable region (SEQ ID NO: 277) encoded by the nucleic acid sequence shown below in SEQ ID NO: 278, a long heavy chain variable region (SEQ ID NO: 276) encoded by the nucleic acid sequence shown below in SEQ ID NO: 196, and a light chain variable region (SEQ ID NO: 46) encoded by the nucleic acid sequence shown in SEQ ID NO: 48.
The amino acids encompassing the CDRs as defined by Chothia et al. 1989, are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 21B15 antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the 21B15 antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).
The heavy chain CDRs of the 21 B 15 antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 109), FIYYGGNTK (SEQ ID NO: 110) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the 21B15 antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).







21B15 VH nucleotide sequence:


(SEQ ID NO: 47)


CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT


 


GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT


 


ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT


 


CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT


 


CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT


 


AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC


 


CGTCTCG


 


21B15 VH amino acid sequence:


(SEQ ID NO: 44)


Kabat Bold, Chothia underlined


Q V Q L Q E S G P G L V K P S E T L S L T


 


C T V S G S S I S N Y Y W S W I R Q S P G


 


K G L E W I G F I Y Y G G N T K Y N P S L


 


K S R V T I S Q D T S K S Q V S L T M S S


 


V T A A E S A V Y F C A R A S C S G G Y C


 


I L D Y W G Q G T L V T V S


 


21B15 VH short nucleotide sequence:


(SEQ ID NO: 278)


CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT


 


GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT


 


ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT


 


CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT


 


CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT


 


AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC


 


CGT


 


21B15 VH short amino acid sequence:


(SEQ ID NO: 277)


Kabat Bold, Chothia underlined


Q V Q L Q E S G P G L V K P S E T L S L T


 


C T V S G S S I S N Y Y W S W I R Q S P G


 


K G L E W I G F I Y Y G G N T K Y N P S L


 


K S R V T I S Q D T S K S Q V S L T M S S


 


V T A A E S A V Y F C A R A S C S G G Y C


 


I L D Y W G Q G T L V T


 


21B15 VH long nucleotide sequence:


(SEQ ID NO: 196)


CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT


 


GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT


 


ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT


 


CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT


 


CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT


 


AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC


 


CGTCTCGAGC


 


21B15 VH long amino acid sequence:


(SEQ ID NO: 276)


Kabat Bold, Chothia underlined


Q V Q L Q E S G P G L V K P S E T L S L T


 


C T V S G S S I S N Y Y W S W I R Q S P G


 


K G L E W I G F I Y Y G G N T K Y N P S L


 


K S R V T I S Q D T S K S Q V S L T M S S


 


V T A A E S A V Y F C A R A S C S G G Y C


 


I L D Y W G Q G T L V T V S S


 


21B15 VL nucleotide sequence:


(SEQ ID NO: 48)


GACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAA


 


ATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCT


 


GCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTG


 


CAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGA


 


GGGACCAGGGTGGATATCAAAC


 


21B15 VL amino acid sequence:


(SEQ ID NO: 292)


Kabat Bold, Chothia underlined


D I Q V T Q S P S S L S A S V G D R V T I


 


T C R A S Q N I Y K Y L N W Y Q Q R P G K


 


A P K G L I S A A S G L Q S G V P S R F S


 


G S G S G T D F T L T I T S L Q P E D F A


 


T Y Y C Q Q S Y S P P L T F G G G T R V D


 


I K






The TCN-031 (23K12) antibody includes a heavy chain variable region (SEQ ID NO: 50) encoded by the nucleic acid sequence shown below in SEQ ID NO: 49, a short heavy chain variable region (SEQ ID NO: 236) encoded by the nucleic acid sequence shown below in SEQ ID NO: 244, a long heavy chain variable region (SEQ ID NO: 195) encoded by the nucleic acid sequence shown below in SEQ ID NO: 235, and a light chain variable region (SEQ ID NO: 52) encoded by the nucleic acid sequence shown in SEQ ID NO: 51.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the TCN-031 (23K12) antibody have the following sequences per Kabat definition: SNYMS (SEQ ID NO: 103), VIYSGGSTYYADSVK (SEQ ID NO: 105) and CLSRMRGYGLDV (SEQ ID NO: 107). The light chain CDRs of the TCN-031 (23K12) antibody have the following sequences per Kabat definition: RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA (SEQ ID NO: 96).
The heavy chain CDRs of the TCN-031 (23K12) antibody have the following sequences per Chothia definition: GFTVSSN (SEQ ID NO: 112), VIYSGGSTY (SEQ ID NO: 113) and CLSRMRGYGLDV (SEQ ID NO: 107). The light chain CDRs of the TCN-031 (23K12) antibody have the following sequences per Chothia definition: RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA (SEQ ID NO: 96).







TCN-031 (23K12) VH nucleotide sequence:


(SEQ ID NO: 49)


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC


 


CCTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACA


 


TGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT


 


ATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATT


 


CTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACA


 


GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGC


 


AGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCAC


 


CGTCTCG


 


TCN-031 (23K12) VH amino acid sequence:


(SEQ ID NO: 50)


Kabat Bold, Chothia underlined


E V Q L V E S G G G L V Q P G G S L R I S


 


C A A S G F T V S S N Y M S W V R Q A P G


 


K G L E W V S V I Y S G G S T Y Y A D S V


 


K G R F S F S R D N S K N T V F L Q M N S


 


L R A E D T A V Y Y C A R C L S R M R G Y


 


G L D V W G Q G T T V T V S


 


TCN-031 (23K12) VH short nucleotide sequence:


(SEQ ID NO: 244)


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC


 


CCTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACA


 


TGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT


 


ATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATT


 


CTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACA


 


GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGC


 


AGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCAC


 


CGT


 


TCN-031 (23K12) VH short amino acid sequence:


(SEQ ID NO: 236)


Kabat Bold, Chothia underlined


E V Q L V E S G G G L V Q P G G S L R I S


 


C A A S G F T V S S N Y M S W V R Q A P G


 


K G L E W V S V I Y S G G S T Y Y A D S V


 


K G R F S F S R D N S K N T V F L Q M N S


 


L R A E D T A V Y Y C A R C L S R M R G Y


 


G L D V W G Q G T T V T V S


 


TCN-031 (23K12) VH long nucleotide sequence:


(SEQ ID NO: 195)


GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC


 


CCTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACA


 


TGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT


 


ATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATT


 


CTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACA


 


GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGC


 


AGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCAC


 


CGTCTCGAGC


 


TCN-031 (23K12) VH long amino acid sequence:


(SEQ ID NO: 235)


Kabat Bold, Chothia underlined


E V Q L V E S G G G L V Q P G G S L R I S


 


C A A S G F T V S S N Y M S W V R Q A P G


 


K G L E W V S V I Y S G G S T Y Y A D S V


 


K G R F S F S R D N S K N T V F L Q M N S


 


L R A E D T A V Y Y C A R C L S R M R G Y


 


G L D V W G Q G T T V T V S S


 


TCN-031 (23K12) VL nucleotide sequence:


(SEQ ID NO: 51)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAA


 


ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCT


 


GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTG


 


CAACCTACTACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGG


 


ACCAAGCTGGAGATCAAA


 


TCN-031 (23K12) VL amino acid sequence:


(SEQ ID NO: 52)


Kabat Bold, Chothia underlined


D I Q M T Q S P S S L S A S V G D R V T I


 


T C R T S Q S I S S Y L N W Y Q Q K P G K


 


A P K L L I Y A A S S L Q S G V P S R F S


 


G S G S G T D F T L T I S G L Q P E D F A


 


T Y Y C Q Q S Y S M P A F G Q G T K L E I


 


K






The 3241_G23 antibody (also referred to herein as G23) includes a heavy chain variable region (SEQ ID NO: 116) encoded by the nucleic acid sequence shown below in SEQ ID NO: 115, and a light chain variable region (SEQ ID NO: 118) encoded by the nucleic acid sequence shown in SEQ ID NO: 117.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the G23 antibody have the following sequences per Kabat definition: GGGYSWN (SEQ ID NO: 179), FMFHSGSPRYNPTLKS (SEQ ID NO: 180) and VGQMDKYYAMDV (SEQ ID NO: 181). The light chain CDRs of the G23 antibody have the following sequences per Kabat definition: RASQSIGAYVN (SEQ ID NO: 184), GASNLQS (SEQ ID NO: 185) and QQTYSTPIT (SEQ ID NO: 186).
The heavy chain CDRs of the G23 antibody have the following sequences per Chothia definition: GGPVSGGG (SEQ ID NO: 182), FMFHSGSPR (SEQ ID NO: 183) and VGQMDKYYAMDV (SEQ ID NO: 181). The light chain CDRs of the G23 antibody have the following sequences per Chothia definition: RASQSIGAYVN (SEQ ID NO: 184), GASNLQS (SEQ ID NO: 185) and QQTYSTPIT (SEQ ID NO: 186).







3241_G23 VH nucleotide sequence


(SEQ ID NO: 115)


CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC


 


CCTGTCCCTCACTTGCACTGTCTCTGGTGGCCCCGTCAGCGGTGGTGGTT


 


ACTCCTGGAACTGGATCCGCCAACGCCCAGGACAGGGCCTGGAGTGGGTT


 


GGGTTCATGTTTCACAGTGGGAGTCCCCGCTACAATCCGACCCTCAAGAG


 


TCGAATTACCATCTCAGTCGACACGTCTAAGAACCTGGTCTCCCTGAAGC


 


TGAGCTCTGTGACGGCCGCGGACACGGCCGTGTATTTTTGTGCGCGAGTG


 


GGGCAGATGGACAAGTACTATGCCATGGACGTCTGGGGCCAAGGGACCAC


 


GGTCACCGTCTCGAGC


 


3241_G23 VH amino acid sequence


(SEQ ID NO: 116)


Kabat Bold, Chothia underlined


QVQLQQSGPGLVKPSQTLSLTCTVSGGPVSGGGYSWNWIRQRPGQGLEWV


 


GFMFHSGSPRYNPTLKSRITISVDTSKNLVSLKLSSVTAADTAVYFCARV


 


GQMDKYYAMDVWGQGTTVTVSS


 


3241_G23 VL nucleotide sequence


(SEQ ID NO: 117)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTTCCTCTGTCGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGGCGCCTATGTAA


 


ATTGGTATCAACAGAAAGCAGGGAAAGCCCCCCAGGTCCTGATCTTTGGT


 


GCTTCCAATTTACAAAGCGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGACTTTG


 


CAACTTACTTCTGTCAACAGACTTACAGTACCCCGATCACCTTCGGCCAA


 


GGGACACGACTGGAGATTAAACG


 


3241_G23 VL amino acid sequence


(SEQ ID NO: 118)


Kabat Bold, Chothia underlined


DIQMTQSPSSLSSSVGDRVTITCRASQSIGAYVNWYQQKAGKAPQVLIFG


 


ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTYSTPITFGQ


 


GTRLEIK






The 3244_I10 antibody (also referred to herein as I10) includes a heavy chain variable region (SEQ ID NO: 120) encoded by the nucleic acid sequence shown below in SEQ ID NO: 119, and a light chain variable region (SEQ ID NO: 122) encoded by the nucleic acid sequence shown in SEQ ID NO: 121.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the I10 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDAKFSGSYYVAS (SEQ ID NO: 189). The light chain CDRs of the I10 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
The heavy chain CDRs of the I10 antibody have the following sequences per Chothia definition: GGSITS (SEQ ID NO: 190), FFYNGGSTK (SEQ ID NO: 191) and HDAKFSGSYYVAS (SEQ ID NO: 189). The light chain CDRs of the I10 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).







3244_I10 VH nucleotide sequence


(SEQ ID NO: 119)


CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACAC


 


CCTGGCCCTCACTTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACT


 


GGAGCTGGATCCGGCAACCCCCAGGGAGGGGACTGGACTGGATCGGATTC


 


TTCTATAACGGCGGAAGCACCAAGTACAATCCCTCCCTCAAGAGTCGAGT


 


CACCATTTCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGACCT


 


CTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGCC


 


AAATTTAGTGGGAGCTACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGT


 


CACCGTCTCGAGC


 


3244_I10 VH amino acid sequence


(SEQ ID NO: 120)


QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGF


 


FYNGGSTKYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDA


 


KFSGSYYVASWGQGTRVTVSS


 


3244_I10 VL nucleotide sequence


(SEQ ID NO: 121)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA


 


ATTGGTATCAGCAGCAACCTGGGAAAGCCCCTAAGGTCCTCATTTTTGGT


 


GCAACCAACTTGCAAAGTGGGGTCCCATCTCGCTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG


 


CAACTTACTACTGTCAACAGAGTTACAATACCCCCCTCATTTTTGGCCAG


 


GGGACCAAGCTGGAGATCAAACG


 


3244_I10 VL amino acid sequence


(SEQ ID NO: 122)


DIQMTQSPSSLSASVGDRVTISCRASQSISTYLNWYQQQPGKAPKVLIFG


 


ATNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLIFGQ


 


GTKLEIK






The 3243_J07 antibody (also referred to herein as J07) includes a heavy chain variable region (SEQ ID NO: 124) encoded by the nucleic acid sequence shown below in SEQ ID NO: 123, and a light chain variable region (SEQ ID NO: 126) encoded by the nucleic acid sequence shown in SEQ ID NO: 125.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the J07 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDVKFSGSYYVAS (SEQ ID NO: 197). The light chain CDRs of the J07 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
The heavy chain CDRs of the J07 antibody have the following sequences per Chothia definition: GGSITS (SEQ ID NO: 190), FFYNGGSTK (SEQ ID NO: 191) and HDVKFSGSYYVAS (SEQ ID NO: 197). The light chain CDRs of the J07 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).







3243_J07 VH nucleotide sequence


(SEQ ID NO: 123)


CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACAC


 


CCTGGCCCTCACTTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACT


 


GGAGCTGGATCCGGCAACCCCCAGGGAGGGGACTGGACTGGATCGGATTC


 


TTCTATAACGGCGGGAGCACCAAGTACAATCCCTCCCTCAAGAGTCGAGT


 


CACCATATCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGACCT


 


CTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGTC


 


AAATTTAGTGGGAGCTACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGT


 


CACCGTCTCGAGC


 


3243_J07 VH amino acid sequence


(SEQ ID NO: 124)


QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGF


 


FYNGGSTKYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDV


 


KFSGSYYVASWGQGTRVTVSS


 


3243_J07 VL nucleotide sequence


(SEQ ID NO: 125)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA


 


ATTGGTATCAGCAGCAACCTGGGAAAGCCCCTAAGGTCCTGATCTCTGGT


 


GCAACCAACTTGCAAAGTGGGGTCCCATCTCGCTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG


 


CAACTTACTACTGTCAACAGAGTTACAATACCCCCCTCATTTTTGGCCAG


 


GGGACCAAGCTGGAGATCAAACG


 


3243_J07 VL amino acid sequence


(SEQ ID NO: 126)


DIQMTQSPSSLSASVGDRVTISCRASQSISTYLNWYQQQPGKAPKVLISG


 


ATNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLIFGQ


 


GTKLEIK






The 3259_J21 antibody (also referred to herein as J21) includes a heavy chain variable region (SEQ ID NO: 128) encoded by the nucleic acid sequence shown below in SEQ ID NO: 127, and a light chain variable region (SEQ ID NO: 130) encoded by the nucleic acid sequence shown in SEQ ID NO: 129.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the J21 antibody have the following sequences per Kabat definition: SYNWI (SEQ ID NO: 203), HIYDYGRTFYNSSLQS (SEQ ID NO: 204) and PLGILHYYAMDL (SEQ ID NO: 205). The light chain CDRs of the J21 antibody have the following sequences per Kabat definition: RASQSIDKFLN (SEQ ID NO: 208), GASNLHS (SEQ ID NO: 209) and QQSFSVPA (SEQ ID NO: 210).
The heavy chain CDRs of the J21 antibody have the following sequences per Chothia definition: GGSISS (SEQ ID NO: 206), HIYDYGRTF (SEQ ID NO: 207) and PLGILHYYAMDL (SEQ ID NO: 205). The light chain CDRs of the J21 antibody have the following sequences per Chothia definition: RASQSIDKFLN (SEQ ID NO: 208), GASNLHS (SEQ ID NO: 209) and QQSFSVPA (SEQ ID NO: 210).







3259_J21 VH nucleotide sequence


(SEQ ID NO: 127)


CAGGTGCAGCTGCAGGAGTCGGGCCCACGAGTGGTGAGGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCGGGGGGCTCCATCAGTTCTTACAACT


 


GGATTTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGATTGGGCAC


 


ATATATGACTATGGGAGGACCTTCTACAACTCCTCCCTCCAGAGTCGACC


 


TACCATATCTGTAGACGCGTCCAAGAATCAGCTCTCCCTGCGATTGACCT


 


CTGTGACCGCCTCAGACACGGCCGTCTATTACTGTGCGAGACCTCTCGGT


 


ATACTCCACTACTACGCGATGGACCTCTGGGGCCAAGGGACCACGGTCAC


 


CGTCTCGAGC


 


3259_J21 VH amino acid sequence


(SEQ ID NO: 128)


QVQLQESGPRVVRPSETLSLTCTVSGGSISSYNWIWIRQPPGKGLEWIGH


 


IYDYGRTFYNSSLQSRPTISVDASKNQLSLRLTSVTASDTAVYYCARPLG


 


ILHYYAMDLWGQGTTVTVSS


 


3259_J21 VL nucleotide sequence


(SEQ ID NO: 129)


GACATCCAGATGACCCAGTCTCCATTATCCGTGTCTGTATCTGTCGGGGA


 


CAGGGTCACCATCGCTTGCCGGGCAAGTCAGAGTATTGACAAGTTTTTAA


 


ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGGT


 


GCCTCCAATTTGCACAGTGGGGCCCCATCAAGGTTCAGTGCCAGTGGGTC


 


TGGGACAGACTTCACTCTAACAATCACCAATATACAGACTGAAGATTTCG


 


CAACTTACCTCTGTCAACAGAGTTTCAGTGTCCCCGCTTTCGGCGGAGGG


 


ACCAAGGTTGAGATCAAACG


 


3259_J21 VL amino acid sequence


(SEQ ID NO: 130)


DIQMTQSPLSVSVSVGDRVTIACRASQSIDKFLNWYQQKPGKAPKLLIYG


 


ASNLHSGAPSRFSASGSGTDFTLTITNIQTEDFATYLCQQSFSVPAFGGG


 


TKVEIK






The 3245_O19 antibody (also referred to herein as O19) includes a heavy chain variable region (SEQ ID NO: 132) encoded by the nucleic acid sequence shown below in SEQ ID NO: 131, and a light chain variable region (SEQ ID NO: 134) encoded by the nucleic acid sequence shown in SEQ ID NO: 133.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the O19 antibody have the following sequences per Kabat definition: STYMN (SEQ ID NO: 211), VFYSETRTYYADSVKG (SEQ ID NO: 212) and VQRLSYGMDV (SEQ ID NO: 213). The light chain CDRs of the O19 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GASTLQS (SEQ ID NO: 217) and QQTYSIPL (SEQ ID NO: 218).
The heavy chain CDRs of the O19 antibody have the following sequences per Chothia definition: GLSVSS (SEQ ID NO: 214), VFYSETRTY (SEQ ID NO: 215) and VQRLSYGMDV (SEQ ID NO: 213). The light chain CDRs of the O19 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GASTLQS (SEQ ID NO: 217) and QQTYSIPL (SEQ ID NO: 218).







3245_O19 VH nucleotide sequence


(SEQ ID NO: 131)


GAGGTGCAACTGGTGGAGTCTGGAGGGGGCTTGGTCCAGCCTGGGGGGTC


 


CCTGAGACTCTCCTGTACGGCCTCTGGGTTAAGTGTCAGTTCCACCTACA


 


TGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTCTCAGTT


 


TTTTATAGTGAGACCAGGACGTACTACGCAGACTCCGTGAAGGGCCGATT


 


CACCGTCTCCAGACACAATTCCAACAACACGCTCTATCTTCAGATGAACA


 


GCCTGAGAGTTGAAGACACGGCCGTGTATTATTGTGCGAGAGTCCAGAGA


 


TTGTCGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC


 


GAGC


 


3245_O19 VH amino acid sequence


(SEQ ID NO: 132)


EVQLVESGGGLVQPGGSLRLSCTASGLSVSSTYMNWVRQAPGKGLEWVSV


 


FYSETRTYYADSVKGRFTVSRHNSNNTLYLQMNSLRVEDTAVYYCARVQR


 


LSYGMDVWGQGTTVTVSS


 


3245_O19 VL nucleotide sequence


(SEQ ID NO: 133)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA


 


ATTGGTATCAGAAGAGACCAGGGAAAGCCCCTAAACTCCTGGTCTATGGT


 


GCATCCACTTTGCAGAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCGCCAGTCTGCAACCTGAAGATTCTG


 


CAACTTACTACTGTCAACAGACTTACAGTATCCCCCTCTTCGGCCAGGGG


 


ACACGGCTGGAGATTAAACG


 


3245_O19 VL amino acid sequence


(SEQ ID NO: 134)


DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQKRPGKAPKLLVYG


 


ASTLQSGVPSRFSGSGSGTDFTLTIASLQPEDSATYYCQQTYSIPLFGQG


 


TRLEIK






The 3244_H04 antibody (also referred to herein as H04) includes a heavy chain variable region (SEQ ID NO: 136) encoded by the nucleic acid sequence shown below in SEQ ID NO: 135, and a light chain variable region (SEQ ID NO: 138) encoded by the nucleic acid sequence shown in SEQ ID NO: 137.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the H04 antibody have the following sequences per Kabat definition: STYMN (SEQ ID NO: 211), VFYSETRTYYADSVKG (SEQ ID NO: 212) and VQRLSYGMDV (SEQ ID NO: 213). The light chain CDRs of the H04 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GASSLQS (SEQ ID NO: 226) and QQTYSIPL (SEQ ID NO: 218).
The heavy chain CDRs of the H04 antibody have the following sequences per Chothia definition: GLSVSS (SEQ ID NO: 214), VFYSETRTY (SEQ ID NO: 215) and VQRLSYGMDV (SEQ ID NO: 213). The light chain CDRs of the H04 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GASSLQS (SEQ ID NO: 226) and QQTYSIPL (SEQ ID NO: 218).







3244_H04 VH nucleotide sequence


(SEQ ID NO: 135)


GAGGTGCAGCTGGTGGAATCTGGAGGGGGCTTGGTCCAGCCTGGGGGGTC


 


CCTGAGACTCTCCTGTACAGCCTCTGGGTTAAGCGTCAGTTCCACCTACA


 


TGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTCTCAGTT


 


TTTTATAGTGAAACCAGGACGTATTACGCAGACTCCGTGAAGGGCCGATT


 


CACCGTCTCCAGACACAATTCCAACAACACGCTGTATCTTCAAATGAACA


 


GCCTGAGAGCTGAAGACACGGCCGTGTATTATTGTGCGAGAGTCCAGAGA


 


CTGTCATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC


 


GAGC


 


3244_H04 VH amino acid sequence


(SEQ ID NO: 136)


EVQLVESGGGLVQPGGSLRLSCTASGLSVSSTYMNWVRQAPGKGLEWVSV


 


FYSETRTYYADSVKGRFTVSRHNSNNTLYLQMNSLRAEDTAVYYCARVQR


 


LSYGMDVWGQGTTVTVSS


 


3244_H04 VL nucleotide sequence


(SEQ ID NO: 137)


GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA


 


ATTGGTATCAGAAGAGACCAGGGAAAGCCCCTAAACTCCTGGTCTATGGT


 


GCATCCAGTTTGCAGAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCGCCAGTCTGCAACCTGAAGATTCTG


 


CAGTTTATTACTGTCAACAGACTTACAGTATCCCCCTCTTCGGCCAGGGG


 


ACACGACTGGAGATTAAACG


 


3244_H04 VL amino acid sequence


(SEQ ID NO: 138)


DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQKRPGKAPKLLVYG


 


ASSLQSGVPSRFSGSGSGTDFTLTIASLQPEDSAVYYCQQTYSIPLFGQG


 


TRLEIK






The 3136_G05 antibody (also referred to herein as G05) includes a heavy chain variable region (SEQ ID NO: 140) encoded by the nucleic acid sequence shown below in SEQ ID NO: 139, and a light chain variable region (SEQ ID NO: 142) encoded by the nucleic acid sequence shown in SEQ ID NO: 141.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the G05 antibody have the following sequences per Kabat definition: SDFWS (SEQ ID NO: 228), YVYNRGSTKYSPSLKS (SEQ ID NO: 229) and NGRSSTSWGIDV (SEQ ID NO: 230). The light chain CDRs of the 3136_G05 antibody have the following sequences per Kabat definition: RASQSISTYLH (SEQ ID NO: 233), AASSLQS (SEQ ID NO: 234) and QQSYSPPLT (SEQ ID NO: 63).
The heavy chain CDRs of the 3136_G05 antibody have the following sequences per Chothia definition: GGSISS (SEQ ID NO: 206), YVYNRGSTK (SEQ ID NO: 232) and NGRSSTSWGIDV (SEQ ID NO: 230). The light chain CDRs of the 3136_G05 antibody have the following sequences per Chothia definition: RASQSISTYLH (SEQ ID NO: 233), AASSLQS (SEQ ID NO: 234) and QQSYSPPLT (SEQ ID NO: 63).







3136_G05 VH nucleotide sequence


(SEQ ID NO: 139)


CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCGGAGAC


 


CCTGTCCCTCACCTGCAGTGTCTCTGGTGGCTCCATTAGTAGTGATTTCT


 


GGAGTTGGATCCGACAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT


 


GTCTATAACAGAGGGAGCACTAAGTACAGTCCCTCCCTCAAGAGTCGAGT


 


CACCATATCAGCAGACATGTCCAAGAACCAGTTTTCCCTGAATATGAGTT


 


CTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAAAAATGGTCGA


 


AGTAGCACCAGTTGGGGCATCGACGTCTGGGGCAAAGGGACCACGGTCAC


 


CGTCTCGAGC


 


3136_G05 VH amino acid sequence


(SEQ ID NO: 140)


QVQLQESGPGLVKPSETLSLTCSVSGGSISSDFWSWIRQPPGKGLEWIGY


 


VYNRGSTKYSPSLKSRVTISADMSKNQFSLNMSSVTAADTAVYYCAKNGR


 


SSTSWGIDVWGKGTTVTVSS


 


3136_G05 VL nucleotide sequence


(SEQ ID NO: 141)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGA


 


CAGACTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAC


 


ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCT


 


GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTAGATC


 


AGGAACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGATGACTTTG


 


CAACTTACTACTGTCAACAGAGTTACAGTCCCCCCCTCACTTTCGGCCCT


 


GGGACCAAAGTGGATATGAAACG


 


3136_G05 VL amino acid sequence


(SEQ ID NO: 142)


DIQMTQSPSSLSASVGDRLTITCRASQSISTYLHWYQQKPGKAPKLLIYA


 


ASSLQSGVPSRFSGSRSGTDFTLTISSLQPDDFATYYCQQSYSPPLTFGP


 


GTKVDMK






The 3252_C13 antibody (also referred to herein as C13) includes a heavy chain variable region (SEQ ID NO: 144) encoded by the nucleic acid sequence shown below in SEQ ID NO: 143, and a light chain variable region (SEQ ID NO: 146) encoded by the nucleic acid sequence shown in SEQ ID NO: 145.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the C13 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), YIYNRGSTKYTPSLKS (SEQ ID NO: 237) and HVGGHTYGIDY (SEQ ID NO: 238). The light chain CDRs of the C13 antibody have the following sequences per Kabat definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
The heavy chain CDRs of the C13 antibody have the following sequences per Chothia definition: GASISS (SEQ ID NO: 239), YIYNRGSTK (SEQ ID NO: 240) and HVGGHTYGIDY (SEQ ID NO: 238). The light chain CDRs of the C13 antibody have the following sequences per Chothia definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).







3252_C13 VH nucleotide sequence


(SEQ ID NO: 143)


CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTGCCTCCATCAGTAGTGACTACT


 


GGAGCTGGATCCGGCTGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT


 


ATCTATAATAGAGGGAGTACCAAGTACACCCCCTCCCTGAAGAGTCGAGT


 


CACCATATCACTAGACACGGCCGAGAACCAGTTCTCCCTGAGGCTGAGGT


 


CGGTGACCGCCGCAGACACGGCCATCTATTACTGTGCGAGACATGTAGGT


 


GGCCACACCTATGGAATTGATTACTGGGGCCAGGGAACCCTGGTCACCGT


 


CTCGAGC


 


3252_C13 VH amino acid sequence


(SEQ ID NO: 144)


QVQLQESGPGLVKPSETLSLTCTVSGASISSDYWSWIRLPPGKGLEWIGY


 


IYNRGSTKYTPSLKSRVTISLDTAENQFSLRLRSVTAADTAIYYCARHVG


 


GHTYGIDYWGQGTLVTVSS


 


3252_C13 VL nucleotide sequence


(SEQ ID NO: 145)


GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCCTCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAACTATTTAA


 


ATTGGTATCAACACAAACCTGGGGAAGCCCCCAAGCTCCTGAACTATGCT


 


GCGTCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCAGTCTTCAACCTGAAGATTTTG


 


CCACTTACTACTGTCAACAGAGTTACAATACTCCGATCACCTTCGGCCAA


 


GGGACACGACTGGAAATTAAACG


 


3252_C13 VL amino acid sequence


(SEQ ID NO: 146)


DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQHKPGEAPKLLNYA


 


ASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSYNTPITFGQ


 


GTRLEIK






The 3259_J06 antibody (also referred to herein as J06) includes a heavy chain variable region (SEQ ID NO: 148) encoded by the nucleic acid sequence shown below in SEQ ID NO: 147, and a light chain variable region (SEQ ID NO: 150) encoded by the nucleic acid sequence shown in SEQ ID NO: 149.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the J06 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), YIYNRGSTKYTPSLKS (SEQ ID NO: 237) and HVGGHTYGIDY (SEQ ID NO: 238). The light chain CDRs of the J06 antibody have the following sequences per Kabat definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
The heavy chain CDRs of the J06 antibody have the following sequences per Chothia definition: GASISS (SEQ ID NO: 239), YIYNRGSTK (SEQ ID NO: 240) and HVGGHTYGIDY (SEQ ID NO: 238). The light chain CDRs of the J06 antibody have the following sequences per Chothia definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).







3255_J06 VH nucleotide sequence


(SEQ ID NO: 147)


CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCTGGTGCCTCCATCAGTAGTGACTACT


 


GGAGCTGGATCCGGCTGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT


 


ATCTATAATAGAGGGAGTACCAAGTACACCCCCTCCCTGAAGAGTCGAGT


 


CACCATATCACTAGACACGGCCGAGAACCAGTTCTCCCTGAGGCTGAGGT


 


CGGTGACCGCCGCAGACACGGCCGTCTATTACTGTGCGAGACATGTGGGT


 


GGCCACACCTATGGAATTGATTACTGGGGCCAGGGAACCCTGGTCACCGT


 


CTCGAGC


 


3255_J06 VH amino acid sequence


(SEQ ID NO: 148)


QVQLQESGPGLVKPSETLSLTCTVSGASISSDYWSWIRLPPGKGLEWIGY


 


IYNRGSTKYTPSLKSRVTISLDTAENQFSLRLRSVTAADTAVYYCARHVG


 


GHTYGIDYWGQGTLVTVSS


 


3255_J06 VL nucleotide sequence


(SEQ ID NO: 149)


GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCCTCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAACTATTTAA


 


ATTGGTATCAACACAAACCTGGGGAAGCCCCCAAGCTCCTGAACTATGCT


 


GCGTCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATC


 


TGGGACAGATTTCACTCTCAGCATCAGCGGTCTTCAACCTGAAGATTTTG


 


CCACTTACTACTGTCAACAGAGCTACAATACTCCGATCACCTTCGGCCCA


 


GGGACACGACTGGAAATTAAACG


 


3255_J06 VL amino acid sequence


(SEQ ID NO: 150)


DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQHKPGEAPKLLNYA


 


ASSLQSGVPSRFSASGSGTDFTLSISGLQPEDFATYYCQQSYNTPITFGP


 


GTRLEIK






The 3410_I23 antibody (also referred to herein as I23) includes a heavy chain variable region (SEQ ID NO: 152) encoded by the nucleic acid sequence shown below in SEQ ID NO: 151, and a light chain variable region (SEQ ID NO: 154) encoded by the nucleic acid sequence shown in SEQ ID NO: 153.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 3410_I23 antibody have the following sequences per Kabat definition: SYSWS (SEQ ID NO: 252), YLYYSGSTKYNPSLKS (SEQ ID NO: 253) and TGSESTTGYGMDV (SEQ ID NO: 254). The light chain CDRs of the 3410_I23 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), AASSLHS (SEQ ID NO: 258) and QQSYSPPIT (SEQ ID NO: 259).
The heavy chain CDRs of the 3410_I23 antibody have the following sequences per Chothia definition: GDSISS (SEQ ID NO: 255), YLYYSGSTK (SEQ ID NO: 256) and TGSESTTGYGMDV (SEQ ID NO: 254). The light chain CDRs of the 3410_I23 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), AASSLHS (SEQ ID NO: 258) and QQSYSPPIT (SEQ ID NO: 259).







3420_I23 VH nucleotide sequence


(SEQ ID NO: 151)


CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCGTCACCTGCAAAGTCTCTGGTGACTCCATCAGTAGTTATTCCT


 


GGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGGTTGGCTAT


 


TTGTATTATAGTGGGAGCACCAAGTACAACCCCTCCCTCAAGAGTCGAAC


 


CACCATATCAGTAGACACGTCCACGAACCAGTTGTCCCTGAAGTTGAGTT


 


TTGTGACCGCCGCGGACACGGCCGTGTATTTCTGTGCGAGAACCGGCTCG


 


GAATCTACTACCGGCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGT


 


CACCGTCTCGAGC


 


3420_I23 VH amino acid sequence


(SEQ ID NO: 152)


QVQLQESGPGLVKPSETLSVTCKVSGDSISSYSWSWIRQPPGKGLEWVGY


 


LYYSGSTKYNPSLKSRTTISVDTSTNQLSLKLSFVTAADTAVYFCARTGS


 


ESTTGYGMDVWGQGTTVTVSS


 


3420_I23 VL nucleotide sequence


(SEQ ID NO: 153)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA


 


ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT


 


GCATCCAGTTTGCACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCGCTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG


 


CAACTTACTACTGTCAACAGAGTTACAGTCCCCCGATCACCTTCGGCCAA


 


GGGACACGACTGGAGATTAAACG


 


3420_I23 VL amino acid sequence


(SEQ ID NO: 154)


DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYA


 


ASSLHSGVPSRFSGSGSGTDFALTISSLQPEDFATYYCQQSYSPPITFGQ


 


GTRLEIK






The 3139_P23 antibody (also referred to herein as P23) includes a heavy chain variable region (SEQ ID NO: 156) encoded by the nucleic acid sequence shown below in SEQ ID NO: 155, and a light chain variable region (SEQ ID NO:158) encoded by the nucleic acid sequence shown in SEQ ID NO:157.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the P23 antibody have the following sequences per Kabat definition: NSFWG (SEQ ID NO: 260), YVYNSGNTKYNPSLKS (SEQ ID NO: 261) and HDDASHGYSIS (SEQ ID NO: 262). The light chain CDRs of the 3139_P23 antibody have the following sequences per Kabat definition: RASQTISTYLN (SEQ ID NO: 265), AASGLQS (SEQ ID NO: 61) and QQSYNTPLT (SEQ ID NO: 267).
The heavy chain CDRs of the 3139_P23 antibody have the following sequences per Chothia definition: GGSISN (SEQ ID NO: 263), YVYNSGNTK (SEQ ID NO: 264) and HDDASHGYSIS (SEQ ID NO: 262). The light chain CDRs of the 3139_P23 antibody have the following sequences per Chothia definition: RASQTISTYLN (SEQ ID NO: 265), AASGLQS (SEQ ID NO: 61) and QQSYNTPLT (SEQ ID NO: 267).







3139_P23 VH nucleotide sequence


(SEQ ID NO: 155)


CAGGTGCAGCTGCAGGAGTCGGGCCCAAGACTGGTGAAGCCTTCGGAGAG


 


CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATTAGTAATTCCTTCT


 


GGGGCTGGATCCGGCAGCCCCCAGGGGAGGGACTGGAGTGGATTGGTTAT


 


GTCTATAACAGTGGCAACACCAAGTACAATCCCTCCCTCAAGAGTCGAGT


 


CACCATTTCGCGCGACACGTCCAAGAGTCAACTCTACATGAAGCTGAGGT


 


CTGTGACCGCCGCTGACACGGCCGTGTACTACTGTGCGAGGCATGACGAC


 


GCAAGTCATGGCTACAGCATCTCCTGGGGCCACGGAACCCTGGTCACCGT


 


CTCGAGC


 


3139_P23 VH amino acid sequence


(SEQ ID NO: 156)


QVQLQESGPRLVKPSESLSLTCTVSGGSISNSFWGWIRQPPGEGLEWIGY


 


VYNSGNTKYNPSLKSRVTISRDTSKSQLYMKLRSVTAADTAVYYCARHDD


 


ASHGYSISWGHGTLVTVSS


 


3139_P23 VL nucleotide sequence


(SEQ ID NO: 157)


GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAGTACTTATTTAA


 


ATTGGTATCAACAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT


 


GCATCCGGTTTGCAAAGTGGAGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCAGTCTTCAACCTGAAGATTTTG


 


CAACTTACTTCTGTCAACAGAGTTACAATACTCCCCTGACGTTCGGCCAA


 


GGGACCAAGGTGGAAATCAAA


 


3139_P23 VL amino acid sequence


(SEQ ID NO: 158)


DIQMTQSPSSLSASVGDRVTITCRASQTISTYLNWYQQKSGKAPKLLIYA


 


ASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSYNTPLTFGQ


 


GTKVEIK






The 3248_P18 antibody (also referred to herein as P18) includes a heavy chain variable region (SEQ ID NO: 160) encoded by the nucleic acid sequence shown below in SEQ ID NO: 159, and a light chain variable region (SEQ ID NO: 162) encoded by the nucleic acid sequence shown in SEQ ID NO: 161.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 3248_P18 antibody have the following sequences per Kabat definition: AYHWS (SEQ ID NO: 268), HIFDSGSTYYNPSLKS (SEQ ID NO: 269) and PLGSRYYYGMDV (SEQ ID NO: 270). The light chain CDRs of the 3248_P18 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASTLQN (SEQ ID NO: 274) and QQSYSVPA (SEQ ID NO: 275).
The heavy chain CDRs of the 3248_P18 antibody have the following sequences per Chothia definition: GGSISA (SEQ ID NO: 271), HIFDSGSTY (SEQ ID NO: 272) and PLGSRYYYGMDV (SEQ ID NO: 270). The light chain CDRs of the 3248_P18 antibody have the following sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273), GASTLQN (SEQ ID NO: 274) and QQSYSVPA (SEQ ID NO: 275).







3248_P18 VH nucleotide sequence


(SEQ ID NO: 159)


CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC


 


CCTGTCCCTCACCTGCACTGTCTCGGGTGGCTCCATCAGTGCTTACCACT


 


GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGCAC


 


ATCTTTGACAGTGGGAGCACTTACTACAACCCCTCCCTTAAGAGTCGAGT


 


CACCATATCACTAGACGCGTCCAAGAACCAGCTCTCCCTGAGATTGACCT


 


CTGTGACCGCCTCAGACACGGCCATATATTACTGTGCGAGACCTCTCGGG


 


AGTCGGTACTATTACGGAATGGACGTCTGGGGCCAAGGGACCACGGTCAC


 


CGTCTCGAGC


 


3248_P18 VH amino acid sequence


(SEQ ID NO: 160)


QVQLQESGPGLVKPSETLSLTCTVSGGSISAYHWSWIRQPPGKGLEWIGH


 


IFDSGSTYYNPSLKSRVTISLDASKNQLSLRLTSVTASDTAIYYCARPLG


 


SRYYYGMDVWGQGTTVTVSS


 


3248_P18 VL nucleotide sequence


(SEQ ID NO: 161)


GACATCCAGATGACCCAGTCTCCGTCCTCCCTGTCTGCATCTGTCGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCAGGTATTTAA


 


ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT


 


GCCTCCACTTTGCAAAATGGGGCCCCATCAAGGTTCAGCGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTCCG


 


CAACTTACCTCTGTCAACAGAGTTACAGTGTCCCTGCTTTCGGCGGAGGA


 


ACCAAGGTGGAGGTCAAA


 


3248_P18 VL amino acid sequence (SEQ ID NO: 162)


DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYG


 


ASTLQNGAPSRFSGSGSGTDFTLTISSLQPEDSATYLCQQSYSVPAFGGG


 


TKVEVK






The 3253_P10 antibody (also referred to herein as P10) includes a heavy chain variable region (SEQ ID NO: 164) encoded by the nucleic acid sequence shown below in SEQ ID NO: 163, and a light chain variable region (SEQ ID NO: 166) encoded by the nucleic acid sequence shown in SEQ ID NO: 165.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 3253_P10 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDAKFSGSYYVAS (SEQ ID NO: 189). The light chain CDRs of the 3253_P10 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GATDLQS (SEQ ID NO: 282) and QQSYNTPLI (SEQ ID NO: 194).
The heavy chain CDRs of the 3253_P10 antibody have the following sequences per Chothia definition: GGSITS (SEQ ID NO: 190), FFYNGGSTK (SEQ ID NO: 191) and HDAKFSGSYYVAS (SEQ ID NO: 189). The light chain CDRs of the 3253_P10 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATDLQS (SEQ ID NO: 282) and QQSYNTPLI (SEQ ID NO: 194).







3253_P10 VH nucleotide sequence


(SEQ ID NO: 163)


CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACAC


 


CCTGGCCCTCACTTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACT


 


GGAGCTGGATCCGGCAACCCCCAGGGAGGGGACTGGACTGGATCGGATTC


 


TTCTATAACGGCGGGAGCACCAAGTACAATCCCTCCCTCAAGAGTCGAGT


 


CACCATATCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGACCT


 


CTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGCC


 


AAATTTAGTGGGAGCTACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGT


 


CACCGTCTCGAGC


 


3253_P10 VH amino acid sequence


(SEQ ID NO: 164)


QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGF


 


FYNGGSTKYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDA


 


KFSGSYYVASWGQGTRVTVSS


 


3253_P10 VL nucleotide sequence


(SEQ ID NO: 165)


GACATCCAGATGACCCAGTCTCCCTCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA


 


ATTGGTATCAGCAGCAACCTGGGAAAGCCCCTAAGGTCCTGATCTCTGGT


 


GCAACCGACTTGCAAAGTGGGGTCCCATCTCGCTTCAGTGGCAGTGGATC


 


TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG


 


CAACTTACTACTGTCAACAGAGTTACAATACCCCCCTCATTTTTGGCCAG


 


GGGACCAAGCTGGAGATCAAA


 


3253_P10 VL amino acid sequence


(SEQ ID NO: 166)


DIQMTQSPSSLSASVGDRVTISCRASQSISTYLNWYQQQPGKAPKVLISG


 


ATDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLIFGQ


 


GTKLEIK






The 3260_D19 antibody (also referred to herein as D19) includes a heavy chain variable region (SEQ ID NO: 168) encoded by the nucleic acid sequence shown below in SEQ ID NO: 167, and a light chain variable region (SEQ ID NO: 170) encoded by the nucleic acid sequence shown in SEQ ID NO: 169.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 3260_D19 antibody have the following sequences per Kabat definition: DNYIN (SEQ ID NO: 284), VFYSADRTSYADSVKG (SEQ ID NO: 285) and VQKSYYGMDV (SEQ ID NO: 286). The light chain CDRs of the 3260_D19 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).
The heavy chain CDRs of the 3260_D19 antibody have the following sequences per Chothia definition: GFSVSD (SEQ ID NO: 287), VFYSADRTS (SEQ ID NO: 288) and VQKSYYGMDV (SEQ ID NO: 286). The light chain CDRs of the 3260_D19 antibody have the following sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).







3260_D19 VH nucleotide sequence







(SEQ ID NO: 167)







GACATGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCCGCCGGGGGGGT


 


CCCTGAGACTCTCCTGCGCAGCCTCTGGGTTTTCCGTCAGTGACAACTA


 


CATAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGACTGGGTCTCA


 


GTCTTTTATAGTGCTGATAGAACATCCTACGCAGACTCCGTGAAGGGCC


 


GATTCACCGTCTCCAGCCACGATTCCAAGAACACAGTGTACCTTCAAAT


 


GAACAGTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTGCGAGAGTT


 


CAGAAGTCCTATTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA


 


CCGTCTCGAGC


 


3260_D19 VH amino acid sequence







(SEQ ID NO: 168)







DMQLVESGGGLVPPGGSLRLSCAASGFSVSDNYINWVRQAPGKGLDWVS


 


VFYSADRTSYADSVKGRFTVSSHDSKNTVYLQMNSLRAEDTAVYYCAR


 


VQKSYYGMDVWGQGTTVTVSS


 


3260_D19 VL nucleotide sequence







(SEQ ID NO: 169)







GGCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG


 


ACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTT


 


AAATTGGTATCTGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCT


 


GGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCACTG


 


GGTCTGGGACAGAATTCACTCTCACCATCAGCAGTTTGCAACCTGAAGA


 


TTTTGCAACTTACTACTGTCAACAGACTTTCAGTATCCCTCTTTTTGGC


 


CAGGGGACCAAGGTGGAGATCAAA


 


3260_D19 VL amino acid sequence







(SEQ ID NO: 170)







GIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYLQKPGKAPKLLIS


 


GASSLQSGVPSRFSGTGSGTEFTLTISSLQPEDFATYYCQQTFSIPLFG


 


QGTKVEIK






The 3362_B11 antibody (also referred to herein as B11) includes a heavy chain variable region (SEQ ID NO: 172) encoded by the nucleic acid sequence shown below in SEQ ID NO: 171, and a light chain variable region (SEQ ID NO: 174) encoded by the nucleic acid sequence shown in SEQ ID NO: 173.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the B11 antibody have the following sequences per Kabat definition: SGAYYWT (SEQ ID NO: 293), YIYYSGNTYYNPSLKS (SEQ ID NO: 294) and AASTSVLGYGMDV (SEQ ID NO: 295). The light chain CDRs of the B11 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), AASSLQS (SEQ ID NO: 234) and QQSYSTPLT (SEQ ID NO: 300).
The heavy chain CDRs of the B11 antibody have the following sequences per Chothia definition: GDSITSGA (SEQ ID NO: 296), YIYYSGNTY (SEQ ID NO: 297) and AASTSVLGYGMDV (SEQ ID NO: 295). The light chain CDRs of the B11 antibody have the following sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273), AASSLQS (SEQ ID NO: 234) and QQSYSTPLT (SEQ ID NO: 300).







3362_B11 VH nucleotide sequence







(SEQ ID NO: 171)







CAGGTGCAGCTGCAGGCGTCGGGCCCAGGACTGGTGAAGCCTTCAGAGA


 


CCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCACCAGTGGTGC


 


TTACTACTGGACCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGG


 


ATTGGGTACATCTATTACAGTGGGAACACCTACTACAACCCGTCCCTCA


 


AGAGTCGAGTTACCATATCACTAGACACGTCTAAGAACCAGTTCTCCCT


 


GAAGGTGAACTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCG


 


CGAGCTGCTTCGACTTCAGTGCTAGGATACGGTATGGACGTCTGGGGCC


 


AAGGGACCACGGTCACCGTCTCGAGC


 


3362_B11 VH amino acid sequence







(SEQ ID NO: 172)







QVQLQASGPGLVKPSETLSLTCTVSGDSITSGAYYWTWIRQHPGKGLEW


 


IGYIYYSGNTYYNPSLKSRVTISLDTSKNQFSLKVNSVTAADTAVYYCA


 


RAASTSVLGYGMDVWGQGTTVTVSS


 


3362_B11 VL nucleotide sequence







(SEQ ID NO: 173)







GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG


 


ACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTT


 


AAATTGGTATCAGCAGGAACCAGGGAAGGCCCCTAAGCTCCTGGTCTAT


 


GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTG


 


GATCTGGGACAGATTTCACTCTCACCATAAGCAGTCTTCAACCTGAAGA


 


TTTTGCAACTTACTACTGTCAACAGAGTTATAGTACCCCCCTCACCTTC


 


GGCCAAGGGACACGACTGGAGATTAAA


 


3362_B11 VH amino acid sequence







(SEQ ID NO: 174)







DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQEPGKAPKLLVY


 


AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF


 


GQGTRLEIK






The 3242_P05 antibody (also referred to herein as P05) includes a heavy chain variable region (SEQ ID NO: 176) encoded by the nucleic acid sequence shown below in SEQ ID NO: 175, and a light chain variable region (SEQ ID NO: 178) encoded by the nucleic acid sequence shown in SEQ ID NO: 177.
The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 3242_P05 antibody have the following sequences per Kabat definition: VSDNYIN (SEQ ID NO: 301), VFYSADRTSYADSVKG (SEQ ID NO: 285) and VQKSYYGMDV (SEQ ID NO: 286). The light chain CDRs of the 3242_P05 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).
The heavy chain CDRs of the 3242_P05 antibody have the following sequences per Chothia definition: SGFSV (SEQ ID NO: 304), VFYSADRTS (SEQ ID NO: 288) and VQKSYYGMDV (SEQ ID NO: 286). The light chain CDRs of the 3242_P05 antibody have the following sequences per Chothia definition: The light chain CDRs of the 3242_P05 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).







3242_P05 VH nucleotide sequence







(SEQ ID NO: 175)







GACATGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCCGCCGGGGGGGT


 


CCCTGAGACTCTCCTGCGCAGCCTCTGGGTTTTCCGTCAGTGACAACTA


 


CATAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGACTGGGTCTCA


 


GTCTTTTATAGTGCTGATAGAACATCCTACGCAGACTCCGTGAAGGGCC


 


GATTCACCGTCTCCAGCCACGATTCCAAGAACACAGTGTACCTTCAAAT


 


GAACAGTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTGCGAGAGTT


 


CAGAAGTCCTATTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA


 


CCGTCTCGAGC


 


_P05 VH amino acid sequence







(SEQ ID NO: 176)







DMQLVESGGGLVPPGGSLRLSCAASGFSVSDNYINWVRQAPGKGLDWVS


 


VFYSADRTSYADSVKGRFTVSSHDSKNTVYLQMNSLRAEDTAVYYCARV


 


QKSYYGMDVWGQGTTVTVSS


 


3242_P05 VL nucleotide sequence







(SEQ ID NO: 177)







GGCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG


 


ACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTT


 


AAATTGGTATCTGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCT


 


GGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCACTG


 


GGTCTGGGACAGAATTCACTCTCACCATCAGCAGTTTGCAACCTGAAGA


 


TTTTGCAACTTACTACTGTCAACAGACTTTCAGTATCCCTCTTTTTGGC


 


CAGGGGACCAAGGTGGAGATCAAA


 


3242_P05 VL amino acid sequence







(SEQ ID NO: 178)







GIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYLQKPGKAPKLLIS


 


GASSLQSGVPSRFSGTGSGTEFTLTISSLQPEDFATYYCQQTFSIPLFG


 


QGTKVEIK






HuM2e antibodies of the invention also include antibodies that include a heavy chain variable amino acid sequence that is at least 90%, 92%, 95%, 97% 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 44, 277, 276, 50, 236, 235, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, or 176. and/or a light chain variable amino acid that is at least 90%, 92%, 95%, 97% 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 46, 52, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178.
Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, or 3242_P05.
The heayy chain of a M2e antibody is derived from a germ line V (variable) gene such as, for example, the IgHV4 or the IgHV3 germline gene.
The M2e antibodies of the invention include a variable heavy chain (VH) region encoded by a human IgHV4 or the IgHV3 germline gene sequence. An IgHV4 germline gene sequence is shown, e.g., in Accession numbers L10088, M29812, M95114, X56360 and M95117. An IgHV3 germline gene sequence is shown, e.g., in Accession numbers X92218, X70208, Z27504, M99679 and AB019437. The M2e antibodies of the invention include a VH region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgHV4 or the IgHV3 germline gene sequence. Preferably, the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgHV4 or the IgHV3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the IgHV4 or the IgHV3 germline gene sequence. The VH region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VH region encoded by the IgHV4 or the IgHV3 VH germline gene sequence. Preferably, the amino acid sequence of VH region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgHV4 or the IgHV3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by the IgHV4 or the IgHV3 germline gene sequence. The M2e antibodies of the invention also include a variable light chain (VL) region encoded by a human IgKV1 germline gene sequence. A human IgKV1 VL germline gene sequence is shown, e.g., Accession numbers X59315, X59312, X59318, J00248, and Y14865. Alternatively, the M2e antibodies include a VL region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgKV1 germline gene sequence. Preferably, the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgKV1 germline gene sequence, and more preferably, at least 98%, 99% homologous to the IgKV1 germline gene sequence. The VL region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VL region encoded the IgKV1 germline gene sequence. Preferably, the amino acid sequence of VL region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgKV1 germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by e the IgKV1 germline gene sequence.
HA Antibodies I
The HA antibodies of the invention may also be capable of specifically binding to one or more fragments of influenza virus H5N1, such as the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and cellular release, or membrane proteins (M1 and M2). In a specific embodiment, the HA antibodies of the invention are capable of specifically binding to the HA molecule of H5N1 strains. They may be capable of specifically binding to the HA1 and/or HA2 subunit of the HA molecule. They may be capable of specifically binding to linear or structural and/or conformational epitopes on the HA1 and/or HA2 subunit of the HA molecule. The HA molecule may be purified from viruses or recombinantly produced and optionally isolated before use. Alternatively, HA may be expressed on the surface of cells.
For diagnostic purposes, the HA antibodies may also be capable of specifically binding to proteins not present on the surface of H5N1 including the nucleoprotein, the nucleocapsid structural protein, polymerases (PA, PB and PB2), and non-structural proteins (NS1 and NS2). The nucleotide and/or amino acid sequence of proteins of various H5N1 strains can be found in the GenBank-database, NCBI Influenza Virus Sequence Database, Influenza Sequence Database (ISD), EMBL-database and/or other databases. It is well within the reach of the skilled person to find such sequences in the respective databases. In another embodiment the HA antibodies of the invention are capable of specifically binding to a fragment of the above-mentioned proteins and/or polypeptides, wherein the fragment at least includes an antigenic determinant recognized by the HA antibodies of the invention. An “antigenic determinant” as used herein is a moiety that is capable of binding to an HA antibody of the invention with sufficiently high affinity to form a detectable antigen-antibody complex. As used herein, the terms “antigenic determinant” and “epitope” are equivalents. The HA antibodies of the invention may or may not be capable of specifically binding to the extracellular part of HA (also called herein soluble HA (sHA)).
The HA antibodies of the invention can be intact immunoglobulin molecules such as polyclonal or monoclonal antibodies or the HA antibodies can be antigen-binding fragments including, but not limited to, Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, and (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to influenza virus H5N1 strains or a fragment thereof. In a preferred embodiment the HA antibodies are human monoclonal antibodies.
HA antibodies can be used in non-isolated or isolated form. Furthermore, the HA antibodies can be used alone or in a mixture including at least one HA antibody (or variant or fragment thereof). Thus, HA antibodies can be used in combination, e.g., as a pharmaceutical composition comprising two or more antibodies of the invention, variants or fragments thereof. For example, antibodies having different, but complementary activities can be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect, but alternatively, antibodies having identical activities can also be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect. Optionally, the mixture further includes at least one other therapeutic agent. Preferably, the therapeutic agent such as, e.g., M2 inhibitors (e.g., amantidine, rimantadine) and/or neuraminidase inhibitors (e.g., zanamivir, oseltamivir) is useful in the prophylaxis and/or treatment of an influenza virus H5N1 infection.
Typically, HA antibodies can bind to their binding partners, i.e. influenza virus H5N1 or fragments thereof, with an affinity constant (Kd-value) that is lower than 0.2×10−4 M, 1.0×10−5 M, 1.0×10−6 M, 1.0×10−7 M, preferably lower than 1.0×10−8 M, more preferably lower than 1.0×10−9 M, more preferably lower than 1.0×10 −10 M, even more preferably lower than 1.0×10−11 M, and in particular lower than 1.0×10−12 M. The affinity constants can vary for antibody isotypes. For example, affinity binding for an IgM isotype refers to a binding affinity of at least about 1.0×10−7 M. Affinity constants can for instance be measured using surface plasmon resonance, for example using the BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden).
HA antibodies may bind to influenza virus H5N1 or a fragment thereof in soluble form such as for instance in a sample or in suspension or may bind to influenza virus H5N1 or a fragment thereof bound or attached to a carrier or substrate, e.g., microtiter plates, membranes and beads, etc. Carriers or substrates may be made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, or Teflon, etc. The surface of such supports may be solid or porous and of any convenient shape. Furthermore, the HA antibodies may bind to influenza virus H5N1 in purified/isolated or non purified/non-isolated form.
HA antibodies exhibit neutralizing activity. Neutralizing activity can for instance be measured as described in International Patent Application PCT/EP2007/059356 (Publication No. WO 2008/028946, the contents of which are incorporated herein in their entirety). Alternative assays measuring neutralizing activity are described in for instance WHO Manual on Animal Influenza Diagnosis and Surveillance, Geneva: World Health Organization, 2005, version 2002.5.
The invention relates to an isolated human HA antibody that recognizes and binds to an epitope in the HA2 subunit of the influenza haemagglutinin protein (HA), characterized in that said HA antibody has neutralizing activity against an influenza virus, for instance, including HA of the H5 subtype. Examples of influenza strains that contain such a HA of the H5 subtype and that are important strains in view of pandemic threats are H5N1, H5N2, H5N8, and H5N9. Particularly preferred are HA antibodies that at least neutralize the H5N1 influenza strain. Preferably, HA antibodies do not depend on an epitope in the HA1 subunit of the HA protein for binding to said HA protein.
Definitions
The term “human HA antibody” describes an intact immunoglobulin including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, or to an antigen-binding and/or variable domain comprising fragment of an immunoglobulin that competes with the intact immunoglobulin for specific binding to the binding partner of the immunoglobulin, e.g. H5N1. Regardless of structure, the antigen binding fragment binds with the same antigen that is recognized by the intact immunoglobulin. An antigen-binding fragment can comprise a peptide or polypeptide comprising an amino acid sequence of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 contiguous amino acid residues of the amino acid sequence of the HA antibody.
The term “HA antibody”, includes all immunoglobulin classes and subclasses known in the art. Depending on the amino acid sequence of the constant domain of their heavy chains, HA antibodies can be divided into the five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.
Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the (poly)peptide, etc. The above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineered by recombinant DNA techniques. The methods of production are well known in the art and are described, for example, in Antibodies: A Laboratory Manual, Edited by: E. Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which is incorporated herein by reference. An HA antibody or antigen-binding fragment thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or they may be different.
With respect to HA antibodies, the term “complementarity determining regions” (CDR) as used herein means sequences within the variable regions of HA antibodies, such as immunoglobulins, that usually contribute to a large extent to the antigen binding site which is complementary in shape and charge distribution to the epitope recognized on the antigen. The CDR regions of HA antibodies can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e.g., by solubilization in SDS. Epitopes of HA antibodies may also consist of posttranslational modifications of proteins.
The term “functional variant”, as used herein, refers to an HA antibody that includes a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parental HA antibody and that is still capable of competing for binding to the binding partner, e.g. H5N1, with the parental HA antibody. In other words, the modifications in the amino acid and/or nucleotide sequence of the parental HA antibody do not significantly affect or alter the binding characteristics of the HA antibody encoded by the nucleotide sequence or containing the amino acid sequence, i.e. the antibody is still able to recognize and bind its target. The functional variant may have conservative sequence modifications including nucledtide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may include natural as well as non-natural nucleotides and amino acids.
Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). It will be clear to the skilled artisan that other classifications of amino acid residue families than the one used above can also be employed. Furthermore, a HA antibody functional variant may have non-conservative amino acid substitutions, e.g., replacement of an amino acid with an amino acid residue having different structural or chemical properties. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art.
A mutation in a nucleotide sequence can be a single alteration made at a locus (a point mutation), such as transition or transversion mutations, or alternatively, multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleotide sequence. The mutations may be performed by any suitable method known in the art.
The term “human”, when applied to HA antibodies, refers to molecules that are either directly derived from a human or based upon a human sequence. When an HA antibody is derived from or based on a human sequence and subsequently modified, it is still to be considered human as used throughout the specification. In other words, the term human, when applied to HA antibodies is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences or based on variable or constant regions occurring in a human or human lymphocyte and modified in some form. Thus, the human HA antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences, contain substitutions and/or deletions (e.g., mutations introduced by for instance random or site-specific mutagenesis in vitro or by somatic mutation in vivo). “Based on” as used herein refers to the situation that a nucleic acid sequence may be exactly copied from a template, or with minor mutations, such as by error-prone PCR methods, or synthetically made matching the template exactly or with minor modifications. Semi-synthetic molecules based on human sequences are also considered to be human as used herein.
Single Chain HA Antibodies
The heavy chain of an HA antibody is derived from a germ line V (variable) gene such as, for example, the VH1 or VH3 germline gene (see, Tomlinson I M, Williams S C, Ignatovitch O, Corbett S J, Winter G. V-BASE Sequence Directory. Cambridge, United Kingdom: MRC Centre for Protein Engineering (1997)). The HA antibodies of the invention include a VH region that is encoded by a nucleic acid sequence that is at least 80% homologous to the VH1 or VH3 germline gene sequence. Preferably, the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the VH1 or VH3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the VH1 or VH3 germline gene sequence. The VH region of the HA antibody is at least 80% homologous to the amino acid sequence of the VH region encoded by the VH1 or VH3 VH germline gene sequence. Preferably, the amino acid sequence of VH region of the HA antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the VH1 or VH3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by the VH1 or VH3 germline gene sequence.
In certain aspects of the invention the VH1 germline gene is VH1 (1-2), VH1 (1-18), VH1 (3-23), or VH1 (1-69). In other aspects of the invention the VH3 germline gene is VH3 (3-21)
The HA antibodies of the invention also include a variable light chain (VL) region encoded by a human germline gene sequence selected from the group consisting of VKI, VKII, VKIII, VKIV, VL1, VL2, and VL3 (see, Tomlinson I M, Williams S C, Ignatovitch O, Corbett S J, Winter G. V-BASE Sequence Directory. Cambridge, United Kingdom: MRC Centre for Protein Engineering (1997)). Alternatively, the HA antibodies include a VL region that is encoded by a nucleic acid sequence that is at least 80% homologous to the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or VL3. Preferably, the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or VL3, and more preferably, at least 98%, 99% homologous to the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or VL3. The VL region of the HA antibody is at least 80% homologous to the amino acid sequence of the VL region encoded the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or VL3. Preferably, the amino acid sequence of VL region of the HA antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or VL3, and more preferably, at least 98%, 99% homologous to the sequence encoded by the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or VL3.
In certain aspects of the invention the VKI germline gene is VKI (A20), the VKII germline gene is VKII (A3), the VKIII germline gene is VKIII (A27), and the VKIV germline gene is VKIV (B3). In other aspects of the invention, the VL1 germline gene is VL1 (V1-13), VL1 (V1-16), VL1 (V1-17), or. VL1 (V1-19). Alternatively, the VL2 germline gene is VL2 (V1-3) or VL2 (V1-4). Furthermore, the VL3 germline gene is VL3 (V2-14).
Specific combinations of a VH- and HL-locus are provided for each HA antibody described below.
The CDR regions of the HA antibodies of the invention were determined according to Kabat et al. (1991) as described in Sequences of Proteins of Immunological Interest. In certain embodiments of the invention, HA antibodies contain two, three, four, five or all six CDR regions as disclosed herein. Preferably, HA antibodies contain at least two of the CDRs disclosed herein.
The SC06-141 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 309) and a light chain variable region (SEQ ID NO: 310) encoded by the nucleic acid sequence shown in SEQ ID NO: 311 and the amino acid sequence shown in SEQ ID NO: 312. The VH-locus is VH1 (1-18) and the VL locus is HKIV (B3).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-141 antibody have the following CDR sequences: GYYVY (HCDR1, SEQ ID NO: 247), WISAYNGNTNYAQKFQG (HCDR2, SEQ ID NO: 248) and SRSLDV (HCDR3, SEQ ID NO: 568). The light chain CDRs of the SC06-141 antibody have the following CDR sequences: KSSQSVLYSSNNKNYLA (LCDR1, SEQ ID NO: 569), WASTRES (LCDR2, SEQ ID NO: 570) and QQYYSTPLT (LCDR3, SEQ ID NO: 289).







SC06-141 nucleotide sequence







(SEQ ID NO: 311)








gaggtccagc tggtgcagtc tggggctgag gtgaagaagc
60


ctggggcctc agtgaaggtc


 


tcctgcaagg cttctgggta caccttcacc ggctactatg
120


tgtactgggt gcgacaggcc


 


cctggacaag ggcttgagtg gatgggatgg atcagcgctt
180


acaatggtaa cacaaactat


 


gcacagaagt tccagggcag agtcacgatt accgcggaca
240


aatccacgag cacagcctac


 


atggagctga gcagcctgag atctgaagac acggctgtgt
300


attactgtgc gagaagtaga


 


tccctggacg tctggggcca agggaccacg gtcaccgtct
360


cgagcggtac gggcggttca


 


ggcggaaccg gcagcggcac tggcgggtcg acggatgttg
420


tgatgactca gtctccagac


 


tccctggctg tgtctctggg cgagagggcc accatcaact
480


gcaagtccag ccagagtgtt


 


ttatacagct ccaacaataa gaactactta gcttggtacc
540


agcagaaacc aggacagcct


 


cctaagctgc tcatttactg ggcatctacc cgggaatccg
600


gggtccctga ccgattcagt


 


ggcagcgggt ctgggacaga tttcactctc accatcagca
660


gcctgcaggc tgaagatgtg


 


gcagtttatt actgtcagca atattatagt actcctctca
720


ctttcggcgg agggaccaaa


 


gtggatatca aacgt
735


 







SC06-141 amino acid sequence







(SEQ ID NO: 312)







EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMG


 


WISAYNGNTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR


 


SRSLDVWGQGTTVTVSSGTGGSGGTGSGTGGSTDVVMTQSPDSLAVSLG


 


ERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVP


 


DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVDIKR


 


SC06-141 VH amino acid sequence







(SEQ ID NO: 309)







EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMG


 


WISAYNGNTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR


 


SRSLDVWGQGTTVTVSS


 


SC06-141 VL amino acid sequence







(SEQ ID NO: 310)







DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP


 


PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYY


 


STPLTFGGGTKVDIKR






The SC06-255 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 313) and a light chain variable region (SEQ ID NO: 314) encoded by the nucleic acid sequence shown in SEQ ID NO: 315 and the amino acid sequence shown in SEQ ID NO: 316. The VH-locus is VH1 (1-69) and the VL locus is VL1 (V1-16).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-255 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-255 antibody have the following CDR sequences: SGSTFNIGSNAVD (LCDR1, SEQ ID NO: 574), SNNQRPS (LCDR2, SEQ ID NO: 575) and AAWDDILNVPV (LCDR3, SEQ ID NO: 576).







SC06-255 nucleotide sequence







(SEQ ID NO: 315)








gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaagtc


 


tcttgcaagg cttctggagg ccccttccgc agctatgcta
120


tcagctgggt gcgacaggcc


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta
180


tttttggtac aacaaaatac


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg
240


atttcgcggg cacagtttac


 


atggagctga gcagcctgcg atctgaggac acggccatgt
300


actactgtgc gaaacatatg


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag
360


ggaccacggt caccgtctcg


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg
420


gcgggtcgac gtcctatgtg


 


ctgactcagc caccctcagc gtctgggacc cccgggcaga
480


gggtcaccat ctcttgttct


 


ggaagcacgt tcaacatcgg aagtaatgct gtagactggt
540


accggcagct cccaggaacg


 


gcccccaaac tcctcatcta tagtaataat cagcggccct
600


caggggtccc tgaccgattc


 


tctggctcca ggtctggcac ctcagcctcc ctggccatca
660


gtgggctcca gtctgaggat


 


gaggctgatt attactgtgc agcatgggat gacatcctga
720


atgttccggt attcggcgga


 


gggaccaagc tgaccgtcct aggt
744


 







SC06-255 amino acid sequence







(SEQ ID NO: 316)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSA


 


SGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIYSNNQRPSGV


 


PDRFSGSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPVFGGGTKLT


 


VLG


 


SC06-255 VH amino acid sequence







(SEQ ID NO: 313)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSS


 


SC06-255 VL amino acid sequence







(SEQ ID NO: 314)







SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLI


 


YSNNQRPSGVPDRFSGSRSGTSASLAISGLQSEDEADYYCAAWDDILNV


 


PVFGGGTKLTVLG






The SC06-257 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 317) and a light chain variable region (SEQ ID NO: 318) encoded by the nucleic acid sequence shown in SEQ ID NO: 319 and the amino acid sequence shown in SEQ ID NO: 320. The VH-locus is VH1 (1-69) and the VL locus is VL2 (V1-4).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-257 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-257 antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDR1, SEQ ID NO: 577), EVSNRPS (LCDR2, SEQ ID NO: 578) and SSYTSSSTY (LCDR3, SEQ ID NO: 579).







SC06-257 nucleotide sequence







(SEQ ID NO: 319)








caggtccagc tggtgcagtc tggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaagtc


 


tcttgcaagg cttctggagg ccccttccgc agctatgcta
120


tcagctgggt gcgacaggcc


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta
180


tttttggtac aacaaaatac


 


gcaccgaagt tccaggcag agtcacgatt accgcggacg
240


atttcgcggg cacagtttac


 


atggagctga gcagcctgcg atctgaggac acggccatgt
300


actactgtgc gaaacatatg


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag
360


ggaccacggt caccgtctcg


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg
420


gcgggtcgac gcagtctgcc


 


ctgactcagc ctgccgccgt gtctgggtct cctggacagt
480


cgatcaccat ctcctgcact


 


ggaaccagca gtgacgttgg tggttataac tatgtctcct
540


ggtaccaaca gcacccaggc


 


aaagccccca aactcatgat ttatgaggtc agtaatcggc
600


cctcaggggt ttctaatcgc


 


ttctctggct ccaagtctgg caacacggcc tccctgacca
660


tctctgggct ccaggctgag


 


gacgaggctg attattactg cagctcatat acaagcagca
720


gcacttatgt cttcggaact


 


gggaccaagg tcaccgtcct aggt
744


 


SC06-257 amino acid sequence







(SEQ ID NO: 320)







QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTQSALTQPAAV


 


SGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSG


 


VSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVT


 


VLG


 


SC06-257 VH amino acid sequence







(SEQ ID NO: 317)







QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSS


 


SC06-257 VL amino acid sequence







(SEQ ID NO: 318)







QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLM


 


IYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSST


 


YVFGTGTKVTVLG






The SC06-260 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 321) and a light chain variable region (SEQ ID NO: 322) encoded by the nucleic acid sequence shown in SEQ ID NO: 323 and the amino acid sequence shown in SEQ ID NO: 324. The VH-locus is VH1 (1-69) and the VL locus is VL1 (V1-17).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-260 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-260 antibody have the following CDR sequences: SGSRSNVGDNSVY (LCDR1, SEQ ID NO: 580), KNTQRPS (LCDR2, SEQ ID NO: 581) and VAWDDSVDGYV (LCDR3, SEQ ID NO: 582).







SC06-260 nucleotide sequence







(SEQ ID NO: 323)








gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaagtc


 


tcttgcaagg cttctggagg ccccttccgc agctatgcta
120


tcagctgggt gcgacaggcc


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta
180


tttttggtac aacaaaatac


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg
240


atttcgcggg cacagtttac


 


atggagctga gcagcctgcg atctgaggac acggccatgt
300


actactgtgc gaaacatatg


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag
360


ggaccacggt caccgtctcg


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg
420


gcgggtcgac gtcctatgtg


 


ctgactcagc caccctcagt ctctgggacc cccgggcaga
480


gggtcaccat ctcttgctct


 


ggaagccgct ccaacgtcgg agataattct gtatattggt
540


atcaacacgt cccagaaatg


 


gcccccaaac tcctcgtcta taagaatact caacggccct
600


caggagtccc tgcccggttt


 


tccggctcca agtctggcac ttcagcctcc ctggccatca
660


ttggcctcca gtccggcgat


 


gaggctgatt attattgtgt ggcatgggat gacagcgtag
720


atggctatgt cttcggatct


 


gggaccaagg tcaccgtcct aggt
744


 


SC06-260 amino acid sequence







(SEQ ID NO: 324)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSV


 


SGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVYKNTQRPSGV


 


PARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYVFGSGTKVT


 


VLG


 


SC06-260 VH amino acid sequence







(SEQ ID NO: 321)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSS


 


SC06-260 VL amino acid sequence







(SEQ ID NO: 322)







SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLV


 


YKNTQRPSGVPARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDG


 


YVFGSGTKVTVLG






The SC06-261 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 325) and a light chain variable region (SEQ ID NO: 326) encoded by the nucleic acid sequence shown in SEQ ID NO: 327 and the amino acid sequence shown in SEQ ID NO: 328. The VH-locus is VH1 (1-69) and the VL locus is VL1 (V1-19).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-261 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-261 antibody have the following CDR sequences: SGSSSNIGNDYVS (LCDR1, SEQ ID NO: 583), DNNKRPS (LCDR2, SEQ ID NO: 584) and ATWDRRPTAYVV (LCDR3, SEQ ID NO: 585).







SC06-261 nucleotide sequence







(SEQ ID NO: 327).








gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaagtc


 


tcttgcaagg cttctggagg ccccttccgc agctatgcta
120


tcagctgggt gcgacaggcc


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta
180


tttttggtac aacaaaatac


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg
240


atttcgcggg cacagtttac


 


atggagctga gcagcctgcg atctgaggac acggccatgt
300


actactgtgc gaaacatatg


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag
360


ggaccacggt caccgtctcg


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg
420


gcgggtcgac gcagtctgtg


 


ttgacgcagc cgccctcagt gtctgcggcc ccaggacaga
480


aggtcaccat ctcctgctct


 


ggaagcagct ccaacattgg gaatgattat gtatcctggt
540


accagcagct cccaggaaca


 


gcccccaaac tcctcattta tgacaataat aagcgaccct
600


cagggattcc tgaccgattc


 


tctggctcca agtctggcac gtcagccacc ctgggcatca
660


ccggactcca gactggggac


 


gaggccaact attactgcgc aacatgggat cgccgcccga
720


ctgcttatgt tgtcttcggc


 


ggagggacca agctgaccgt cctaggt
747


 


SC06-261 amino acid sequence







(SEQ ID NO: 328)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTQSVLTQPPSV


 


SAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGI


 


PDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKL


 


TVLG


 


SC06-261 VH amino acid sequence







(SEQ ID NO: 325)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSS


 


SC06-261 VL amino acid sequence







(SEQ ID NO: 326)







SVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIY


 


DNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAY


 


VVFGGGTKLTVLG






The SC06-262 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 329) and a light chain variable region (SEQ ID NO: 330) encoded by the nucleic acid sequence shown in SEQ ID NO: 331 and the amino acid sequence shown in SEQ ID NO: 332. The VH-locus is VH1 (1-69) and the VL locus is VKI (A20).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-262 antibody have the following CDR sequences: GSAIS (HCDR1, SEQ ID NO: 586), GISPLFGTTNYAQKFQG (HCDR2, SEQ ID NO: 587) and GPKYYSEYMDV (HCDR3, SEQ ID NO: 588). The light chain CDRs of the SC06-262 antibody have the following CDR sequences: RASQGISSYLA (LCDR1, SEQ ID NO: 589), DASTLRS (LCDR2, SEQ ID NO: 590) and QRYNSAPPI (LCDR3, SEQ ID NO: 591).







SC06-262 nucleotide sequence







(SEQ ID NO: 331)








caggtacagc tgcagcagtc aggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaggtc


 


tcctgcaagg tttccggagt cattttcagc ggcagtgcga
120


tcagctgggt gcgacaggcc


 


cctggacaag gccttgagtg gatgggaggg atcagccctc
180


tctttggcac aacaaattac


 


gcacaaaagt tccagggcag agtcacgatt accgcggacc
240


aatccacgaa cacaacctac


 


atggaggtga acagcctgag atatgaggac acggccgtgt
300


atttctgtgc gcgaggtcca


 


aaatattaca gtgagtacat ggacgtctgg ggcaaaggga
360


ccacggtcac cgtctcgagc


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
420


ggtcgacgga catccagatg


 


acccagtctc catcctccct gtctgcatct gtaggagaca
480


gagtcaccat cacttgccgg


 


gcgagtcagg gcattagcag ttatttagcc tggtatcagc
540


agaagccagg gaaagttcct


 


acactcctga tctatgatgc atccactttg cgatcagggg
600


tcccatctcg cttcagtggc


 


agtggatctg cgacagattt cactctcacc atcagcagcc
660


tgcagcctga agatgttgca


 


acttattact gtcaaaggta taacagtgcc cccccgatca
720


ccttcggcca agggacacga


 


ctggagatta aacgt
735


 


SC06-262 amino acid sequence







(SEQ ID NO: 332)







QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMG


 


GISPLFGTTNYAQKFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCAR


 


GPKYYSEYMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTDIQMTQSPSSL


 


SASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYDASTLRSGVPS


 


RFSGSGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFGQGTRLEIKR


 


SC06-262 VH amino acid sequence







(SEQ ID NO: 329)







QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMG


 


GISPLFGTTNYAQKFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCAR


 


GPKYYSEYMDVWGKGTTVTVSS


 


SC06-262 VL amino acid sequence







(SEQ ID NO: 330)







DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIY


 


DASTLRSGVPSRFSGSGSATDFTLTISSLQPEDVATYYCQRYNSAPPIT


 


FGQGTRLEIKR






The SC06-268 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 333) and a light chain variable region (SEQ ID NO: 334) encoded by the nucleic acid sequence shown in SEQ ID NO: 335 and the amino acid sequence shown in SEQ ID NO: 336. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-268 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIMGMFGTTNYAQKFQG (HCDR2, SEQ ID NO: 592) and SSGYYPEYFQD (HCDR3, SEQ ID NO: 593). The light chain CDRs of the SC06-268 antibody have the following CDR sequences: SGHKLGDKYVS (LCDR1, SEQ ID NO: 594), QDNRRPS (LCDR2, SEQ ID NO: 595) and QAWDSSTA (LCDR3, SEQ ID NO: 596).







SC06-268 nucleotide sequence







(SEQ ID NO: 335)








caggtccagc tggtacagtc tggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaggtc


 


tcctgcaagg cttctggagg caccttcagt agttatgcta
120


tcagctgggt gcgacaggcc


 


cctggacaag ggcttgagtg gatgggagga atcatgggta
180


tgtttggcac aactaactac


 


gcacagaagt tccagggcag agtcacgatt accgcggacg
240


aattcacgag cgcagcctac


 


atggagctga ggagcctgag atctgaggac acggccgtct
300


actactgtgc gaggtctagt


 


ggttattacc ccgaatactt ccaggactgg ggccagggca
360


ccctggtcac cgtctcgagc


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
420


ggtcgacgca gtctgtgctg


 


actcagccac cctcagagtc cgtgtcccca ggacagacag
480


ccagcgtcac ctgctctgga


 


cataaattgg gggataaata tgtttcgtgg tatcagcaga
540


agccaggcca gtcccctgta


 


ttactcatct atcaagataa caggcggccc tcagggatcc
600


ctgagcgatt cataggctcc


 


aactctggga acacagccac tctgaccatc agcgggaccc
660


aggctctgga tgaggctgac


 


tattactgtc aggcgtggga cagcagcact gcggttttcg
720


gcggagggac caagctgacc


 


gtcctaggt
729


 


SC06-268 amino acid sequence







(SEQ ID NO: 336)







QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG


 


GIMGMFGTTNYAQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCAR


 


SSGYYPEYFQDWGQGTLVTVSSGTGGSGGTGSGTGGSTQSVLTQPPSES


 


VSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQDNRRPSGIPER


 


FIGSNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLG


 


SC06-268 VH amino acid sequence







(SEQ ID NO: 333)







QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG


 


GIMGMFGTTNYAQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCAR


 


SSGYYPEYFQDWGQGTLVTVSS


 


SC06-268 VL amino acid sequence







(SEQ ID NO: 334)







QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQ


 


DNRRPSGIPERFIGSNSGNTATLTISGTQALDEADYYCQAWDSSTAVFG


 


GGTKLTVLG






The SC06-272 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 337) and a light chain variable region (SEQ ID NO: 338) encoded by the nucleic acid sequence shown in SEQ ID NO: 339 and the amino acid sequence shown in SEQ ID NO: 340. The VH-locus is VH1 (1-69) and the VL locus is VL2 (V1-3).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-272 antibody have the following CDR sequences: SYAIT (HCDR1, SEQ ID NO: 597), GIIGMFGSTNYAQNFQG (HCDR2, SEQ ID NO: 598) and STGYYPAYLHH (HCDR3, SEQ ID NO: 599). The light chain CDRs of the SC06-272 antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDR1, SEQ ID NO: 577), DVSKRPS (LCDR2, SEQ ID NO: 601) and SSYTSSSTHV (LCDR3, SEQ ID NO: 602).







SC06-272 nucleotide sequence







(SEQ ID NO: 339)








cagatgcagc tggtgcagtc tggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaggtc


 


tcctgcaagg cttctggagg caccttctcc agttatgcta
120


tcacctgggt gcgacaggcc


 


cctggacaag ggcttgagtg gatgggaggg atcatcggta
180


tgtttggttc aacaaactac


 


gcacagaact tccagggcag agtcacgatt accgcggacg
240


aatccacgag cacagcctac


 


atggagctga gcagcctcag atctgaggac acggccgtgt
300


attactgtgc gagaagtact


 


ggttattacc ctgcatacct ccaccactgg ggccagggca
360


ccctggtcac cgtctcgagc


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
420


ggtcgacgca gtctgccctg


 


actcagcctc gctcagtgtc cgggtctcct ggacagtcag
480


tcaccatctc ctgcactgga


 


accagcagtg atgttggtgg ttataactat gtctcctggt
540


accaacagca cccaggcaaa


 


gcccccaaac tcatgattta tgatgtcagt aagcggccct
600


caggggtccc tgatcgcttc


 


tctggctcca agtctggcaa cacggcctcc ctgaccatct
660


ctgggctcca ggctgaggat


 


gaggctgatt attactgcag ctcatataca agcagcagca
720


ctcatgtctt cggaactggg


 


accaaggtca ccgtcctagg t
741


 


SC06-272 amino acid sequence







(SEQ ID NO: 340)







QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMG


 


GIIGMFGSTYAQNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARS


 


TGYYPAYLHHWGQGTLVTVSSGTGGSGGTGSGTGGSTQSALTQPRSVSG


 


SPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPD


 


RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVLG


 


SC06-272 VH amino acid sequence







(SEQ ID NO: 337)







QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMG


 


GIIGMFGSTNYAQNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR


 


STGYYPAYLHHWGQGTLVTVSS


 


SC06-272 VL amino acid sequence







(SEQ ID NO: 338)







QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLM


 


IYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSST


 


HVFGTGTKVTVLG






The SC06-296 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 341) and a light chain variable region (SEQ ID NO: 342) encoded by the nucleic acid sequence shown in SEQ ID NO: 343 and the amino acid sequence shown in SEQ ID NO: 344. The VH-locus is VH1 (1-2) and the VL locus is VKIII (A27).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-296 antibody have the following CDR sequences: SYYMH (HCDR1, SEQ ID NO: 603), WINPNSGGTNYAQKFQG (HCDR2, SEQ ID NO: 604) and EGKWGPQAAFDI (HCDR3, SEQ ID NO: 605). The light chain CDRs of the SC06-296 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), DASSRAT (LCDR2, SEQ ID NO: 607) and QQYGSSLW (LCDR3, SEQ ID NO: 608).







SC06-296 nucleotide sequence







(SEQ ID NO: 343)








gaggtgcagc tggtggagac cggggctgag gtgaagaagc
60


ctggggcctc agtgaaggtt


 


tcctgcaagg catctggata caccttcacc agctactata
120


tgcactgggt gcgacaggcc


 


cctggacaag ggcttgagtg gatgggatgg atcaacccta
180


acagtggtgg cacaaactat


 


gcacagaagt ttcagggcag ggtcaccatg accagggaca
240


cgtccatcag cacagcctac


 


atggagctga gcaggctgag atctgacgac acggccgtgt
300


attactgtgc gagagagggg


 


aaatggggac ctcaagcggc ttttgatatc tggggccaag
360


ggacaatggt caccgtctcg


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg
420


gcgggtcgac ggaaattgtg


 


atgacgcagt ctccaggcac cctgtctttg tctccagggg
480


aaagagccac cctctcctgc


 


agggccagtc agagtgttag cagcagctac ttagcctggt
540


accagcagaa acctggccag


 


gctcccaggc tcctcatcta tgatgcatcc agcagggcca
600


ctgacatccc agacaggttc


 


agtggcagtg ggtctgggac agacttcact ctcaccatca
660


gcagactgga gcctgaagat


 


tttgcagtgt attactgtca gcagtatggt agctcacttt
720


ggacgttcgg ccaagggacc


 


aaggtggaga tcaaacgt
738


 


SC06-296 amino acid sequence







(SEQ ID NO: 344)







EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG


 


WINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR


 


EGKWGPQAAFDIWGQGTMVTVSSGTGGSGGTGSGTGGSTEIVMTQSPGT


 


LSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATDI


 


PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFGQGTKVEIKR


 


SC06-296VH amino acid sequence







(SEQ ID NO: 341)







EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG


 


WINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR


 


EGKWGPQAAFDIWGQGTMVTVSS


 


SC06-296 VL amino acid sequence







(SEQ ID NO: 342)







EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLI


 


YDASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWT


 


FGQGTKVEIKR






The SC06-301 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 345) and a light chain variable region (SEQ ID NO: 346) encoded by the nucleic acid sequence shown in SEQ ID NO: 347 and the amino acid sequence shown in SEQ ID NO: 348. The VH-locus is VH1 (3-23) and the VL locus is VKII (A3).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-301 antibody have the following CDR sequences: IYAMS (HCDR1, SEQ ID NO: 609), AISSSGDSTYYADSVKG (HCDR2, SEQ ID NO: 610) and AYGYTFDP (HCDR3, SEQ ID NO: 611). The light chain CDRs of the SC06-301 antibody have the following CDR sequences: RSSQSLLHSNGYNYLD (LCDR1, SEQ ID NO: 612), LGSNRAS (LCDR2, SEQ ID NO: 613) and MQALQTPL (LCDR3, SEQ ID NO: 614).







SC06-301 nucleotide sequence







(SEQ ID NO: 347)








gaggtgcagc tggtagagtc tgggggaggc ttggtacagc
60


ctggggggtc cctgagactc


 


tcctgtgcag cctctggatt cacctttagc atctatgcca
120


tgagctgggt ccgccaggca


 


ccagggaagg ggctggagtg ggtctcagct attagtagta
180


gtggtgatag cacatactac


 


gcagactccg tgaagggccg gttcaccatc tccagagaca
240


acgccaggaa cacgctgtat


 


ctgcaaatga acagtctgag agccgaggac acggctgtgt
300


attactgtgc gagagcgtat


 


ggctacacgt tcgacccctg gggccaggga accctggtca
360


ccgtctcgag cggtacgggc


 


ggttcaggcg gaaccggcag cggcactggc gggtcgacgg
420


aaattgtgct gactcagtct


 


ccactctccc tgcccgtcac ccctggagag ccggcctcca
480


tctcctgcag gtctagtcag


 


agcctcctgc atagtaatgg atacaactat ttggattggt
540


acctgcagaa gccagggcag


 


tctccacagc tcctgatcta tttgggttct aatcgggcct
600


ccggggtccc tgacaggttc


 


agtggcagtg gatcaggcac agattttaca ctgaaaatca
660


gcagagtgga ggctgaggat


 


gttggggttt attactgcat gcaagctcta caaactcccc
720


tcactttcgg cggagggacc


 


aaggtggaga tcaaacgt
738


 


SC06-301 amino acid sequence







(SEQ ID NO: 348)







EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVS


 


AISSSGDSTYYADSVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCAR


 


AYGYTFDPWGQGTLVTVSSGTGGSGGTGSGTGGSTEIVLTQSPLSLPVT


 


PGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGV


 


PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR


 


SC06-301 VH amino acid sequence







(SEQ ID NO: 345)







EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVS


 


AISSSGDSTYYADSVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCAR


 


AYGYTFDPWGQGTLVTVSS


 


SC06-301 VL amino acid sequence







(SEQ ID NO: 346)







EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSP


 


QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQ


 


TPLTFGGGTKVEIKR






The SC06-307 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 349) and a light chain variable region (SEQ ID NO: 350) encoded by the nucleic acid sequence shown in SEQ ID NO: 351 and the amino acid sequence shown in SEQ ID NO: 352. The VH-locus is VH3 (3-21) and the VL locus is VKIII (A27).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-307 antibody have the following CDR sequences: SYSMN (HCDR1, SEQ ID NO: 615), SISSSSSYIYYVDSVKG (HCDR2, SEQ ID NO: 616) and GGGSYGAYEGFDY (HCDR3, SEQ ID NO: 617). The light chain CDRs of the SC06-307 antibody have the following CDR sequences: RASQRVSSYLA (LCDR1, SEQ ID NO: 618), GASTRAA (LCDR2, SEQ ID NO: 619) and QQYGRTPLT (LCDR3, SEQ ID NO: 620).







SC06-307 nucleotide sequence







(SEQ ID NO: 351)








caggtccagc tggtgcagtc tgggggaggc ctggtcaagc
60


ctggggggtc cctgagactc


 


tcctgtgcag cctctggatt caccttcagt agctatagca
120


tgaactgggt ccgccaggct


 


ccagggaagg ggctggagtg ggtctcatcc attagtagta
180


gtagtagtta catatactac


 


gtagactcag tgaagggccg attcaccatc tccagagaca
240


acgccaagaa ctcactgtat


 


ctgcaaatga acagcctgag agccgaggac acggctgtgt
300


attactgtgc gagaggtggt


 


gggagctacg gggcctacga aggctttgac tactggggcc
360


agggcaccct ggtcaccgtc


 


tcgagcggta cgggcggttc aggcggaacc ggcagcggca
420


ctggcgggtc gacggaaatt


 


gtgctgactc agtctccagg caccctgtct ttgtctccag
480


gggaaagagc caccctctcc


 


tgcagggcca gtcagcgtgt tagcagctac ttagcctggt
540


accaacagaa acctggccag


 


gctcccaggc tcctcatcta tggtgcatcc accagggccg
600


ctggcatccc agacaggttc


 


agtggcagtg ggtctgggac agacttcact ctcaccatca
660


gcagactgga gcctgaagat


 


tctgcagtgt attactgtca gcagtatggt aggacaccgc
720


tcactttcgg cggagggacc


 


aaggtggaga tcaaacgt
738


 


SC06-307 amino acid sequence







(SEQ ID NO: 352)







QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVS


 


SISSSSSYIYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR


 


GGGSYGAYEGFDYWGQGTLVTVSSGTGGSGGTGSGTGGSTEIVLTQSPG


 


TLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYGASTRAAGI


 


PDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGGGTKVEIKR


 


SC06-307 VH amino acid sequence







(SEQ ID NO: 349)







QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVS


 


SISSSSSYIYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR


 


GGGSYGAYEGFDYWGQGTLVTVSS


 


SC06-307 VL amino acid sequence







(SEQ ID NO: 350)







EIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIY


 


GASTRAAGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTF


 


GGGTKVEIKR






The SC06-310 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 353) and a light chain variable region (SEQ ID NO: 354) encoded by the nucleic acid sequence shown in SEQ ID NO: 355 and the amino acid sequence shown in SEQ ID NO: 356. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-310 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-310 antibody have the following CDR sequences: GGNNIGSKSVH (LCDR1, SEQ ID NO: 621), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDHAV (LCDR3, SEQ ID NO: 623).







SC06-310 nucleotide sequence







(SEQ ID NO: 355)








gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
60


ctgggtcctc ggtgaaagtc


 


tcttgcaagg cttctggagg ccccttccgc agctatgcta
120


tcagctgggt gcgacaggcc


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta
180


tttttggtac aacaaaatac


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg
240


atttcgcggg cacagtttac


 


atggagctga gcagcctgcg atctgaggac acggccatgt
300


actactgtgc gaaacatatg


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag
360


ggaccacggt caccgtctcg


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg
420


gcgggtcgac gtcctatgtg


 


ctgactcagc caccctcggt gtcagtggcc ccaggacaga
480


cggccaggat tacctgtggg


 


ggaaacaaca ttggaagtaa aagtgtgcac tggtaccagc
540


agaagccagg ccaggcccct


 


gtgctggtcg tctatgatga tagcgaccgg ccctcaggga
600


tccctgagcg attctctggc


 


tccaactctg ggaacacggc caccctgacc atcagcaggg
660


tcgaagccgg ggatgaggcc


 


gactattact gtcaggtgtg ggatagtagt agtgatcatg
720


ctgtgttcgg aggaggcacc


 


cagctgaccg tcctcggt
738


 


SC06-310 amino acid sequence







(SEQ ID NO: 356)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSV


 


SVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPE


 


RFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLG


 


SC06-310 VH amino acid sequence







(SEQ ID NO: 353)







EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMG


 


GIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAK


 


HMGYQVRETMDVWGKGTTVTVSS


 


SC06-310 VL amino acid sequence







(SEQ ID NO: 354)







SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYD


 


DSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAV


 


FGGGTQLTVLG






The SC06-314 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 357) and a light chain variable region (SEQ ID NO: 358) encoded by the nucleic acid sequence shown in SEQ ID NO: 359 and the amino acid sequence shown in SEQ ID NO: 360. The VH-locus is VH1 (1-69) and the VL locus is VL1 (V1-17).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-314 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-314 antibody have the following CDR sequences: SGSSSNIGSNYVY (LCDR1, SEQ ID NO: 624), RDGQRPS (LCDR2, SEQ ID NO: 625) and ATWDDNLSGPV (LCDR3, SEQ ID NO: 626).








SC06-314 nucleotide sequence









(SEQ ID NO: 359)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc
60



 


tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac
180


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac
240


 


atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gtcctatgtg
420


 


ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct
480


 


ggaagcagct ccaacatcgg aagtaattat gtatactggt accagcagct cccaggcacg
540


 


gcccccaaac tcctcatcta tagggatggt cagcggccct caggggtccc tgaccgattc
600


 


tctggctcca agtctggcac ctcagcctcc ctggccatca gtggactccg gtccgatgat
660


 


gaggctgatt attactgtgc aacatgggat gacaacctga gtggtccagt attcggcgga
720


 


gggaccaagc tgaccgtcct aggt
744


 


SC06-314 amino acid sequence








(SEQ ID NO: 360)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSG


 


TGGSGGTGSGTGGSTSYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLI


 


YRDGQRPSGVPDRFSGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPVFGGGTKLTVLG


 


SC06-314 VH amino acid sequence








(SEQ ID NO: 357)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYA



 


PKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS


 


SC06-314 VL amino acid sequence








(SEQ ID NO: 358)









SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKWYRDGQRPSGVPDRF



 


SGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPVFGGGTKLTVLG






The SC06-323 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 361) and a light chain variable region (SEQ ID NO: 362) encoded by the nucleic acid sequence shown in SEQ ID NO: 363 and the amino acid sequence shown in SEQ ID NO: 364. The VH-locus is VH1 (1-69) and the VL locus is VKIII (A27).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-323 antibody have the following CDR sequences: SYGIS (HCDR1, SEQ ID NO: 627), DIIGMFGSTNYAQNFQG (HCDR2, SEQ ID NO: 628) and SSGYYPAYLPH (HCDR3, SEQ ID NO: 629). The light chain CDRs of the SC06-323 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSPRT (LCDR3, SEQ ID NO: 632).








SC06-323 nucleotide sequence









(SEQ ID NO: 363)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc cagggtcctc ggtgaaggtc
60



 


tcctgtaagg cctctggagg caccttctcc agctatggta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggagac atcatcggta tgtttggttc aacaaactac
180


 


gcacagaact tccagggcag actcacgatt accgcggacg aatccacgag cacagcctac
240


 


atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaagtagt
300


 


ggttattacc ctgcatacct cccccactgg ggccagggca ccttggtcac cgtctcgagc
360


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgga aattgtgttg
420


 


acccagtctc caggcaccct gtctttgtct ccaggggaaa gagccaccct ctcctgcagg
480


 


gccagtcaga gtgttagcag cagctactta gcctggtacc agcagaaacc tggccaggct
540


 


cccaggctcc tcatctatgg tgcatccagc agggccactg gcatcccaga caggttcagt
600


 


ggcagtgggt ctgggacaga cttcactctc accatcagca gactggagcc tgaagatttt
660


 


gcagtgtatt actgtcagca gtatggtagc tcacccagaa ctttcggcgg agggaccaag
720


 


gtggagatca aacgt
735


 


SC06-323 amino acid sequence








(SEQ ID NO: 364)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGDIIGMFGSTNYA



 


QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSSGTG


 


GSGGTGSGTGGSTEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY


 


GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR


 


SC06-323 VH amino acid sequence








(SEQ ID NO: 361)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGDIIGMFGSTNYA



 


QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSS


 


SC06-323 VL amino acid sequence








(SEQ ID NO: 362)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS



 


GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR






The SC06-325 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 365) and a light chain variable region (SEQ ID NO: 366) encoded by the nucleic acid sequence shown in SEQ ID NO: 367 and the amino acid sequence shown in SEQ ID NO: 368. The VH-locus is VH1 (1-69) and the VL locus is VL2 (V1-4).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-325 antibody have the following CDR sequences: FYSMS (HCDR1, SEQ ID NO: 633), GIIPMFGTTNYAQKFQG (HCDR2, SEQ ID NO: 634) and GDKGIYYYYMDV (HCDR3, SEQ ID NO: 635). The light chain CDRs of the SC06-325 antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDR1, SEQ ID NO: 577), EVSNRPS (LCDR2, SEQ ID NO: 578) and SSYTSSSTLV (LCDR3, SEQ ID NO: 636).








SC06-325 nucleotide sequence









(SEQ ID NO: 367)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc cggggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttcagc ttctattcta tgagctgggt gcgacaggcc
120


 


cctggacaag gacttgagtg gatgggaggg atcatcccta tgtttggtac aacaaactac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggtcg aatccacgag cacagcctac
240


 


atggaggtga gcagcctgag atctgaggac acggccgttt attactgtgc gagaggtgat
300


 


aagggtatct actactacta catggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gcagtctgcc
420


 


ctgactcagc ctgcctccgt gtctgggtct cctggacagt cgatcaccat ctcctgcact
480


 


ggaaccagca gtgacgttgg tggttataac tatgtctcct ggtaccaaca gcacccaggc
540


 


aaagccccca aactcatgat ttatgaggtc agtaatcggc cctcaggggt ttctaatcgc
600


 


ttctctggct ccaagtctgg caacacggcc tccctgacca tctctgggct ccaggctgag
660


 


gacgaggctg attattactg cagctcatat acaagcagca gcactcttgt cttcggaact
720


 


gggaccaagg tcaccgtcct aggt
744


 


SC06-325 amino acid sequence








(SEQ ID NO: 368)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFGTTNYA



 


QKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGDKGIYYYYMDVWGKGTTVTVSSG


 


TGGSGGTGSGTGGSTQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK


 


LMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLG


 


SC06-325 VH amino acid sequence








(SEQ ID NO: 365)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFGTTNY



 


AQKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGDKGIYYYYMDVWGKGTTVTVSS


 


SC06-325 VL amino acid sequence








(SEQ ID NO: 366)









QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSN



 


RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLG






The SC06-327 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 369) and a light chain variable region (SEQ ID NO: 370) encoded by the nucleic acid sequence shown in SEQ ID NO: 371 and the amino acid sequence shown in SEQ ID NO: 372. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-327 antibody have the following CDR sequences: THAIS (SEQ ID NO: 637), GIIAIFGTANYAQKFQG (SEQ ID NO: 638) and GSGYHISTPFDN (SEQ ID NO: 639). The light chain CDRs of the SC06-327 antibody have the following CDR sequences: GGNNIGSKGVH (SEQ ID NO: 640), DDSDRPS (SEQ ID NO: 622) and QVWDSSSDHVV (SEQ ID NO: 642).








SC06-327 nucleotide sequence









(SEQ ID NO: 371)










gaggtgcagc tggtggagac cggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cctctggagg caccttcagg acccatgcta tcagttgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcgcta tcttcggaac agcaaactac
180


 


gcacagaagt tccagggcag aatcacgatt accgcggacg aatccacgag tacagcctac
240


 


atggagctga gcagcctgag atctgaggac acggccgtgt atttctgtgc gagaggcagt
300


 


ggttatcata tatcgacacc ctttgacaac tggggccagg gaaccctggt caccgtctcg
360


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gtcctatgtg
420


 


ctgactcagc caccctcggt gtcagtggcc ccaggacaga cggccaggat tacctgtggg
480


 


ggaaacaaca ttggaagtaa aggtgtgcac tggtaccagc agaagcctgg ccaggcccct
540


 


gtgctggtcg tctatgatga tagcgaccgg ccctcaggga tccctgagcg attctctggc
600


 


tccaactctg ggaacacggc caccctgacc atcagcaggg tcgaagccgg ggatgaggcc
660


 


gactattact gtcaggtgtg ggatagtagt agtgatcatg tggtattcgg cggagggacc
720


 


aagctgaccg tcctaggt
738


 


SC06-327 amino acid sequence








(SEQ ID NO: 372)









EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGGIIAIFGTANYA



 


QKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGSGYHISTPFDNWGQGTLVTVSSG


 


TGGSGGTGSGTGGSTSYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLV


 


VYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG


 


SC06-327 VH amino acid sequence








(SEQ ID NO: 369)









EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGGIIAIFGTANYA



 


QKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGSGYHISTPFDNWGQGTLVTVSS


 


SC06-327 VL amino acid sequence








(SEQ ID NO: 370)









SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG






The SC06-328 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 373) and a light chain variable region (SEQ ID NO: 374) encoded by the nucleic acid sequence shown in SEQ ID NO: 375 and the amino acid sequence shown in SEQ ID NO: 376. The VH-locus is VH1 (1-69) and the VL locus is VKIII (A27).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-328 antibody have the following CDR sequences: GYAIS (HCDR1, SEQ ID NO: 643), GIIPIFGTTNYAQKFQG (HCDR2, SEQ ID NO: 644) and VKDGYCTLTSCPVGWYFDL (HCDR3, SEQ ID NO: 645). The light chain CDRs of the SC06-328 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSLT (LCDR3, SEQ ID NO: 648).








SC06-328 nucleotide sequence









(SEQ ID NO: 375)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggaca catcttcagc ggctatgcaa tcagttgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac aacaaactac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacc aatccacgag cacagcctac
240


 


atggacctga gcaacttgag atctgaggac acggccgtct attactgtgc gagagtgaaa
300


 


gatggatatt gtactcttac cagctgccct gtcggctggt acttcgatct ctggggccgt
360


 


ggcaccctgg tcactgtctc gagcggtacg ggcggttcag gcggaaccgg cagcggcact
420


 


ggcgggtcga cggaaattgt gatgacgcag tctccaggca ccctgtcttt gtctccaggg
480


 


gaaagagcca ccctctcgtg cagggccagt cagagtgtta gcagcagcta cttagcctgg
540


 


taccagcaga aacctggcca ggctcccagg ctcctcatct ttggtgcctc cagcagggcc
600


 


actggcatcc cagacaggtt cagtggcagt gggtctggga cagacttcac tctcaccatc
660


 


agcagactgg agcctgaaga ttttgcagtg tattactgtc agcagtatgg tagctcactc
720


 


actttcggcg gagggaccaa gctggagatc aaacgt
756


 


SC06-328 amino acid sequence








(SEQ ID NO: 376)









EVALVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGGIIPIFGTTNYAQ



 


KFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVKDGYCTLTSCPVGWYFDLWGRGTL


 


VTVSSGTGGSGGTGSGTGGSTEIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ


 


QKPGQAPRLLIFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLT


 


FGGGTKLEIKR


 


SC06-328 VH amino acid sequence








(SEQ ID NO: 373)









EVALVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGGIIPIFGTTNYA



 


QKFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVKDGYCTLTSCPVGWYFDLWGR


 


GTLVTVSS


 


SC06-328 VL amino acid sequence








(SEQ ID NO: 374)









EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASSRATG



 


IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGGGTKLEIKR






The SC06-329 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 377) and a light chain variable region (SEQ ID NO: 378) encoded by the nucleic acid sequence shown in SEQ ID NO: 379 and the amino acid sequence shown in SEQ ID NO: 380. The VH-locus is VH1 (1-69) and the VL locus is VKIII (A27).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-329 antibody have the following CDR sequences: SNSIS (HCDR1, SEQ ID NO: 649), GIFALFGTTDYAQKFQG (HCDR2, SEQ ID NO: 650) and GSGYTTRNYFDY (HCDR3, SEQ ID NO: 651). The light chain CDRs of the SC06-329 antibody have the following CDR sequences: RASQSVSSNYLG (LCDR1, SEQ ID NO: 652), GASSRAS (LCDR2, SEQ ID NO: 653) and QQYGSSPLT (LCDR3, SEQ ID NO: 654).








SC06-329 nucleotide sequence









(SEQ ID NO: 379)










gaggtccagc tggtacagtc tggggctgag gttaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg catcttcaga agcaattcta tcagttgggt gcgacaggcc
120


 


cctgggcaag ggcttgagtg gatgggaggg atcttcgctc ttttcggaac aacagactac
180


 


gcgcagaagt tccagggcag agtcacgatt accgcggacg aatcttcgac cacagtctac
240


 


ctggagctga gtagcctgac atctgaggac acggccgttt attactgtgc gagaggcagt
300


 


ggctacacca cacgcaacta ctttgactac tggggccagg gcaccctggt caccgtctcg
360


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac ggaaattgtg
420


 


ctgactcagt ctccaggcac cctgtctttg tctccagggg aaagagccac actctcctgc
480


 


agggccagtc agagtgttag cagcaactac ttaggctggt accagcagaa acctggccag
540


 


gctcccaggc tcctgatcta tggtgcatcc agcagggcca gtggcatccc agacaggttc
600


 


agtggcggtg ggtctgggac agacttcact ctcaccatca gcagactgga gcctgaagat
660


 


tttgcagtgt attactgtca gcagtatggt agctcacccc tcactttcgg cggagggacc
720


 


aaggtggaga tcaaacgt
738


 


SC06-329 amino acid sequence








(SEQ ID NO: 380)









EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGGIFALFGTTDYAQ



 


KFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGSGYTTRNYFDYWGQGTLVTVSSGTG


 


GSGGTGSGTGGSTEIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWTQQKPGQAPRLLIY


 


GASSRASGIPDRFSGGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR


 


SC06-329 VH amino acid sequence








(SEQ ID NO: 377)









EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGGIFALFGTTDYA



 


QKFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGSGYTTRNYFDYWGQGTLVTVSS


 


SC06-329 VL amino acid sequence








(SEQ ID NO: 378)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWTQQKPGQAPRLLIYGASSRASGIPDRFS



 


GGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR






The SC06-331 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 381) and a light chain variable region (SEQ ID NO: 382) encoded by the nucleic acid sequence shown in SEQ ID NO: 383 and the amino acid sequence shown in SEQ ID NO: 384. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-331antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 571), GIIGMFGTANYAQKFQG (HCDR2, SEQ ID NO: 655) and GNYYYESSLDY (HCDR3, SEQ ID NO: 656). The light chain CDRs of the SC06-331 antibody have the following CDR sequences: GGNNIGSKSVH (LCDR1, SEQ ID NO: 621), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDH (LCDR3, SEQ ID NO: 657).








SC06-331 nucleotide sequence









(SEQ ID NO: 383)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcggta tgttcggtac agcaaactac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatttacgag cacagcctac
240


 


atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggaaat
300


 


tattactatg agagtagtct cgactactgg ggccagggaa ccctggtcac cgtctcgagc
360


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgca gtctgtcgtg
420


 


acgcagccgc cctcggtgtc agtggcccca ggacagacgg ccaggattac ctgtggggga
480


 


aacaacattg gaagtaaaag tgtgcactgg taccagcaga agccaggcca ggcccctgtg
540


 


ctggtcgtct atgatgatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc
600


 


aactctggga acacggccac cctgaccatc agcagggtcg aagccgggga tgaggccgac
660


 


tattactgtc aggtgtggga tagtagtagt gatcattatg tcttcggaac tgggaccaag
720


 


gtcaccgtcc taggt
735


 


SC06-331 amino acid sequence








(SEQ ID NO: 384)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIGMFGTANYA



 


QKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGNYYYESSLDYWGQGTLVTVSSGT


 


GGSGGTGSGTGGSTQSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVV


 


YDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG


 


SC06-331 VH amino acid sequence








(SEQ ID NO: 381)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIGMFGTANY



 


AQKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGNYYYESSLDYWGQGTLVTVSS


 


SC06-331 VL amino acid sequence








(SEQ ID NO: 382)









QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG






The SC06-332 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 385) and a light chain variable region (SEQ ID NO: 386) encoded by the nucleic acid sequence shown in SEQ ID NO: 387 and the amino acid sequence shown in SEQ ID NO: 388. The VH-locus is VH1 (1-69) and the VL locus is VKI (A20).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-332 antibody have the following CDR sequences: NFAIN (HCDR1, SEQ ID NO: 658), GIIAVFGTTKYAHKFQG (HCDR2, SEQ ID NO: 659) and GPHYYSSYMDV (HCDR3, SEQ ID NO: 660). The light chain CDRs of the SC06-332 antibody have the following CDR sequences: RASQGISTYLA (LCDR1, SEQ ID NO: 661), AASTLQS (LCDR2, SEQ ID NO: 662) and QKYNSAPS (LCDR3, SEQ ID NO: 663).








SC06-332 nucleotide sequence









(SEQ ID NO: 387)










caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtaaaggtc
60



 


tcctgcaagg cttctggagg ccccttccgc aattttgcta tcaactgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcgctg tctttgggac gacaaagtac
180


 


gcacataagt tccagggcag agtcaccatc accgcggacg actccacaaa tacagcttac
240


 


atggagctgg gcagcctgaa atctgaggac acggccgtgt attactgtgc gagaggtccc
300


 


cactactact cctcctacat ggacgtctgg ggcgaaggga ccacggtcac cgtctcgagc
360


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgga catccagttg
420


 


acccagtctc catcctccct gtctgcatct gtaggagaca gagtcaccat cacttgccgg
480


 


gcgagtcagg gcattagcac ttatttagcc tggtatcagc agaaacccgg gaaagttcct
540


 


aaactcctga tctatgctgc atccactttg caatcagggg tcccatctcg gttcagtggc
600


 


agtggatctg ggacagattt cactctcacc atcagcagcc tgcagcctga agatgttgca
660


 


acttattact gtcaaaagta taacagtgcc ccttctttcg gccctgggac caaagtggat
720


 


atcaaacgt
729


 


SC06-332 amino acid sequence








(SEQ ID NO: 388)









QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKYA



 


HKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGPHYYSSYMDVWGEGTTVTVSSGT


 


GGDGGTGSGTGGSTDIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIY


 


AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKR


 


SC06-332 VH amino acid sequence








(SEQ ID NO: 385)









QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKY



 


AHKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGPHYYSSYMDVWGEGTTVTVSS


 


SC06-332 VL amino acid sequence








(SEQ ID NO: 386)









DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFS



 


GSGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKR






The SC06-334 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 389) and a light chain variable region (SEQ ID NO: 390) encoded by the nucleic acid sequence shown in SEQ ID NO: 391 and the amino acid sequence shown in SEQ ID NO: 392. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-334 antibody have the following CDR sequences: SNAVS (HCDR1, SEQ ID NO: 664), GILGVFGSPSYAQKFQG (HCDR2, SEQ ID NO: 665) and GPTYYYSYMDV (HCDR3, SEQ ID NO: 666). The light chain CDRs of the SC06-334 antibody have the following CDR sequences: GGNNIGRNSVH (LCDR1, SEQ ID NO: 667), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWHSSSDHYV (LCDR3, SEQ ID NO: 669).








SC06-334 nucleotide sequence









(SEQ ID NO: 391)










gaggtgcagc tggtggagac tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


ccctgcaaat cttctggaag ccccttcagg agtaatgctg tcagctgggt gcgacaggcc
120


 


cccggacaag ggcttgagtg ggtgggagga atcctcggtg tctttggttc accaagctac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatccaccaa cacagtccac
240


 


atggagctga gaggtttgag atctgaggac acggccgtgt attattgtgc gagaggtcct
300


 


acctactact actcctacat ggacgtctgg ggcaaaggga ccacggtcac cgtctcgagc
360


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgtc ctatgtgctg
420


 


actcagccac cctcggagtc agtggcccca ggacagacgg ccaggattac ctgtggggga
480


 


aataacattg gaagaaatag tgtgcactgg tatcagcaga agccaggcca ggcccctgtg
540


 


ctggtcgtgt atgatgatag cgaccggccc tcagggatcc ctgagcgatt ttctggctcc
600


 


aagtctggga acacggccac cctgattatc agcagggtcg aagtcgggga tgaggccgac
660


 


tactactgtc aggtgtggca tagtagtagt gatcattatg tcttcggaac tgggaccaag
720


 


gtcaccgtcc taggt
735


 


SC06-334 amino acid sequence








(SEQ ID NO: 392)









EVALVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGGILGVFGSPSYA



 


QKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKGTTVTVSSG


 


TGGSGGTGSGTGGSTSYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVV


 


YDDSDRPSGIPERFSGSKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLG


 


SC06-334 VH amino acid sequence








(SEQ ID NO: 389)









EVALVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGGILGVFGSPSYA



 


QKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKGTTVTVSS


 


SC06-334 VL amino acid sequence








(SEQ ID NO: 390)









SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLG






The SC06-336 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 393) and a light chain variable region (SEQ ID NO: 394) encoded by the nucleic acid sequence shown in SEQ ID NO: 395 and the amino acid sequence shown in SEQ ID NO: 396. The VH-locus is VH1 (1-69) and the VL locus is VKIII (A27).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-336 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 670), GIFGMFGTANYAQKFQG (HCDR2, SEQ ID NO: 671) and SSGYYPQYFQD (HCDR3, SEQ ID NO: 672). The light chain CDRs of the SC06-336 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSSLT (LCDR3, SEQ ID NO: 308).








SC06-336 nucleotide sequence









(SEQ ID NO: 395)










cagatgcagc tggtacaatc tggagctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcttcggta tgtttgggac agcaaactac
180


 


gcgcagaagt tccagggcag agtcacgatt accgcggacg aattcacgag cgcggcctac
240


 


atggagctga gcagcctggg atctgaggac acggccatgt attactgtgc gaggtctagt
300


 


ggttattacc cccaatactt ccaggactgg ggccagggca ccctggtcac cgtctcgagc
360


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgga aattgtgatg
420


 


acacagtctc caggcaccct gtctttgtct ccagggcaaa gagccaccct ctcctgcagg
480


 


gccagtcaga gtgttagcag cagctactta gcctggtacc agcagaaacc tggccaggct
540


 


cccagactcc tcatgtatgg tgcatccagc agggccactg gcatcccaga caggttcagt
600


 


ggcagtgggt ctgggacaga cttcactctc accatcagca gactggagcc tgaagatttt
660


 


gcagtgtatt actgtcagca gtatggtagc tcatcgctca ctttcggcgg agggaccaag
720


 


ctggagatca aacgt
735


 


SC06-336 amino acid sequence








(SEQ ID NO: 396)









QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIFGMFGTANY



 


AQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSSGYYPQYFQDWGQGTLVTVSSG


 


TGGSGGTGSGTGGSTEIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRL


 


LMYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKR


 


SC06-336 VH amino acid sequence








(SEQ ID NO: 393)









QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIFGMFGTAN



 


YAQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSSGYYPQYFQDWGQGTLVTVSS


 


SC06-336 VL amino acid sequence








(SEQ ID NO: 394)









EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYGASSRATGIPDR



 


FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKR






The SC06-339 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 397) and a light chain variable region (SEQ ID NO: 398) encoded by the nucleic acid sequence shown in SEQ ID NO: 399 and the amino acid sequence shown in SEQ ID NO: 400. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-339 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 303), GIIAIFHTPKYAQKFQG (HCDR2, SEQ ID NO: 306) and GSTYDFSSGLDY (HCDR3, SEQ ID NO: 725). The light chain CDRs of the SC06-339 antibody have the following CDR sequences: GGNNIGSKSVH (LCDR1, SEQ ID NO: 621), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDHVV (LCDR3, SEQ ID NO: 642).








SC06-339 nucleotide sequence









(SEQ ID NO: 399)










gaggtgcagc tggtggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg catcttcaac agttatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggc atcatcgcta tctttcatac accaaagtac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa cacagcctac
240


 


atggaactga gaagcctgaa atctgaggac acggccctgt attactgtgc gagagggtcc
300


 


acttacgatt tttcgagtgg ccttgactac tggggccagg gaaccctggt caccgtctcg
360


 


agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gcaggcaggg
420


 


ctgactcagc caccctcggt gtcagtggcc ccaggacaga cggccaggat tacctgtggg
480


 


ggaaacaaca ttggaagtaa aagtgtgcac tggtaccagc agaagccagg ccaggcccct
540


 


gtcctagtcg tctatgatga tagcgaccgg ccctcaggga tccctgagcg attctctggc
600


 


tccaactctg ggaacacggc caccctgacc atcagcaggg tcgaagccgg ggatgaggcc
660


 


gactattact gtcaggtgtg ggatagtagt agtgatcatg tggtattcgg cggagggacc
720


 


aagctgaccg tcctaggt
738


 


SC06-339 amino acid sequence








(SEQ ID NO: 400)









EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGGIIAIFHTPKYAQ



 


KFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGSTYDFSSGLDYWGQGTLVTVSSGTG


 


GSGGTGSGTGGSTQAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVY


 


DDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG


 


SC06-339 VH amino acid sequence








(SEQ ID NO: 397)









EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGGIIAIFHTPKYA



 


QKFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGSTYDFSSGLDYWGQGTLVTVSS


 


SC06-339 VL amino acid sequence








(SEQ ID NO: 398)









QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG






The SC06-342 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 401) and a light chain variable region (SEQ ID NO: 402) encoded by the nucleic acid sequence shown in SEQ ID NO: 403 and the amino acid sequence shown in SEQ ID NO: 404. The VH-locus is VH1 (1-69) and the VL locus is VKIV (B3).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-342 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 251), GVIPIFRTANYAQNFQG (HCDR2, SEQ ID NO: 249) and LNYHDSGTYYNAPRGWFDP (HCDR3, SEQ ID NO: 246). The light chain CDRs of the SC06-342 antibody have the following CDR sequences: KSSQSILNSSNNKNYLA (LCDR1, SEQ ID NO: 245), WASTRES (LCDR2, SEQ ID NO: 570) and QQYYSSPPT (LCDR3, SEQ ID NO: 250).








SC06-342 nucleotide sequence









(SEQ ID NO: 403)










caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg cttcttcagc agctatgcta tcagctgggt gcgccaggcc
120


 


cctggacaag gacttgagtg gatggggggg gtcatcccta tctttcgtac agcaaactac
180


 


gcacagaact tccagggcag agtcaccatt accgcggacg aattcacatc gtatatggag
240


 


ctgagcagcc tgagatctga cgacacggcc gtgtattact gtgcgaggtt gaattaccat
300


 


gattcgggga cttattataa cgccccccgg ggctggttcg acccctgggg ccagggaacc
360


 


ctggtcaccg tctcgagcgg tacgggcggt tcaggcggaa ccggcagcgg cactggcggg
420


 


tcgacggaca tccagatgac ccagtctcca gactccctgg ctgtgtctct gggcgagaag
480


 


gccaccatca actgcaagtc cagccagagt attttaaaca gctccaacaa taagaactac
540


 


ttagcttggt accagcagaa accaggacag cctcctaagc tgctcattta ctgggcatct
600


 


acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact
660


 


ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat
720


 


agtagtccgc cgacgttcgg ccaagggacc aaggtggaaa tcaaacgt
768


 


SC06-342 amino acid sequence








(SEQ ID NO: 404)









QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGGVIPIFRTANYA



 


QNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYHDSGTYYNAPRGWFDPWGQGTLV


 


TVSSGTGGSGGTGSGTGGSTDIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQ


 


QKPGQPPKLIAYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPPTFGQ


 


GTKVEIKR


 


SC06-342 VH amino acid sequence








(SEQ ID NO: 401)









QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGGVIPIFRTANYA



 


QNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYHDSGTYYNAPRGWFDPWGQGT


 


LVTVSS


 


SC06-342 VL amino acid sequence








(SEQ ID NO: 402)









DIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPPKLLIYWASTRES



 


GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPPTFGQGTKVEIKR






The SC06-343 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 405) and a light chain variable region (SEQ ID NO: 406) encoded by the nucleic acid sequence shown in SEQ ID NO: 407 and the amino acid sequence shown in SEQ ID NO: 408. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-343 antibody have the following CDR sequences: YYAMS (HCDR1, SEQ ID NO: 242), GISPMFGTTTYAQKFQG (HCDR2, SEQ ID NO: 307) and SSNYYDSVYDY (HCDR3, SEQ ID NO: 290). The light chain CDRs of the SC06-343 antibody have the following CDR sequences: GGHNIGSNSVH (LCDR1, SEQ ID NO: 224), DNSDRPS (LCDR2, SEQ ID NO: 223) and QVWGSSSDH (LCDR3, SEQ ID NO: 227).








SC06-343 nucleotide sequence









(SEQ ID NO: 407)










caggtccagc tggtgcagtc tggagctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagt caccttcagt tactatgcta tgagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggagga atcagcccta tgtttgggac aacaacctac
180


 


gcacagaagt tccagggcag agtcacgatt actgcggacg actccacgag tacagcctac
240


 


atggaggtga ggagcctgag atctgaggac acggccgtgt attactgtgc gagatcttcg
300


 


aattactatg atagtgtata tgactactgg ggccagggaa ccctggtcac cgtctcgagc
360


 


ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgca gtctgtcgtg
420


 


acgcagccgc cctcggagtc agtggcccca ggacagacgg ccaggattac ctgtggggga
480


 


cataacattg gaagtaatag tgtgcactgg taccagcaga agccaggcca ggcccctgtg
540


 


ctggtcgtgt atgataatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc
600


 


aactctggga acacggccac cctgaccatc agcagggtcg aagccgggga tgaggccgac
660


 


tattactgtc aggtgtgggg tagtagtagt gaccattatg tcttcggaac tgggaccaag
720


 


gtcaccgtcc taggt
735


 


SC06-343 amino acid sequence








(SEQ ID NO: 408)









QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGGISPMFGTTTY



 


AQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVSSG


 


TGGSGGTGSGTGGSTQSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVV


 


YDNSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLG


 


SC06-343 VH amino acid sequence








(SEQ ID NO: 405)









QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGGISPMFGTTT



 


YAQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVSS


 


SC06-343 VL amino acid sequence








(SEQ ID NO: 406)









QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDNSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLG






The SC06-344 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 409) and a light chain variable region (SEQ ID NO: 410) encoded by the nucleic acid sequence shown in SEQ ID NO: 411 and the amino acid sequence shown in SEQ ID NO: 412. The VH-locus is VH1 (1-69) and the VL locus is VL1 (V1-13).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SC06-344 antibody have the following CDR sequences: NYAMS (HCDR1, SEQ ID NO: 222), GIIAIFGTPKYAQKFQG (HCDR2, SEQ ID NO: 221) and IPHYNFGSGSYFDY (HCDR3, SEQ ID NO: 220). The light chain CDRs of the SC06-344 antibody have the following CDR sequences: TGSSSNIGAGYDVH (LCDR1, SEQ ID NO: 219), GNSNRPS (LCDR2, SEQ ID NO: 231) and GTWDSSLSAYV (LCDR3, SEQ ID NO: 280).








SC06-344 nucleotide sequence









(SEQ ID NO: 411)










caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgagagtc
60



 


tcctgcaagg cttctggaag catcttcaga aactatgcta tgagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcgcta tttttgggac accaaagtac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatcgacgag cactgtctac
240


 


atggaactga gcggactgag atctgaggac acggccatgt attactgtgc gaggattccc
300


 


cactataatt ttggttcggg gagttatttc gactactggg gccagggaac cctggtcacc
360


 


gtctcgagcg gtacgggcgg ttcaggcgga accggcagcg gcactggcgg gtcgacgact
420


 


gtgttgacac agccgccctc agtgtctggg gccccagggc agagggtcac catctcctgc
480


 


actgggagca gctccaacat cggggcaggt tatgatgtac actggtacca gcagcttcca
540


 


ggaacagccc ccaaactcct catctatggt aacagcaatc ggccctcagg ggtccctgac
600


 


cgattctctg gctccaagtc tggcacgtca gccaccctgg gcatcaccgg actccagact
660


 


ggggacgagg ccgattatta ctgcggaaca tgggatagca gcctgagtgc ttatgtcttc
720


 


ggaactggga ccaaggtcac cgtcctaggt
750


 


SC06-344 amino acid sequence








(SEQ ID NO: 412)









QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGGIIAIFGTPKYA



 


QKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVTVSS


 


GTGGSGGTGSGTGGSTTVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK


 


LLIYGNSNRPSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVT


 


VLG


 


SC06-344 VH amino acid sequence








(SEQ ID NO: 409)









QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGGIIAIFGTPKY



 


AQKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVT


 


VSS


 


SC06-344 VL amino acid sequence








(SEQ ID NO: 410)









TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDR



 


FSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLG






IgG HA Antibodies

The CR6141 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 199) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 279 and the heavy chain amino acid sequence shown in SEQ ID NO: 413. The CR6141 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 414) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 415 and the light chain amino acid sequence shown in SEQ ID NO: 416.








CR6141 Heavy Chain nucleotide sequence









(SEQ ID NO: 279)










gaggtccagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc
60



 


tcctgcaagg cttctgggta caccttcacc ggctactatg tgtactgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat
180


 


gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac
240


 


atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagaagtaga
300


 


tccctggacg tctggggcca agggaccacg gtcaccgtct cgagtgctag caccaagggc
360


 


cccagcgtgt tccccctggc ccccagcagc aagagcacca gcggcggcac agccgccctg
420


 


ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgagctggaa cagcggcgcc
480


 


ttgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct gtacagcctg
540


 


agcagcgtgg tgaccgtgcc cagcagcagc ctgggcaccc agacctacat ctgcaacgtg
600


 


aaccacaagc ccagcaacac caaggtggac aaacgcgtgg agcccaagag ctgcgacaag
660


 


acccacacct gccccccctg ccctgccccc gagctgctgg gcggaccctc cgtgttcctg
720


 


ttccccccca agcccaagga caccctcatg atcagccgga cccccgaggt gacctgcgtg
780


 


gtggtggacg tgagccacga ggaccccgag gtgaagttca actggtacgt ggacggcgtg
840


 


gaggtgcaca acgccaagac caagccccgg gaggagcagt acaacagcac ctaccgggtg
900


 


gtgagcgtgc tcaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaag
960


 


gtgagcaaca aggccctgcc tgcccccatc gagaagacca tcagcaaggc caagggccag
1020


 


ccccgggagc cccaggtgta caccctgccc cccagccggg aggagatgac caagaaccag
1080


 


gtgtccctca cctgtctggt gaagggcttc taccccagcg acatcgccgt ggagtgggag
1140


 


agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc
1200


 


agcttcttcc tgtacagcaa gctcaccgtg gacaagagcc ggtggcagca gggcaacgtg
1260


 


ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgagc
1320


 


ctgagccccg gcaag
1335


 


CR6141 Heavy Chain amino acid sequence








(SEQ ID NO: 413)









EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGWISAYNGNTN



 


YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSRSLDVWGQGTTVTVSSASTKGP


 


SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV


 


TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL


 


FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV


 


VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ


 


VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV


 


FSCSVMHEALHNHYTQKSLSLSPGK


 


CR6141 VH amino acid sequence








(SEQ ID NO: 199)









EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGWISAYNGNTN



 


YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSRSLDVWGQGTTVTVSS


 


CR6141 Light Chain nucleotide sequence








(SEQ ID NO: 415)










gatgttgtga tgactcagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc
60



 


atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct
120


 


tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg
180


 


gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc
240


 


atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtact
300


 


cctctcactt tcggcggagg gaccaaagtg gatatcaaac gtgcggccgc acccagcgtg
360


 


ttcatcttcc ccccctccga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg
420


 


ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag
480


 


agcggcaaca gccaggagag cgtgaccgag caggacagca aggactccac ctacagcctg
540


 


agcagcaccc tcaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag
600


 


gtgacccacc agggcctgag cagccccgtg accaagagct tcaaccgggg cgagtgt
657


 


CR6141 Light Chain amino acid sequence








(SEQ ID NO: 416)









DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES



 


GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVDIKRAAAPSVFIFPPS


 


DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK


 


ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC


 


CR6141 VL amino acid sequence








(SEQ ID NO: 414)









DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES



 


GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVDIKR






The CR6255 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 417) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 418 and the heavy chain amino acid sequence shown in SEQ ID NO: 419. The CR6255 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 420) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 421 and the light chain amino acid sequence shown in SEQ ID NO: 422.








CR6255 Heavy Chain nucleotide sequence









(SEQ ID NO: 418)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc
60



 


tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac
180


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac
240


 


atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6255 Heavy Chain amino acid sequence









(SEQ ID NO: 419)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS


 


SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG


 


GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY


 


NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE


 


EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR


 


WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK


 


CR6255 VH amino acid sequence








(SEQ ID NO: 417)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKY



 


APKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS


 


CR6255 Light Chain nucleotide sequence








(SEQ ID NO: 421)










tcctatgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc
60



 


tcttgttctg gaagcacgtt caacatcgga agtaatgctg tagactggta ccggcagctc
120


 


ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct
180


 


gaccgattct ctggctccag gtctggcacc tcagcctccc tggccatcag tgggctccag
240


 


tctgaggatg aggctgatta ttactgtgca gcatgggatg acatcctgaa tgttccggta
300


 


ttcggcggag ggaccaagct gaccgtccta ggtgcggccg caggccagcc caaggccgct
360


 


cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg
420


 


gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc
480


 


agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac
540


 


gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc
600


 


tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660


 








CR6255 Light Chain amino acid sequence









(SEQ ID NO: 422)









SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIYSNNQRPSGVPDRFS



 


GSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPVFGGGTKLTVLGAAAGQPKAAPSVTLF


 


PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6255 VL amino acid sequence









(SEQ ID NO: 420)









SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIYSNNQRPSGVPDRFS



 


GSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPVFGGGTKLTVLG






The CR6257 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 423) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 424 and the heavy chain amino acid sequence shown in SEQ ID NO: 425. The CR6257 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 426) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 427 and the light chain amino acid sequence shown in SEQ ID NO: 428.








CR6257 Heavy Chain nucleotide sequence









(SEQ ID NO: 424)










caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc
60



 


tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac
180


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac
240


 


atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6257 Heavy Chain amino acid sequence









(SEQ ID NO: 425)









QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKY



 


APKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVS


 


SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL


 


YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF


 


PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY


 


NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE


 


EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR


 


WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK


 


CR6257 VH amino acid sequence








(SEQ ID NO: 423)









QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKY



 


APKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS


 


CR6257 Light Chain nucleotide sequence








(SEQ ID NO: 427)










cagtctgccc tgactcagcc tgccgccgtg tctgggtctc ctggacagtc gatcaccatc
60



 


tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag
120


 


cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt
180


 


tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240


 


caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cacttatgtc
300


 


ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc caaggccgct
360


 


cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg
420


 


gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc
480


 


agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac
540


 


gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc
600


 


tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660


 








CR6257 Light Chain amino acid sequence








(SEQ ID NO: 428)









QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSN



 


RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVLGAAAGQPKAAPSVTL


 


FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYS CQVTHEGSTVEKTVAPTECS


 


CR6257 VL amino acid sequence








(SEQ ID NO: 426)









QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSN



 


RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVLG






The CR6260 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 429) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 430 and the heavy chain amino acid sequence shown in SEQ ID NO: 431. The CR6260 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 432) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 433 and the light chain amino acid sequence shown in SEQ ID NO: 434.








CR6260 Heavy Chain nucleotide sequence









(SEQ ID NO: 430)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc
60



 


tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac
180


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac
240


 


atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6260 Heavy Chain amino acid sequence









(SEQ ID NO: 431)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6260 VH amino acid sequence








(SEQ ID NO: 429)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS


 


CR6260 Light Chain nucleotide sequence








(SEQ ID NO: 433)










tcctatgtgc tgactcagcc accctcagtc tctgggaccc ccgggcagag ggtcaccatc
60



 


tcttgctctg gaagccgctc caacgtcgga gataattctg tatattggta tcaacacgtc
120


 


ccagaaatgg cccccaaact cctcgtctat aagaatactc aacggccctc aggagtccct
180


 


gcccggtttt ccggctccaa gtctggcact tcagcctccc tggccatcat tggcctccag
240


 


tccggcgatg aggctgatta ttattgtgtg gcatgggatg acagcgtaga tggctatgtc
300


 


ttcggatctg ggaccaaggt caccgtccta ggtgcggccg caggccagcc caaggccgct
360


 


cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg
420


 


gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc
480


 


agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac
540


 


gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc
600


 


tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660


 








CR6260 Light Chain amino acid sequence









(SEQ ID NO: 434)









SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVYKNTQRPSGVP



 


ARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYVFGSGTKVTVLGAAAGQPKAAP


 


SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY


 


AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6260 VL amino acid sequence








(SEQ ID NO: 432)









SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVYKNTQRPSGVP



 


ARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYVFGSGTKVTVLG






The CR6261 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 435) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 436 and the heavy chain amino acid sequence shown in SEQ ID NO: 437. The CR6261 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 438) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 439 and the light chain amino acid sequence shown in SEQ ID NO: 440.








CR6261 Heavy Chain nucleotide sequence









(SEQ ID NO: 436)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc
60



 


tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac
180


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac
240


 


atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6261 Heavy Chain amino acid sequence









(SEQ ID NO: 437)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6261 VH amino acid sequence








(SEQ ID NO: 435)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS


 


CR6261 Light Chain nucleotide sequence








(SEQ ID NO: 439)










cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc
60



 


tcctgctctg gaagcagctc caacattggg aatgattatg tatcctggta ccagcagctc
120


 


ccaggaacag cccccaaact cctcatttat gacaataata agcgaccctc agggattcct
180


 


gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240


 


actggggacg aggccaacta ttactgcgca acatgggatc gccgcccgac tgcttatgtt
300


 


gtcttcggcg gagggaccaa gctgaccgtc ctaggtgcgg ccgcaggcca gcccaaggcc
360


 


gctcccagcg tgaccctgtt ccccccctcc tccgaggagc tgcaggccaa caaggccacc
420


 


ctggtgtgcc tcatcagcga cttctaccct ggcgccgtga ccgtggcctg gaaggccgac
480


 


agcagccccg tgaaggccgg cgtggagacc accaccccca gcaagcagag caacaacaag
540


 


tacgccgcca gcagctacct gagcctcacc cccgagcagt ggaagagcca ccggagctac
600


 


agctgccagg tgacccacga gggcagcacc gtggagaaga ccgtggcccc caccgagtgc
660


 








CR6261 Light Chain amino acid sequence









(SEQ ID NO: 440)









QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS



 


GSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVLGAAAGQPKAAPSVT


 


LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS


 


LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6261VL amino acid sequence








(SEQ ID NO: 438)









QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS



 


GSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVLG






The CR6262 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 441) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 442 and the heavy chain amino acid sequence shown in SEQ ID NO: 443. The CR6262 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 444) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 445 and the light chain amino acid sequence shown in SEQ ID NO: 446.








CR6262 Heavy Chain nucleotide sequence









(SEQ ID NO: 442)










caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg tttccggagt cattttcagc ggcagtgcga tcagctgggt gcgacaggcc
120


 


cctggacaag gccttgagtg gatgggaggg atcagccctc tctttggcac aacaaattac
180


 


gcacaaaagt tccagggcag agtcacgatt accgcggacc aatccacgaa cacaacctac
240


 


atggaggtga acagcctgag atatgaggac acggccgtgt atttctgtgc gcgaggtcca
300


 


aaatattaca gtgagtacat ggacgtctgg ggcaaaggga ccacggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 








CR6262 Heavy Chain amino acid sequence









(SEQ ID NO: 443)









QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGGISPLFGTTNYAQ



 


KFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGPKYYSEYMDVWGKGTTVTVSSAST


 


KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS


 


SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV


 


LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL


 


HNHYTQKSLSLSPGK


 


CR6262 VH amino acid sequence








(SEQ ID NO: 441)









QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGGISPLFGTTNYAQ



 


KFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGPKYYSEYMDVWGKGTTVTVSS


 


CR6262 Light Chain nucleotide sequence








(SEQ ID NO: 445)










gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60



 


atcacttgcc gggcgagtca gggcattagc agttatttag cctggtatca gcagaagcca
120


 


gggaaagttc ctacactcct gatctatgat gcatccactt tgcgatcagg ggtcccatct
180


 


cgcttcagtg gcagtggatc tgcgacagat ttcactctca ccatcagcag cctgcagcct
240


 


gaagatgttg caacttatta ctgtcaaagg tataacagtg cccccccgat caccttcggc
300


 


caagggacac gactggagat taaacgtgcg gccgcaccca gcgtgttcat cttccccccc
360


 


tccgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac
420


 


ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag
480


 


gagagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctcacc
540


 


ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc
600


 


ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt
642


 








CR6262 Light Chain amino acid sequence









(SEQ ID NO: 446)









DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYDASTLRSGVPSRFSG



 


SGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFGQGTRLEIKRAAAPSVFIFPPSDEQLKSGT


 


ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK


 


VYACEVTHQGLSSPVTKSFNRGEC


 


CR6262 VL amino acid sequence








(SEQ ID NO: 444)









DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYDASTLRSGVPSRFSG



 


SGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFGQGTRLEIKR






The CR6268 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 447) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 448 and the heavy chain amino acid sequence shown in SEQ ID NO: 449. The CR6268 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 450) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 451 and the light chain amino acid sequence shown in SEQ ID NO: 452.








CR6268 Heavy Chain nucleotide sequence









(SEQ ID NO: 448)










caggtccagc tggtacagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttcagt agttatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggagga atcatgggta tgtttggcac aactaactac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aattcacgag cgcagcctac
240


 


atggagctga ggagcctgag atctgaggac acggccgtct actactgtgc gaggtctagt
300


 


ggttattacc ccgaatactt ccaggactgg ggccagggca ccctggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 








CR6268 Heavy Chain amino acid sequence









(SEQ ID NO: 449)









QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIMGMFGTTNY



 


AQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCARSSGYYPEYFQDWGQGTLVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6268 VH amino acid sequence








(SEQ ID NO: 447)









QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIMGMFGTTNY



 


AQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCARSSGYYPEYFQDWGQGTLVTVSS


 


CR6268 Light Chain nucleotide sequence








(SEQ ID NO: 451)










cagtctgtgc tgactcagcc accctcagag tccgtgtccc caggacagac agccagcgtc
60



 


acctgctctg gacataaatt gggggataaa tatgtttcgt ggtatcagca gaagccaggc
120


 


cagtcccctg tattactcat ctatcaagat aacaggcggc cctcagggat ccctgagcga
180


 


ttcataggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctctg
240


 


gatgaggctg actattactg tcaggcgtgg gacagcagca ctgcggtttt cggcggaggg
300


 


accaagctga ccgtcctagg tgcggccgca ggccagccca aggccgctcc cagcgtgacc
360


 


ctgttccccc cctcctccga ggagctgcag gccaacaagg ccaccctggt gtgcctcatc
420


 


agcgacttct accctggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag
480


 


gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc
540


 


tacctgagcc tcacccccga gcagtggaag agccaccgga gctacagctg ccaggtgacc
600


 


cacgagggca gcaccgtgga gaagaccgtg gcccccaccg agtgcagc
648


 


CR6268 Light Chain amino acid sequence








(SEQ ID NO: 452)









QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQDNRRPSGIPERFIG



 


SNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLGAAAGQPKAAPSVTLFPPS


 


SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPE


 


QWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6268 VL amino acid sequence








(SEQ ID NO: 450)









QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQDNRRPSGIPERFIG



 


SNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLG






The CR6272 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 453) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 454 and the heavy chain amino acid sequence shown in SEQ ID NO: 455. The CR6272 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 456) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 457 and the light chain amino acid sequence shown in SEQ ID NO: 458.








CR6272 Heavy Chain nucleotide sequence









(SEQ ID NO: 454)










cagatgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttctcc agttatgcta tcacctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcggta tgtttggttc aacaaactac
180


 


gcacagaact tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac
240


 


atggagctga gcagcctcag atctgaggac acggccgtgt attactgtgc gagaagtact
300


 


ggttattacc ctgcatacct ccaccactgg ggccagggca ccctggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 








CR6272 Heavy Chain amino acid sequence









(SEQ ID NO: 455)









QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGGIIGMFGSTNYA



 


QNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTGYYPAYLHHWGQGTLVTVSSAST


 


KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS


 


SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV


 


LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL


 


HNHYTQKSLSLSPGK


 


CR6272 VH amino acid sequence








(SEQ ID NO: 453)









QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGGIIGMFGSTNYA



 


QNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTGYYPAYLHHWGQGTLVTVSS


 


CR6272 Light Chain nucleotide sequence








(SEQ ID NO: 457)










cagtctgccc tgactcagcc tcgctcagtg tccgggtctc ctggacagtc agtcaccatc
60



 


tcctgcactg gaaccagcag tgatgttggt ggttataact atgtctcctg gtaccaacag
120


 


cacccaggca aagcccccaa actcatgatt tatgatgtca gtaagcggcc ctcaggggtc
180


 


cctgatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240


 


caggctgagg atgaggctga ttattactgc agctcatata caagcagcag cactcatgtc
300


 


ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc caaggccgct
360


 


cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg
420


 


gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc
480


 


agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac
540


 


gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc
600


 


tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660


 








CR6272 Light Chain amino acid sequence









(SEQ ID NO: 458)









QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPD



 


RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVLGAAAGQPKAAPSVTL


 


FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6272 VL amino acid sequence








(SEQ ID NO: 456)









GSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPD



 


RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVLG






The CR696 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 459) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 460 and the heavy chain amino acid sequence shown in SEQ ID NO: 461. The CR6296 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 462) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 463 and the light chain amino acid sequence shown in SEQ ID NO: 464.








CR6296 Heavy Chain nucleotide sequence









(SEQ ID NO: 460)










gaggtgcagc tggtggagac cggggctgag gtgaagaagc ctggggcctc agtgaaggtt
60



 


tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat
180


 


gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac
240


 


atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagagggg
300


 


aaatggggac ctcaagcggc ttttgatatc tggggccaag ggacaatggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6296 Heavy Chain amino acid sequence









(SEQ ID NO: 461)









EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGGTN



 


YAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGKWGPQAAFDIWGQGTMVTV


 


SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG


 


LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL


 


FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV


 


SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL


 


TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV


 


MHEALHNHYTQKSLSLSPGK


 


CR6296 VH amino acid sequence








(SEQ ID NO: 459)









EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGGTN



 


YAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGKWGPQAAFDIWGQGTMVTV


 


SS


 


CR6296 Light Chain nucleotide sequence








(SEQ ID NO: 463)










gaaattgtga tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60



 


ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
120


 


cctggccagg ctcccaggct cctcatctat gatgcatcca gcagggccac tgacatccca
180


 


gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240


 


cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcactttg gacgttcggc
300


 


caagggacca aggtggagat caaacgtgcg gccgcaccca gcgtgttcat cttccccccc
360


 


tccgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac
420


 


ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag
480


 


gagagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctcacc
540


 


ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc
600


 


ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt
642


 


CR6296 Light Chain amino acid sequence








(SEQ ID NO: 464)









EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATDIPDRFS



 


GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFGQGTKVEIKRAAAPSVFIFPPSDEQLKSG


 


TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH


 


KVYACEVTHQGLSSPVTKSFNRGEC


 


CR6296 VL amino acid sequence








(SEQ ID NO: 462)









EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATDIPDRFS



 


GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFGQGTKVEIKR






The CR6301 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 465) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 466 and the heavy chain amino acid sequence shown in SEQ ID NO: 467. The CR6301 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 468) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 469 and the light chain amino acid sequence shown in SEQ ID NO: 470.








CR6301 Heavy Chain nucleotide sequence









(SEQ ID NO: 466)










gaggtgcagc tggtagagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60



 


tcctgtgcag cctctggatt cacctttagc atctatgcca tgagctgggt ccgccaggca
120


 


ccagggaagg ggctggagtg ggtctcagct attagtagta gtggtgatag cacatactac
180


 


gcagactccg tgaagggccg gttcaccatc tccagagaca acgccaggaa cacgctgtat
240


 


ctgcaaatga acagtctgag agccgaggac acggctgtgt attactgtgc gagagcgtat
300


 


ggctacacgt tcgacccctg gggccaggga accctggtca ccgtctcgag tgctagcacc
360


 


aagggcccca gcgtgttccc cctggccccc agcagcaaga gcaccagcgg cggcacagcc
420


 


gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc
480


 


ggcgccttga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac
540


 


agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgc
600


 


aacgtgaacc acaagcccag caacaccaag gtggacaaac gcgtggagcc caagagctgc
660


 


gacaagaccc acacctgccc cccctgccct gcccccgagc tgctgggcgg accctccgtg
720


 


ttcctgttcc cccccaagcc caaggacacc ctcatgatca gccggacccc cgaggtgacc
780


 


tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac
840


 


ggcgtggagg tgcacaacgc caagaccaag ccccgggagg agcagtacaa cagcacctac
900


 


cgggtggtga gcgtgctcac cgtgctgcac caggactggc tgaacggcaa ggagtacaag
960


 


tgcaaggtga gcaacaaggc cctgcctgcc cccatcgaga agaccatcag caaggccaag
1020


 


ggccagcccc gggagcccca ggtgtacacc ctgcccccca gccgggagga gatgaccaag
1080


 


aaccaggtgt ccctcacctg tctggtgaag ggcttctacc ccagcgacat cgccgtggag
1140


 


tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc
1200


 


gacggcagct tcttcctgta cagcaagctc accgtggaca agagccggtg gcagcagggc
1260


 


aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc
1320


 


ctgagcctga gccccggcaa g
1341


 








CR6301 Heavy Chain amino acid sequence









(SEQ ID NO: 467)









EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSAISSSGDSTYYAD



 


SVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAYGYTFDPWGQGTLVTVSSASTKGP


 


SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV


 


TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT


 


LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ


 


DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY


 


PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN


 


HYTQKSLSLSPGK


 


CR6301 VH amino acid sequence








(SEQ ID NO: 465)









EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSAISSSGDSTYYAD



 


SVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAYGYTFDPWGQGTLVTVSS


 


CR6301 Light Chain nucleotide sequence








(SEQ ID NO: 469)










gaaattgtgc tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc
60



 


atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg
120


 


tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc
180


 


tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc
240


 


agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccc
300


 


ctcactttcg gcggagggac caaggtggag atcaaacgtg cggccgcacc cagcgtgttc
360


 


atcttccccc cctccgacga gcagctgaag agcggcaccg ccagcgtggt gtgcctgctg
420


 


aacaacttct acccccggga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc
480


 


ggcaacagcc aggagagcgt gaccgagcag gacagcaagg actccaccta cagcctgagc
540


 


agcaccctca ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg
600


 


acccaccagg gcctgagcag ccccgtgacc aagagcttca accggggcga gtgt
654


 








CR6301 Light Chain amino acid sequence









(SEQ ID NO: 470)









EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP



 


DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKRAAAPSVFIFPPSDE


 


QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA


 


DYEKHKVYACEVTHQGLSSPVTKSFNRGEC


 


CR6301 VL amino acid sequence








(SEQ ID NO: 468)









EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP



 


DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR






The CR6307 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 471) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 472 and the heavy chain amino acid sequence shown in SEQ ID NO: 473. The CR6307 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 474) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 475 and the light chain amino acid sequence shown in SEQ ID NO: 476.








CR6307 Heavy Chain nucleotide sequence









(SEQ ID NO: 472)










caggtccagc tggtgcagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc
60



 


tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct
120


 


ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac
180


 


gtagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240


 


ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagaggtggt
300


 


gggagctacg gggcctacga aggctttgac tactggggcc agggcaccct ggtcaccgtc
360


 


tcgagtgcta gcaccaaggg ccccagcgtg ttccccctgg cccccagcag caagagcacc
420


 


agcggcggca cagccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc
480


 


gtgagctgga acagcggcgc cttgaccagc ggcgtgcaca ccttccccgc cgtgctgcag
540


 


agcagcggcc tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc
600


 


cagacctaca tctgcaacgt gaaccacaag cccagcaaca ccaaggtgga caaacgcgtg
660


 


gagcccaaga gctgcgacaa gacccacacc tgccccccct gccctgcccc cgagctgctg
720


 


ggcggaccct ccgtgttcct gttccccccc aagcccaagg acaccctcat gatcagccgg
780


 


acccccgagg tgacctgcgt ggtggtggac gtgagccacg aggaccccga ggtgaagttc
840


 


aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagccccg ggaggagcag
900


 


tacaacagca cctaccgggt ggtgagcgtg ctcaccgtgc tgcaccagga ctggctgaac
960


 


ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ctgcccccat cgagaagacc
1020


 


atcagcaagg ccaagggcca gccccgggag ccccaggtgt acaccctgcc ccccagccgg
1080


 


gaggagatga ccaagaacca ggtgtccctc acctgtctgg tgaagggctt ctaccccagc
1140


 


gacaccgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc
1200


 


cctgtgctgg acagcgacgg cagcttcttc ctgtacagca agctcaccgt ggacaagagc
1260


 


cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac
1320


 


tacacccaga agagcctgag cctgagcccc ggcaag
1356


 








CR6307 Heavy Chain amino acid sequence









(SEQ ID NO: 473)









QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYVD



 


SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGGSYGAYEGFDYWGQGTLVTVSS


 


ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY


 


SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP


 


PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV


 


LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC


 


LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM


 


HEALHNHYTQKSLSLSPGK


 


CR6307 VH amino acid sequence








(SEQ ID NO: 471)









QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYVD



 


SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGGSYGAYEGFDYWGQGTLVTVSS


 


CR6307 Light Chain nucleotide sequence








(SEQ ID NO: 475)










gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60



 


ctctcctgca gggccagtca gcgtgttagc agctacttag cctggtacca acagaaacct
120


 


ggccaggctc ccaggctcct catctatggt gcatccacca gggccgctgg catcccagac
180


 


aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag actggagcct
240


 


gaagattctg cagtgtatta ctgtcagcag tatggtagga caccgctcac tttcggcgga
300


 


gggaccaagg tggagatcaa acgtgcggcc gcacccagcg tgttcatctt ccccccctcc
360


 


gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc
420


 


cgggaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag
480


 


agcgtgaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctcaccctg
540


 


agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg
600


 


agcagccccg tgaccaagag cttcaaccgg ggcgagtgt
639


 








CR6307 Light Chain amino acid sequence









(SEQ ID NO: 476)









EIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYGASTRAAGIPDRFSG



 


SGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGGGTKVEIKRAAAPSVFIFPPSDEQLKSGT


 


ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK


 


VYACEVTHQGLSSPVTKSFNRGEC


 


CR6307 VL amino acid sequence








(SEQ ID NO: 474)









EIVLYQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYGASTRAAGIPDRFS



 


GSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGGGTKVEIKR






The CR6310 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 477) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 478 and the heavy chain amino acid sequence shown in SEQ ID NO: 479. The CR6310 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 480) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 481 and the light chain amino acid sequence shown in SEQ ID NO: 482.








CR6310 Heavy Chain nucleotide sequence









(SEQ ID NO: 478)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc
60



 


tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac
180


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac
240


 


atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6310 Heavy Chain amino acid sequence









(SEQ ID NO: 479)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6310 VH amino acid sequence








(SEQ ID NO: 477)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS


 


CR6310 Light Chain nucleotide sequence








(SEQ ID NO: 481)










tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt
60



 


acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc
120


 


caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga
180


 


ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg
240


 


gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgc tgtgttcgga
300


 


ggaggcaccc agctgaccgt cctcggtgcg gccgcaggcc agcccaaggc cgctcccagc
360


 


gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc
420


 


ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc
480


 


gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc
540


 


agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag
600


 


gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc
654


 








CR6310 Light Chain amino acid sequence









(SEQ ID NO: 482)









SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG



 


SNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLGAAAGQPKAAPSVTLF


 


PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6310 VL amino acid sequence








(SEQ ID NO: 480)









SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG



 


SNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLG






The CR6314 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 483) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 484 and the heavy chain amino acid sequence shown in SEQ ID NO: 485. The CR6314 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 486) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 487 and the light chain amino acid sequence shown in SEQ ID NO: 488.








CR6314 Heavy Chain nucleotide sequence









(SEQ ID NO: 484)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc
60



 


tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac
180


 


gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac
240


 


atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300


 


gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6314 Heavy Chain amino acid sequence









(SEQ ID NO: 485)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6314 VH amino acid sequence








(SEQ ID NO: 483)









EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP



 


KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS


 


CR6314 Light Chain nucleotide sequence








(SEQ ID NO: 487)










tcctatgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc
60



 


tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccagcagctc
120


 


ccaggcacgg cccccaaact cctcatctat agggatggtc agcggccctc aggggtccct
180


 


gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tggactccgg
240


 


tccgatgatg aggctgatta ttactgtgca acatgggatg acaacctgag tggtccagta
300


 


ttcggcggag ggaccaagct gaccgtccta ggtgcggccg caggccagcc caaggccgct
360


 


cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg
420


 


gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc
480


 


agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac
540


 


gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc
600


 


tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660


 








CR6314 Light Chain amino acid sequence









(SEQ ID NO: 488)









SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRDGQRPSGVPDRFS



 


GSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPVFGGGTKLTVLGAAAQPKAAPSVTLFP


 


PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT


 


PGQWKSHRSYSCQVTHEGSTVEKTVAPTECSG


 


CR6314 VL amino acid sequence








(SEQ ID NO: 486)









SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRDGQRPSGVPDRFS



 


GSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPVFGGGTKLTVLG






The CR6323 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 489) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 490 and the heavy chain amino acid sequence shown in SEQ ID NO: 491. The CR6323 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 492) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 493 and the light chain amino acid sequence shown in SEQ ID NO: 494.








CR6323 Heavy Chain nucleotide sequence









(SEQ ID NO: 490)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc cagggtcctc ggtgaaggtc
60



 


tcctgtaagg cctctggagg caccttctcc agctatggta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggagac atcatcggta tgtttggttc aacaaactac
180


 


gcacagaact tccagggcag actcacgatt accgcggacg aatccacgag cacagcctac
240


 


atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaagtagt
300


 


ggttattacc ctgcatacct cccccactgg ggccagggca ccttggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 








CR6323 Heavy Chain amino acid sequence









(SEQ ID NO: 491)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGDIIGMFGSTNYA



 


QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSSAST


 


KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS


 


SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV


 


LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL


 


HNHYTQKSLSLSPGK


 


CR6323 VH amino acid sequence








(SEQ ID NO: 489)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGDIIGMFGSTNYA



 


QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSS


 


CR6323 Light Chain nucleotide sequence








(SEQ ID NO: 493)










gaaattgtgt tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60



 


ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
120


 


cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca
180


 


gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240


 


cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacccag aactttcggc
300


 


ggagggacca aggtggagat caaacgtgcg gccgcaccca gcgtgttcat cttccccccc
360


 


tccgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac
420


 


ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag
480


 


gagagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctcacc
540


 


ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc
600


 


ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt
642


 








CR6323 Light Chain amino acid sequence









(SEQ ID NO: 494)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS



 


GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKRAAAPSVFIFPPSDEQLKSG


 


TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH


 


KVYACEVTHQGLSSPVTKSFNRGEC


 


CR6323 VL amino acid sequence








(SEQ ID NO: 492)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS



 


GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR






The CR6325 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 495) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 496 and the heavy chain amino acid sequence shown in SEQ ID NO: 497. The CR6325 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 498) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 499 and the light chain amino acid sequence shown in SEQ ID NO: 500.








CR6325 Heavy Chain nucleotide sequence









(SEQ ID NO: 496)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc cggggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttcagc ttctattcta tgagctgggt gcgacaggcc
120


 


cctggacaag gacttgagtg gatgggaggg atcatcccta tgtttggtac aacaaactac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggtcg aatccacgag cacagcctac
240


 


atggaggtga gcagcctgag atctgaggac acggccgttt attactgtgc gagaggtgat
300


 


aagggtatct actactacta catggacgtc tggggcaaag ggaccacggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 








CR6325 Heavy Chain amino acid sequence









(SEQ ID NO: 497)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFGTTNYA



 


QKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGDKGIYYYYMDVWGKGTTVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6325 VH amino acid sequence








(SEQ ID NO: 495)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFGTTNYA



 


QKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGDKGIYYYYMDVWGKGTTVTVSS


 


CR6325 Light Chain nucleotide sequence








(SEQ ID NO: 499)










cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc
60



 


tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag
120


 


cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt
180


 


tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240


 


caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cactcttgtc
300


 


ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc caaggccgct
360


 


cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg
420


 


gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc
480


 


agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac
540


 


gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc
600


 


tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660


 


CR6325 Light Chain amino acid sequence








(SEQ ID NO: 500)









QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNR



 


FSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLGAAAGQPKAAPSVTLF


 


PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6325 VL amino acid sequence








(SEQ ID NO: 498)









QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNR



 


FSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLG






The CR6327 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 501) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 502 and the heavy chain amino acid sequence shown in SEQ ID NO: 503. The CR6327 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 504) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 505 and the light chain amino acid sequence shown in SEQ ID NO: 506.








CR6327 Heavy Chain nucleotide sequence









(SEQ ID NO: 502)










gaggtgcagc tggtggagac cggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cctctggagg caccttcagg acccatgcta tcagttgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcgcta tcttcggaac agcaaactac
180


 


gcacagaagt tccagggcag aatcacgatt accgcggacg aatccacgag tacagcctac
240


 


atggagctga gcagcctgag atctgaggac acggccgtgt atttctgtgc gagaggcagt
300


 


ggttatcata tatcgacacc ctttgacaac tggggccagg gaaccctggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 


CR6327 Heavy Chain amino acid sequence








(SEQ ID NO: 503)









EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGGIIAIFGTANYA



 


QKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGSGYHISTPFDNWGQGTLVTVSSAST


 


KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS


 


SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV


 


LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL


 


HNHYTQKSLSLSPGK


 


CR6327 VH amino acid sequence








(SEQ ID NO: 501)









EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGGIIAIFGTANYA



 


QKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGSGYHISTPFDNWGQGTLVTVSS


 


CR6327 Light Chain nucleotide sequence








(SEQ ID NO: 505)










tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt
60



 


acctgtgggg gaaacaacat tggaagtaaa ggtgtgcact ggtaccagca gaagcctggc
120


 


caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga
180


 


ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg
24 0


 


gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgt ggtattcggc
300


 


ggagggacca agctgaccgt cctaggtgcg gccgcaggcc agcccaaggc cgctcccagc
360


 


gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc
420


 


ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc
480


 


gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc
540


 


agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag
600


 


gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc
654


 


CR6327 Light Chain amino acid sequence








(SEQ ID NO: 506)









SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGAAAGQPKAAPSVTL


 


FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6327 VL amino acid sequence








(SEQ ID NO: 504)









SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG






The CR6328 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 507) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 508 and the heavy chain amino acid sequence shown in SEQ ID NO: 509. The CR6328 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 510) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 511 and the light chain amino acid sequence shown in SEQ ID NO: 512.








CR6328 Heavy Chain nucleotide sequence









(SEQ ID NO: 508)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggaca catcttcagc ggctatgcaa tcagttgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac aacaaactac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacc aatccacgag cacagcctac
240


 


atggacctga gcaacttgag atctgaggac acggccgtct attactgtgc gagagtgaaa
300


 


gatggatatt gtactcttac cagctgccct gtcggctggt acttcgatct ctggggccgt
360


 


ggcaccctgg tcactgtctc gagtgctagc accaagggcc ccagcgtgtt ccccctggcc
420


 


cccagcagca agagcaccag cggcggcaca gccgccctgg gctgcctggt gaaggactac
480


 


ttccccgagc ccgtgaccgt gagctggaac agcggcgcct tgaccagcgg cgtgcacacc
540


 


ttccccgccg tgctgcagag cagcggcctg tacagcctga gcagcgtggt gaccgtgccc
600


 


agcagcagcc tgggcaccca gacctacatc tgcaacgtga accacaagcc cagcaacacc
660


 


aaggtggaca aacgcgtgga gcccaagagc tgcgacaaga cccacacctg ccccccctgc
720


 


cctgcccccg agctgctggg cggaccctcc gtgttcctgt tcccccccaa gcccaaggac
780


 


accctcatga tcagccggac ccccgaggtg acctgcgtgg tggtggacgt gagccacgag
840


 


gaccccgagg tgaagttcaa ctggtacgtg gacggcgtgg aggtgcacaa cgccaagacc
900


 


aagccccggg aggagcagta caacagcacc taccgggtgg tgagcgtgct caccgtgctg
960


 


caccaggact ggctgaacgg caaggagtac aagtgcaagg tgagcaacaa ggccctgcct
1020


 


gcccccatcg agaagaccat cagcaaggcc aagggccagc cccgggagcc ccaggtgtac
1080


 


accctgcccc ccagccggga ggagatgacc aagaaccagg tgtccctcac ctgtctggtg
1140


 


aagggcttct accccagcga catcgccgtg gagtgggaga gcaacggcca gcccgagaac
1200


 


aactacaaga ccaccccccc tgtgctggac agcgacggca gcttcttcct gtacagcaag
1260


 


ctcaccgtgg acaagagccg gtggcagcag ggcaacgtgt tcagctgcag cgtgatgcac
1320


 


gaggccctgc acaaccacta cacccagaag agcctgagcc tgagccccgg caag
1374


 


CR6328 Heavy Chain amino acid sequence








(SEQ ID NO: 509)









EVQLVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGGIIPIFGTTNYAQ



 


KFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVKDGYCTLTSCPVGWYFDLWGRGTL


 


VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ


 


SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS


 


VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY


 


RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ


 


VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS


 


CSVMHEALHNHYTQKSLSLSPGK


 


CR6328 VH amino acid sequence








(SEQ ID NO: 507)









EVQLVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGGIIPIFGTTNYAQ



 


KFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVKDGYCTLTSCPVGWYFDLWGRGTL


 


VTVSS


 


CR6328 Light Chain nucleotide sequence








(SEQ ID NO: 511)










gaaattgtga tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60



 


ctctcgtgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
120


 


cctggccagg ctcccaggct cctcatcttt ggtgcctcca gcagggccac tggcatccca
180


 


gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240


 


cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcactcac tttcggcgga
300


 


gggaccaagc tggagatcaa acgtgcggcc gcacccagcg tgttcatctt ccccccctcc
360


 


gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc
420


 


cgggaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag
480


 


agcgtgaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctcaccctg
540


 


agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg
600


 


agcagccccg tgaccaagag cttcaaccgg ggcgagtgt
639


 


CR6328 Light Chain amino acid sequence








(SEQ ID NO: 512)









EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASSRATGIPDRFS



 


GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGGGTKLEIKRAAAPSVFIFPPSDEQLKSGT


 


ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK


 


VYACEVTHQGLSSPVTKSFNRGEC


 


CR6328 VL amino acid sequence








(SEQ ID NO: 510)









EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASSRATGIPDRFS



 


GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGGGTKLEIKR






The CR6329 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 513) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 514 and the heavy chain amino acid sequence shown in SEQ ID NO: 515. The CR6329 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 516) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 517 and the light chain amino acid sequence shown in SEQ ID NO: 518.








CR6329 Heavy Chain nucleotide sequence









(SEQ ID NO: 514)










gaggtccagc tggtacagtc tggggctgag gttaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg catcttcaga agcaattcta tcagttgggt gcgacaggcc
120


 


cctgggcaag ggcttgagtg gatgggaggg atcttcgctc ttttcggaac aacagactac
180


 


gcgcagaagt tccagggcag agtcacgatt accgcggacg aatcttcgac cacagtctac
240


 


ctggagctga gtagcctgac atctgaggac acggccgttt attactgtgc gagaggcagt
300


 


ggctacacca cacgcaacta ctttgactac tggggccagg gcaccctggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 


CR6329 Heavy Chain amino acid sequence








(SEQ ED NO: 515)









EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGGIFALFGTTDYAQ



 


KFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGSGYTTRNYFDYWGQGTLVTVSSAST


 


KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS


 


SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV


 


LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL


 


HNHYTQKSLSLSPGK


 


CR6329 VH amino acid sequence








(SEQ ID NO: 513)









EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGGIFALFGTTDYAQ



 


KFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGSGYTTRNYFDYWGQGTLVTVSS


 


CR6329 Light Chain nucleotide sequence








(SEQ ID NO: 517)










gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccaca
60



 


ctctcctgca gggccagtca gagtgttagc agcaactact taggctggta ccagcagaaa
120


 


cctggccagg ctcccaggct cctgatctat ggtgcatcca gcagggccag tggcatccca
180


 


gacaggttca gtggcggtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240


 


cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacccct cactttcggc
300


 


ggagggacca aggtggagat caaacgtgcg gccgcaggcc agcccaaggc cgctcccagc
360


 


gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc
420


 


ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc
480


 


gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc
540


 


agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag
600


 


gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc
654


 


CR6329 Light Chain amino acid sequence








(SEQ ID NO: 518)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWYQQKPGQAPRLLIYGASSRASGIPDRFS



 


GGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRAAAGQPKAAPSVTLFPPSS


 


EELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ


 


WKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6329 VL amino acid sequence








(SEQ ID NO: 516)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWYQQKPGQAPRLLIYGASSRASGIPDRFS



 


GGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR






The CR6331 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 519) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 520 and the heavy chain amino acid sequence shown in SEQ ID NO: 521. The CR6331 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 522) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 523 and the light chain amino acid sequence shown in SEQ ID NO: 524.








CR6331 Heavy Chain nucleotide sequence









(SEQ ID NO: 520)










gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcggta tgttcggtac agcaaactac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatttacgag cacagcctac
240


 


atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggaaat
300


 


tattactatg agagtagtct cgactactgg ggccagggaa ccctggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 


CR6331 Heavy Chain amino acid sequence








(SEQ ID NO: 521)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIGMFGTANYA



 


QKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGNYYYESSLDYWGQGTLVTVSSAST


 


KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS


 


SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV


 


LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL


 


HNHYTQKSLSLSPGK


 


CR6331 VH amino acid sequence








(SEQ ID NO: 519)









EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIGMFGTANYA



 


QKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGNYYYESSLDYWGQGTLVTVSS


 


CR6331 Light Chain nucleotide sequence








(SEQ ID NO: 523)










cagtctgtcg tgacgcagcc gccctcggtg tcagtggccc caggacagac ggccaggatt
60



 


acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc
120


 


caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga
180


 


ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg
240


 


gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatta tgtcttcgga
300


 


actgggacca aggtcaccgt cctaggtgcg gccgcaggcc agcccaaggc cgctcccagc
360


 


gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc
420


 


ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc
480


 


gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc
540


 


agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag
600


 


gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc
654


 


CR6331 Light Chain amino acid sequence








(SEQ ID NO: 524)









QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGAAAGQPKAAPSVTL


 


FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6331 VL amino acid sequence








(SEQ ID NO: 522)









QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG






The CR6332 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 525) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 526 and the heavy chain amino acid sequence shown in SEQ ID NO: 527. The CR6332 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 528) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 529 and the light chain amino acid sequence shown in SEQ ID NO: 530.








CR6332 Heavy Chain nucleotide sequence









(SEQ ID NO: 526)










caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtaaaggtc
60



 


tcctgcaagg cttctggagg ccccttccgc aattttgcta tcaactgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcgctg tctttgggac gacaaagtac
180


 


gcacataagt tccagggcag agtcaccatc accgcggacg actccacaaa tacagcttac
240


 


atggagctgg gcagcctgaa atctgaggac acggccgtgt attactgtgc gagaggtccc
300


 


cactactact cctcctacat ggacgtctgg ggcgaaggga ccacggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 


CR6332 Heavy Chain amino acid sequence








(SEQ ID NO: 527)









QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKYA



 


HKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGPHYYSSYMDVWGEGTTVTVSSAS


 


TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL


 


SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK


 


PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT


 


VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV


 


KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE


 


ALHNHYTQKSLSLSPGK


 


CR6332 VH amino acid sequence








(SEQ ID NO: 525)









QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKYA



 


HKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGPHYYSSYMDVWGEGTTVTVSS


 


CR6332 Light Chain nucleotide sequence








(SEQ ID NO: 529)










gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60



 


atcacttgcc gggcgagtca gggcattagc acttatttag cctggtatca gcagaaaccc
120


 


gggaaagttc ctaaactcct gatctatgct gcatccactt tgcaatcagg ggtcccatct
180


 


cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240


 


gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccttcttt cggccctggg
300


 


accaaagtgg atatcaaacg tgcggccgca cccagcgtgt tcatcttccc cccctccgac
360


 


gagcagctga agagcggcac cgccagcgtg gtgtgcctgc tgaacaactt ctacccccgg
420


 


gaggccaagg tgcagtggaa ggtggacaac gccctgcaga gcggcaacag ccaggagagc
480


 


gtgaccgagc aggacagcaa ggactccacc tacagcctga gcagcaccct caccctgagc
540


 


aaggccgact acgagaagca caaggtgtac gcctgcgagg tgacccacca gggcctgagc
600


 


agccccgtga ccaagagctt caaccggggc gagtgt
636


 


CR6332 Light Chain amino acid sequence








(SEQ ID NO: 530)









DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSG



 


SGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKRAAAPSVFIFPPSDEQLKSGTA


 


SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV


 


YACEVTHQGLSSPVTKSFNRGEC


 


CR6332 VL amino acid sequence








(SEQ ID NO: 528)









DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSG



 


SGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKR






The CR6334 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 531) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 532 and the heavy chain amino acid sequence shown in SEQ ID NO: 533. The CR6334 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 534) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 535 and the light chain amino acid sequence shown in SEQ ID NO: 536.








CR6334 Heavy Chain nucleotide sequence









(SEQ ID NO: 532)










gaggtgcagc tggtggagac tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


ccctgcaaat cttctggaag ccccttcagg agtaatgctg tcagctgggt gcgacaggcc
120


 


cccggacaag ggcttgagtg ggtgggagga atcctcggtg tctttggttc accaagctac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatccaccaa cacagtccac
240


 


atggagctga gaggtttgag atctgaggac acggccgtgt attattgtgc gagaggtcct
300


 


acctactact actcctacat ggacgtctgg ggcaaaggga ccacggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 


CR6334 Heavy Chain amino acid sequence








(SEQ ID NO: 533)









EVQLVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGGILGVFGSPSYA



 


QKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKGTTVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6334 VH amino acid sequence








(SEQ ID NO: 531)









EVQLVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGGILGVFGSPSYA



 


QKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKGTTVTVSS


 


CR6334 Light Chain nucleotide sequence








(SEQ ID NO: 535)










tcctatgtgc tgactcagcc accctcggag tcagtggccc caggacagac ggccaggatt
60



 


acctgtgggg gaaataacat tggaagaaat agtgtgcact ggtatcagca gaagccaggc
120


 


caggcccctg tgctggtcgt gtatgatgat agcgaccggc cctcagggat ccctgagcga
180


 


ttttctggct ccaagtctgg gaacacggcc accctgatta tcagcagggt cgaagtcggg
240


 


gatgaggccg actactactg tcaggtgtgg catagtagta gtgatcatta tgtcttcgga
300


 


actgggacca aggtcaccgt cctaggtgcg gccgcaggcc agcccaaggc cgctcccagc
360


 


gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc
420


 


ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc
480


 


gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc
540


 


agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag
600


 


gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc
654


 


CR6334 Light Chain amino acid sequence








(SEQ ID NO: 536)









SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG



 


SKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLGAAAGQPKAAPSVTLFP


 


PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT


 


PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6334 VL amino acid sequence








(SEQ ID NO: 534)









SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG



 


SKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLG






The CR6336 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 537) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 538 and the heavy chain amino acid sequence shown in SEQ ID NO: 539. The CR6336 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 540) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 541 and the light chain amino acid sequence shown in SEQ ID NO: 542.








CR6336 Heavy Chain nucleotide sequence









(SEQ ID NO: 538)










cagatgcagc tggtacaatc tggagctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcttcggta tgtttgggac agcaaactac
180


 


gcgcagaagt tccagggcag agtcacgatt accgcggacg aattcacgag cgcggcctac
240


 


atggagctga gcagcctggg atctgaggac acggccatgt attactgtgc gaggtctagt
300


 


ggttattacc cccaatactt ccaggactgg ggccagggca ccctggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 


CR6336 Heavy Chain amino acid sequence








(SEQ ID NO: 539)









QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIFGMFGTANY



 


AQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSSGYYPQYFQDWGQGTLVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6336 VH amino acid sequence








(SEQ ID NO: 537)









QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIFGMFGTANY



 


AQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSSGYYPQYFQDWGQGTLVTVSS


 


CR6336 Light Chain nucleotide sequence








(SEQ ID NO: 541)










gaaattgtga tgacacagtc tccaggcacc ctgtctttgt ctccagggca aagagccacc
60



 


ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
120


 


cctggccagg ctcccagact cctcatgtat ggtgcatcca gcagggccac tggcatccca
180


 


gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240


 


cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcatcgct cactttcggc
300


 


ggagggacca agctggagat caaacgtgcg gccgcaccca gcgtgttcat cttccccccc
360


 


tccgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac
420


 


ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag
480


 


gagagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctcacc
540


 


ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc
600


 


ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt
642


 


CR6336 Light Chain amino acid sequence








(SEQ ID NO: 542)









EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYGASSRATGIPDRF



 


SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKRAAAPSVFIFPPSDEQLKSG


 


TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH


 


KVYACEVTHQGLSSPVTKSFNRGEC


 


CR6336 VL amino acid sequence








(SEQ ID NO: 540)









EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYGASSRATGIPDRF



 


SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKR






The CR6339 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 543) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 545 and the heavy chain amino acid sequence shown in SEQ ID NO: 546. The CR6339 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 547) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 548 and the light chain amino acid sequence shown in SEQ ID NO: 549.








CR6339 Heavy Chain nucleotide sequence









(SEQ ID NO: 545)










gaggtgcagc tggtggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg catcttcaac agttatgcta tcagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggc atcatcgcta tctttcatac accaaagtac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa cacagcctac
240


 


atggaactga gaagcctgaa atctgaggac acggccctgt attactgtgc gagagggtcc
300


 


acttacgatt tttcgagtgg ccttgactac tggggccagg gaaccctggt caccgtctcg
360


 


agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc
420


 


ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg
480


 


agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc
540


 


agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag
600


 


acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660


 


cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc
720


 


ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc
780


 


cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac
840


 


tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac
900


 


aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc
960


 


aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020


 


agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag
1080


 


gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac
1140


 


atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct
1200


 


gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg
1260


 


tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
1320


 


acccagaaga gcctgagcct gagccccggc aag
1353


 


CR6339 Heavy Chain amino acid sequence








(SEQ ID NO: 546)









EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGGIIAIFHTPKYAQ



 


KFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGSTYDFSSGLDYWGQGTLVTVSSAST


 


KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS


 


SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV


 


LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR


 


WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK


 


CR6339 VH amino acid sequence








(SEQ ID NO: 543)









EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGGIIAIFHTPKYAQ



 


KFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGSTYDFSSGLDYWGQGTLVTVSS


 


CR6339 Light Chain nucleotide sequence








(SEQ ID NO: 548)










caggcagggc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt
60



 


acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc
120


 


caggcccctg tcctagtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga
180


 


ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg
240


 


gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgt ggtattcggc
300


 


ggagggacca agctgaccgt cctaggtgcg gccgcaggcc agcccaaggc cgctcccagc
360


 


gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc
420


 


ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc
480


 


gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc
540


 


agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag
600


 


gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc
654


 


CR6339 Light Chain amino acid sequence








(SEQ ID NO: 549)









QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGAAAGQPKAAPSVTL


 


FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6339 VL amino acid sequence








(SEQ ID NO: 547)









QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS



 


GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG






The CR6342 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 550) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 551 and the heavy chain amino acid sequence shown in SEQ ID NO: 552. The CR6342 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 553) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 554 and the light chain amino acid sequence shown in SEQ ID NO: 555.








CR6342 Heavy Chain nucleotide sequence









(SEQ ID NO: 551)










caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagg cttcttcagc agctatgcta tcagctgggt gcgccaggcc
120


 


cctggacaag gacttgagtg gatggggggg gtcatcccta tctttcgtac agcaaactac
180


 


gcacagaact tccagggcag agtcaccatt accgcggacg aattcacatc gtatatggag
240


 


ctgagcagcc tgagatctga cgacacggcc gtgtattact gtgcgaggtt gaattaccat
300


 


gattcgggga cttattataa cgccccccgg ggctggttcg acccctgggg ccagggaacc
360


 


ctggtcaccg tctcgagtgc tagcaccaag ggccccagcg tgttccccct ggcccccagc
420


 


agcaagagca ccagcggcgg cacagccgcc ctgggctgcc tggtgaagga ctacttcccc
480


 


gagcccgtga ccgtgagctg gaacagcggc gccttgacca gcggcgtgca caccttcccc
540


 


gccgtgctgc agagcagcgg cctgtacagc ctgagcagcg tggtgaccgt gcccagcagc
600


 


agcctgggca cccagaccta catctgcaac gtgaaccaca agcccagcaa caccaaggtg
660


 


gacaaacgcg tggagcccaa gagctgcgac aagacccaca cctgcccccc ctgccctgcc
720


 


cccgagctgc tgggcggacc ctccgtgttc ctgttccccc ccaagcccaa ggacaccctc
780


 


atgatcagcc ggacccccga ggtgacctgc gtggtggtgg acgtgagcca cgaggacccc
840


 


gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc
900


 


cgggaggagc agtacaacag cacctaccgg gtggtgagcg tgctcaccgt gctgcaccag
960


 


gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct gcctgccccc
1020


 


atcgagaaga ccatcagcaa ggccaagggc cagccccggg agccccaggt gtacaccctg
1080


 


ccccccagcc gggaggagat gaccaagaac caggtgtccc tcacctgtct ggtgaagggc
1140


 


ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac
1200


 


aagaccaccc cccctgtgct ggacagcgac ggcagcttct tcctgtacag caagctcacc
1260


 


gtggacaaga gccggtggca gcagggcaac gtgttcagct gcagcgtgat gcacgaggcc
1320


 


ctgcacaacc actacaccca gaagagcctg agcctgagcc ccggcaag
1368


 


CR6342 Heavy Chain amino acid sequence








(SEQ ID NO: 552)









QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGGVIPIFRTANYA



 


QNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYHDSGTYYNAPRGWFDPWGQGTLV


 


TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS


 


SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS


 


VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY


 


RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ


 


VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS


 


CSVMHEALHNHYTQKSLSLSPGK


 


CR6342 VH amino acid sequence








(SEQ ID NO: 550)









QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGGVIPIFRTANYA



 


QNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYHDSGTYYNAPRGWFDPWGQGTLV


 


TVSS


 


CR6342 Light Chain nucleotide sequence








(SEQ ID NO: 554)










gacatccaga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gaaggccacc
60



 


atcaactgca agtccagcca gagtatttta aacagctcca acaataagaa ctacttagct
120


 


tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg
180


 


gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc
240


 


atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtagt
300


 


ccgccgacgt tcggccaagg gaccaaggtg gaaatcaaac gtgcggccgc acccagcgtg
360


 


ttcatcttcc ccccctccga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg
420


 


ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag
480


 


agcggcaaca gccaggagag cgtgaccgag caggacagca aggactccac ctacagcctg
540


 


agcagcaccc tcaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag
600


 


gtgacccacc agggcctgag cagccccgtg accaagagct tcaaccgggg cgagtgt
657


 


CR6342 Light Chain amino acid sequence








(SEQ ID NO: 555)









DIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPPKLLIYWASTRESG



 


VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPPTFGQGTKVEIKRAAAPSVFIFPPSD


 


EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA


 


DYEKHKVYACEVTHQGLSSPVTKSFNRGEC


 


CR6342 VL amino acid sequence








(SEQ ID NO: 553)









DIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPPKLLIYWASTRESG



 


VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPPTFGQGTKVEIKR






The CR6343 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 556) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 557 and the heavy chain amino acid sequence shown in SEQ ID NO: 558. The CR6343 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 559) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 560 and the light chain amino acid sequence shown in SEQ ID NO: 561.








CR6343 Heavy Chain nucleotide sequence









(SEQ ID NO: 557)










caggtccagc tggtgcagtc tggagctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60



 


tcctgcaagg cttctggagt caccttcagt tactatgcta tgagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggagga atcagcccta tgtttgggac aacaacctac
180


 


gcacagaagt tccagggcag agtcacgatt actgcggacg actccacgag tacagcctac
240


 


atggaggtga ggagcctgag atctgaggac acggccgtgt attactgtgc gagatcttcg
300


 


aattactatg atagtgtata tgactactgg ggccagggaa ccctggtcac cgtctcgagt
360


 


gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc
420


 


ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc
480


 


tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc
540


 


ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc
600


 


tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660


 


aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga
720


 


ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc
780


 


gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg
840


 


tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac
900


 


agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag
960


 


gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020


 


aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag
1080


 


atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc
1140


 


gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200


 


ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg
1260


 


cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc
1320


 


cagaagagcc tgagcctgag ccccggcaag
1350


 


CR6343 Heavy Chain amino acid sequence








(SEQ ID NO: 558)









QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGGISPMFGTTTY



 


AQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVSSA


 


STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS


 


LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP


 


KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL


 


TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH


 


EALHNHYTQKSLSLSPGK


 


CR6343 VH amino acid sequence








(SEQ ID NO: 556)









QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGGISPMFGTTTY



 


AQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVSS


 


CR6343 Light Chain nucleotide sequence








(SEQ ID NO: 560)










cagtctgtcg tgacgcagcc gccctcggag tcagtggccc caggacagac ggccaggatt
60



 


acctgtgggg gacataacat tggaagtaat agtgtgcact ggtaccagca gaagccaggc
120


 


caggcccctg tgctggtcgt gtatgataat agcgaccggc cctcagggat ccctgagcga
180


 


ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg
240


 


gatgaggccg actattactg tcaggtgtgg ggtagtagta gtgaccatta tgtcttcgga
300


 


actgggacca aggtcaccgt cctaggtgcg gccgcaggcc agcccaaggc cgctcccagc
360


 


gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc
420


 


ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc
480


 


gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc
540


 


agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag
600


 


gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc
654


 


CR6343 Light Chain amino acid sequence








(SEQ ID NO: 561)









QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDNSDRPSGIPERFSG



 


SNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLGAAAGQPKAAPSVTLF


 


PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL


 


TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6343 VL amino acid sequence








(SEQ ID NO: 559)









QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDNSDRPSGIPERFSG



 


SNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLG






The CR6344 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 562) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 563 and the heavy chain amino acid sequence shown in SEQ ID NO: 564. The CR6344 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 565) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 566 and the light chain amino acid sequence shown in SEQ ID NO: 567.








CR6344 Heavy Chain nucleotide sequence









(SEQ ID NO: 563)










caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgagagtc
60



 


tcctgcaagg cttctggaag catcttcaga aactatgcta tgagctgggt gcgacaggcc
120


 


cctggacaag ggcttgagtg gatgggaggg atcatcgcta tttttgggac accaaagtac
180


 


gcacagaagt tccagggcag agtcacgatt accgcggacg aatcgacgag cactgtctac
240


 


atggaactga gcggactgag atctgaggac acggccatgt attactgtgc gaggattccc
300


 


cactataatt ttggttcggg gagttatttc gactactggg gccagggaac cctggtcacc
360


 


gtctcgagtg ctagcaccaa gggccccagc gtgttccccc tggcccccag cagcaagagc
420


 


accagcggcg gcacagccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg
480


 


accgtgagct ggaacagcgg cgccttgacc agcggcgtgc acaccttccc cgccgtgctg
540


 


cagagcagcg gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc
600


 


acccagacct acatctgcaa cgtgaaccac aagcccagca acaccaaggt ggacaaacgc
660


 


gtggagccca agagctgcga caagacccac acctgccccc cctgccctgc ccccgagctg
720


 


ctgggcggac cctccgtgtt cctgttcccc cccaagccca aggacaccct catgatcagc
780


 


cggacccccg aggtgacctg cgtggtggtg gacgtgagcc acgaggaccc cgaggtgaag
840


 


ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc ccgggaggag
900


 


cagtacaaca gcacctaccg ggtggtgagc gtgctcaccg tgctgcacca ggactggctg
960


 


aacggcaagg agtacaagtg caaggtgagc aacaaggccc tgcctgcccc catcgagaag
1020


 


accatcagca aggccaaggg ccagccccgg gagccccagg tgtacaccct gccccccagc
1080


 


cgggaggaga tgaccaagaa ccaggtgtcc ctcacctgtc tggtgaaggg cttctacccc
1140


 


agcgacatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta caagaccacc
1200


 


ccccctgtgc tggacagcga cggcagcttc ttcctgtaca gcaagctcac cgtggacaag
1260


 


agccggtggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac
1320


 


cactacaccc agaagagcct gagcctgagc cccggcaag
1359


 


CR6344 Heavy Chain amino acid sequence








(SEQ ID NO: 564)









QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGGIIAIFGTPKYA



 


QKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVTVSS


 


ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY


 


SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP


 


PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV


 


LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC


 


LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM


 


HEALHNHYTQKSLSLSPGK


 


CR6344 VH amino acid sequence








(SEQ ID NO: 562)









QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGGIIAIFGTPKYA



 


QKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVTVSS


 


CR6344 Light Chain nucleotide sequence








(SEQ ID NO: 566)










actgtgttga cacagccgcc ctcagtgtct ggggccccag ggcagagggt caccatctcc
60



 


tgcactggga gcagctccaa catcggggca ggttatgatg tacactggta ccagcagctt
120


 


ccaggaacag cccccaaact cctcatctat ggtaacagca atcggccctc aggggtccct
180


 


gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240


 


actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgcttatgtc
300


 


ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc caaggccgct
360


 


cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg
420


 


gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc
480


 


agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac
540


 


gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc
600


 


tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660


 


CR6344 Light Chain amino acid sequence








(SEQ ID NO: 567)









TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRF



 


SGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLGAAAGQPKAAPSVT


 


LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS


 


LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS


 


CR6344 VL amino acid sequence








(SEQ ID NO: 565)









TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRF



 


SGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLG






HA Antibody Epitopes

The invention relates to an isolated human HA antibody that is able to recognize and bind to an epitope in the HA2 subunit of the influenza haemagglutinin protein (HA) (also known as hemagglutinin(HA)), characterized in that the HA antibody has neutralizing activity against an influenza virus 5 including HA of the H5 subtype. Examples of influenza strains that contain such a HA of the H5 subtype and that are important strains in view of pandemic threats are H5N1, H5N2, H5N8, and H5N9. Particularly preferred are HA antibodies that at least neutralize the H5N1 influenza strain. Preferably, an HA antibody of the invention does not depend on an epitope in the HA1 subunit of the HA protein for binding to said HA protein.
A number of the antibodies of the invention (such as CR6307 and CR6323) do not depend on conformational epitopes and recognize the HA2 epitope even in a reduced form (when used in western-blotting). This is an advantage over the antibodies from the art because when a conformational change is induced in the HA protein due to whatever mutation in another part of the protein, such conformational change will not most likely hamper the binding of the antibodies of the present invention to the HA2 epitope, whereas antibodies that do depend on conformation might very well be unable to bind when such mutations occur.
In another preferred embodiment, an HA antibody of the invention also has neutralizing activity against an influenza virus comprising HA of the H1 subtype, and preferably wherein the HA antibody also has neutralizing activity against influenza virus comprising HA of the H2, H6 and/or H9 subtype. The HA antibodies of the invention interact with an epitope present in the HA2 epitopes present in the H5, HI, H2, H6, and H9 subtypes (see, International Patent Application PCT/EP2007/059356, published as WO 2008/028946, the contents of which are incorporated by reference in their entirety), and it has been shown that the HA antibodies of the invention cross-neutralize between influenza subtypes because of this epitope-sharing.
In another preferred aspect of the invention an HA antibody of the invention binds to an epitope that is selected from the group consisting of the amino acid sequence: GVTNKVNSIIDK (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO: 283), GVTNKENSIIDK (SEQ ID NO: 202), GVTNKVNRIIDK (SEQ ID NO: 201), GITNKVNSVIEK (SEQ ID NO: 281), GITNKENSVIEK (SEQ ID NO: 257), GITNKVNSIIDK (SEQ ID NO: 225), and KITSKVNNIVDK (SEQ ID NO: 216). Certain HA antibodies of the invention, CR6261, CR6325, and CR6329 interact with the GVTNKVNSIIDK (SEQ ID NO: 198) epitope present in H5N1, and are not hampered by a mutation in the TGLRN (SEQ ID NO: 200) epitope in HA1 that do influence the binding of C179. Moreover, some HA antibodies, such as CR6307 and CR6323 are not even hampered by a escape mutant, as disclosed in Okuno et al. (1993) with a valine→glutamic acid mutation at position 6 (exemplified by GVTNKENSIIDK (SEQ ID NO: 202)). This epitope is part of an extended alpha helix in the HA2 region. The residues in this putative epitope that are predicted to be most solvent exposed are underlined in bold: GV-TNKENSIIDK (SEQ ID NO: 202). These amino acids would be most accessible to an HA antibody and thus may form the most important region of the epitope. Consistent with this notion the highlighted (bolded) amino acids are absolutely conserved in identity and position in all the sequences presented. This knowledge could be used to predict binding epitopes in influenza subtypes that do not carry the same sequence as above (i.e. H3, H7 and B strains).
HA Antibodies II
The invention provides neutralizing human monoclonal antibodies that bind influenza A virus and inhibit the influenza A virus from infecting a cell. Although neutralizing human monoclonal antibodies of the invention bind epitopes within proteins that are exposed on the surface of an influenza virus, the invention focuses on the relatively invariant Influenza hemagglutinin (HA) protein. A neutralizing MAb raised against an Influenza HA protein, which is maintained in its native conformation, provides a superior therapy for all Influenza A strains because it is not dependent upon small changes to the amino acid sequence.
The Influenza hemagglutinin (HA) protein is responsible for allowing the virus to recognize target cells through binding the monosaccharide sialic acid-containing receptors on the surface of the target cell prior to infection. Moreover, the Influenza HA protein is responsible for allowing entry of the viral genome into the target cell by fusing the host endosomal membrane with the viral membrane.
The Influenza hemagglutinin (HA) protein is a homotrimeric integral membrane glycoprotein found on the surface of the Influenza virus. Using the host cell's protein synthesis machinery, the Influenza HA protein is first synthesized as a single-chain precursor polypeptide (HA0) in the endoplasmic reticulum, where it is also assembled as a homotrimer. The resulting HA homotrimer is subsequently exported to the cell surface via the Golgi network. HA homotrimers located on a cell surface are cleaved by a host-produced protease into two smaller peptide subunits: HA1 and HA2. The HA2 subunit forms a long helical chain anchored to the viral membrane whereas the HA1 subunit tops the HA2 subunit to form a large globule. The cleavage step, which converts the HA0 precursor into the mature HA protein containing HA1 and HA2 subunits, is essential for the viral pathogenicity of Influenza. Structurally, the mature HA protein contains a central α-helix coil resulting in an overall cylindrical shape with three spherical heads. The HA protein, and specifically, the HA1 subunit of the mature HA protein, binds receptors containing glycans with terminal sialic acids on host cells. The way in which sialic acid is connected to galactose, for example, α2-3 linkages as in avian serotypes versus α2-6 linkages as in human serotypes, not only determine species specificity of an Influenza virus, but also prevents cross-species infection. However, within certain serotypes of HA, such as H1 and H3, only two amino acid mutations in the framework sequence are required to convert species specificity from avian to human.
To mediate infection, the Influenza HA protein first binds sialic acid-containing receptors present on the surface of the target cell. Consequently, the target cell membrane endocytoses or engulfs the Influenza virus. Once inside the endosome, and upon the host cell's acidification of that compartment, the Influenza HA protein partially unfolds revealing a very hydrophobic fusion peptide that inserts itself into the endosomal membrane. As the rest of the Influenza HA protein refolds, the fusion protein retracts and fuses the endosomal membrane with the viral membrane. Upon fusion of the cellular and viral membranes, the contents of the virus, including the viral genome, are released in the cytoplasm of the target cell.
At least 16 different Influenza A hemagglutinin serotypes or antigens have been identified: H1-H16. Only HA serotypes H1-H3 normally mediate human Influenza infection. However, Influenza strains thought to infect only certain avian or mammalian species can mutate to infect humans. As described above, only a few amino acids need to change along the length of the entire protein to enable Influenza to cross a species barrier. For instance, a single amino acid change in the sequence of the H5 subtype allowed an avian-specific Influenza strain to become infectious in humans (H5N1). A pandemic arose when an Influenza strain common to swine species, became lethal to humans (H1N1). In contrast to Influenza A, Influenza B and C viruses each contain only one form of HA protein.
Specifically, the invention provides an isolated fully human monoclonal antibody, wherein said monoclonal antibody has the following characteristics: a) binds to an influenza A virus; b) binds to a cell contacted with influenza A; c) binds to an epitope of an influenza A viral protein; and, optionally, d) neutralizes influenza A virus infection. An antibody that does not neutralize influenza A virus infection may be used, for instance, for a conjugate therapy. In certain aspects, this antibody binds to a eukaryotic cell. Moreover, the cell is optionally a human cell.
In another aspect, this antibody is isolated from a B-cell from a human donor. Isolation of a fully human monoclonal antibody of the invention from a B-cell is performed using recombinant methods. Alternatively, or in addition, the isolated fully human monoclonal antibody of the invention is isolated from the supernatant of a plasma cell cultured either in vitro or ex vivo. Plasma cells also known as a differentiated B-cells, plasma B-cells, plasmacytes, or effector B-cells. The fully human monoclonal antibody isolated from either a B-cell or a plasma cell demonstrates neutralizing activity.
Antibodies of the invention bind to an epitope of influenza A viral hemagglutinin (HA) protein. Exemplary HA epitopes to which the antibodies of the invention bind include a hemagglutinin precursor peptide (HA0), a HA1 subunit, a HA2 subunit, a mature protein containing HA1 and HA2, and a recombinant HA polypeptide. Alternatively, antibodies of the invention bind to an epitope within a hemagglutinin precursor peptide (HA0), a HA1 subunit, a HA2 subunit, a mature protein containing HA1 and HA2, or a recombinant HA polypeptide. Recombinant HA polypeptides are encoded, for example, by the sequence of SEQ ID NO: 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, or 744.
Antibodies of the invention bind to an epitope that is linear or non-linear. In certain aspects of the invention, a non-linear epitope is a discontinuous epitope.
An antibody of the invention is TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), or TCN-504 (3251_K17).
The invention further encompasses an antibody that binds the same epitope as TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), or TCN-504 (3251_K17).
The invention provides an isolated fully human monoclonal anti-HA antibody or fragment thereof, wherein said antibody includes a variable heavy chain (VH) region comprising CDR1 and CDR2, wherein the VH region is encoded by a human IGHV1 (or specifically, IGHV1-18, IGHV1-2, IGHV1-69, IGHV1-8), IGHV2 (or specifically, IGHV2-5) , IGHV3 (or specifically, IGHV3-30, IGHV3-33, IGHV3-49, IGHV3-53, 66, IGHV3-7), IGHV4 (or specifically, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61), or IGHV5 (or specifically, IGHV5-51) VH germline sequence or an allele thereof, or a nucleic acid sequence that is homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 VH germline gene sequence or an allele thereof. In one aspect, the nucleic acid sequence that is homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 VH germline sequence is at least 75% homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 VH germline sequence or an allele thereof. Exemplary alleles include, but are not limited to, IGHV1-18*01, IGHV1-2*02, IGHV1-2*04, IGHV1-69*01, IGHV1-69*05, IGHV1-69*06, IGHV1-69*12, IGHV1-8*01, IGHV2-5*10, IGHV3-30-3*01, IGHV3-30*03, IGHV3-30*18, IGHV3-33*05, IGHV3-49*04, IGHV3-53*01, IGHV3-66*03, IGHV3-7*01, IGHV4-31*03, IGHV4-31*06, IGHV4-34*01, IGHV4-34*02, IGHV4-34*03, IGHV4-34*12, IGHV4-39*01, IGHV4-59*01, IGHV4-59*03, IGHV4-61*01, IGHV4-61*08, and IGHV5-51*01.
An antibody of the invention, or specifically, any antibody described herein, may be operably-linked to a therapeutic agent or a detectable label.
The invention further provides a pharmaceutical composition including an antibody described herein and a pharmaceutical carrier. This composition optionally includes an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor. Exemplary anti-viral drugs include, but are not limited to, a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor and an M2 ion channel inhibitor. In one embodiment of the composition, the M2 ion channel inhibitor is amantadine or rimantadine. Alternatively, or in addition, the neuraminidase inhibitor zanamivir or oseltamivir phosphate. The composition may also include a second anti-Influenza A antibody. The second anti-Influenza A antibody is optionally an antibody described herein.
The invention provides a method for stimulating an immune response in a subject, including administering to the subject the pharmaceutical composition described herein.
Moreover, the invention provides a method for the treatment of an Influenza virus infection in a subject, including administering to the subject the pharmaceutical composition described herein. This method further includes administering an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor.
The invention also provides a method for the prevention of an Influenza virus infection in a subject, including administering to the subject the pharmaceutical composition described herein prior to exposure of the subject to Influenza virus or infection. This method further includes administering an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor. This method may be a method of vaccination.
The subject of these methods may have an Influenza infection or is predisposed to developing an Influenza virus infection. Subjects predisposed to developing an Influenza infection, or who may be at elevated risk for contracting an infection, are those subjects with compromised immune systems because of autoimmune disease, those persons receiving immunosuppressive therapy (for example, following organ transplant), those persons afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), certain forms of anemia that deplete or destroy white blood cells, those persons receiving radiation or chemotherapy, or those persons afflicted with an inflammatory disorder. Additionally, subject of extreme young or old age are at increased risk. Any person who comes into physical contact or close physical proximity with an infected individual has an increased risk of developing an Influenza virus infection. Moreover, a subject is at risk of contracting an influenza infection due to proximity to an outbreak of the disease, e.g. subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of Influenza virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.
According to the methods described herein, exemplary anti-viral drugs include, but are not limited to, a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor and an M2 ion channel. In one aspect of these methods, the M2 ion channel inhibitor is amantadine or rimantadine. Alternatively, or in addition, the neuraminidase inhibitor is zanamivir or oseltamivir phosphate.
These methods optionally include administering a second anti-Influenza A antibody. For example, the antibody is administered prior to or after exposure to Influenza virus. In certain aspects of these methods, the antibody is administered at a dose sufficient to promote viral clearance or to eliminate Influenza A infected cells. The second antibody is optionally an antibody described herein
The invention further provides a method for determining the presence of a Influenza virus infection in a subject, including the steps of: (a) contacting a biological sample obtained from the subject with an antibody described herein or the pharmaceutical composition described herein; (b) detecting an amount of the antibody that binds to the biological sample; and (c) comparing the amount of antibody that binds to the biological sample to a control value, and therefrom determining the presence of the Influenza virus in the subject.
The invention provides a vaccine composition including an antibody described herein. This composition optionally contains a pharmaceutical carrier.
Alternatively, the invention provides a vaccine composition including an epitope of an antibody described herein. This composition optionally contains a pharmaceutical carrier.
Vaccines of the invention are multivalent vaccines. The term “multivalent vaccine” is meant to describe a single vaccine that elicits an immune response either to more than one infectious agent, e.g. recombinant homotrimeric HA0 proteins or fragments thereof derived from multiple strains of Influenza A (see, Table 9), or to several different epitopes of a molecule, e.g. a linear and a discontinuous epitope of the same recombinant homotrimeric HA0 protein or fragment thereof derived from a single strain of Influenza A. Alternatively, or in addition, the term multivalent vaccine is meant to describe the administration of a combination of human antibodies raised against more than one infectious agent, e.g. a combination of HuMHA antibodies raised against recombinant homotrimeric HA0 proteins or fragments thereof derived from multiple strains of Influenza A (see, Table 9).
The invention provides a diagnostic kit including an antibody described herein.
The invention provides a prophylactic kit including an antibody described herein or an epitope of an antibody described herein. Alternatively, or in addition, the invention provides a prophylactic kit including a vaccine composition described herein.
In a preferred embodiment, the present invention provides fully human monoclonal antibodies specifically directed against the Influenza hemagglutinin glycoprotein, which neutralize influenza infection. Optionally, the antibody is isolated from a B-cell from a mammalian donor, and preferably, a human donor. In certain embodiments of the invention, the antibody is identified for its ability to bind an intact or whole Influenza virus. Alternatively, or in addition, the antibody is identified isolated for its ability to bind to an epitope of a recombinant homotrimeric Influenza HA0 protein or HA protein(s) isolated from multiple Influenza strains, or made as recombinant proteins such as those influenza A virus strains provided in Table 9. Alternatively, or in addition, the antibody is identified for its ability to inhibit or neutralize virus infection of susceptible eukaryotic cells. Exemplary neutralizing antibodies of this profile include, but are not limited to, those antibodies listed in Table 10. Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as the antibodies provided in Table 10. In certain embodiments, neutralizing human monoclonal antibodies of the invention are anti-HA antibodies. A monoclonal anti-HA antibody of the invention has one or more of the following characteristics: a) binds to an epitope in an HA1 subunit of an Influenza hemagglutinin (HA) protein; b) binds to an epitope in the HA2 subunit of Influenza hemagglutinin (HA) protein; c) binds to an epitope in the extracellular domain of an Influenza hemagglutinin (HA) protein, consisting of an HA1 subunit and an HA2 subunit; d) binds to an epitope of a recombinant homotrimeric Influenza HA0 protein; e) binds to an epitope of an Influenza HA protein expressed on an infected cell; f) binds to an epitope of an Influenza HA protein expressed on a modified cell; g) binds to an Influenza virus; or h) inhibits virus infection of susceptible eukaryotic cells.
Modified cells of the invention are transfected or transformed with a polynucleotide that encodes an Influenza HA protein, or any fragment thereof. The term “Influenza HA protein fragment” is meant to describe any portion of the protein that is smaller or less than the entire protein. Polynucleotides and polypeptides of the invention do not always encode a functional Influenza HA protein.
Infected cells of the invention are mammalian, and preferably human in origin. Specifically, mammalian cells are infected with Influenza A virus in vivo, in vitro, in situ, ex vivo, in culture, and any combination thereof. Cells are infected with active or inactive virions. Exemplary inactive virions display the HA protein on their surfaces, however, they are replication-defective, and therefore, unable to propagate within the cell or subject.
Epitopes of the human monoclonal antibodies of the invention include a transmembrane or integral membrane Influenza A protein. Specifically, epitopes of the human monoclonal antibodies of the invention comprise Influenza hemagglutinin (HA) protein.
Epitopes of the human monoclonal antibodies of the invention include one or more subunits of an influenza hemagglutinin (HA) protein. HA proteins of the invention include hemagglutinin precursor proteins (HA0), the HA1 subunit, the HA2 subunit, the mature protein containing the HA1 and HA2 subunits, and a recombinant HA protein. Recombinant HA proteins contain SEQ ID NO: 726. Exemplary recombinant proteins include but, are not limited to, those proteins described by SEQ ID NO: 727-744.
Epitopes of the human monoclonal antibodies of the invention are linear or non-linear. For instance, a non-linear epitope is discontinuous. Discontinuous epitopes are available for antibody binding only when the Influenza HA protein is maintained in its native homotrimeric conformation. When an antibody binds to a discontinuous epitope, the antibody binds to a three-dimensional surface of the target protein, i.e. the Influenza HA protein, upon which juxtaposed amino acids are alternatively exposed or masked.
Recombinant homotrimeric HA0 proteins of the invention are encoded by, for instance, sequences described by any one of SEQ ID NO: 727-744. In certain embodiments of the invention, the human monoclonal antibodies, or monoclonal anti-HA antibodies, described herein bind membrane-bound or soluble recombinant homotrimeric Influenza HA proteins. Alternatively, the monoclonal anti-HA antibodies described herein bind membrane-bound and soluble recombinant homotrimeric Influenza HA proteins. In certain embodiments of the invention, the monoclonal anti-HA antibodies described herein bind and neutralize Influenza virus subtypes H1, H2, and H3. In other embodiments of the invention, the monoclonal anti-HA antibodies bind Influenza virus subtypes H1, H2, and H3, and neutralize one of these subtypes, such as H1, H2, or H3. In a specific embodiment, the monoclonal anti-HA antibodies bind Influenza subtypes H1N1, H2N2, and H3N2, and neutralize H1N1.
In one aspect, the HA precursor polypeptide (HA0) of the soluble and recombinant homotrimeric Influenza HA protein contains a trimerization domain (foldon) encoded in the phage T4 fibritin. An exemplary trimerization domain isolated from the phage T4 fibritin has the following sequence wherein a thrombin cleavage site is italicized and bolded, a T4 trimerization domain or sequence is underlined, a V5 tag is boxed, and a hexa-histidine (His) tag is bolded:




As used herein, the term “neutralizing antibody” is meant to describe an antibody that inhibits or prevents influenza A infection, inhibits or prevents Influenza A viral entry into a cell, inhibits or prevents influenza replication, inhibits or prevents influenza egress from a host cell, or reduces the Influenza A titer in a cell, biological sample, or subject. In a preferred embodiment, neutralizing antibodies of the invention prevent viral entry into the cytoplasmic compartment of host cells.
The present invention provides fully human monoclonal antibodies that bind influenza virus and neutralize infection. In certain embodiments, the present invention provides fully human monoclonal neutralizing antibodies specific against the Influenza hemagglutinin protein. The antibodies are respectively referred to herein is human monoclonal anti-HA (huMHA) antibodies.
The Influenza hemagglutinin (HA) protein is a homotrimeric integral membrane glycoprotein found on the surface of the Influenza virus. To mimic the native conformation of this homotrimeric protein, the methods of the invention provide an isolated HA protein precursor that is operably-linked to a trimerization or foldon domain from the phage T4 fibritin protein




The resultant recombinant homotrimeric foldon HA protein not only retains the native Influenza hemagglutinin homotrimeric conformation, but also becomes soluble, i.e. the protein is no longer bound to a viral or cellular membrane. Specifically, these recombinant HA homotrimeric proteins lack an integral membrane or transmembrane domain. In certain embodiments, these recombinant HA homotrimeric proteins include HA1 and HA2 subunits as well as a trimerization domain, the resultant recombinant HA homotrimeric protein containing between 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600 amino acids (aa) or any length of amino acids in between. Preferably, these recombinant HA homotrimeric proteins contain between 565-575 amino acids (aa). Recombinant HA homotrimeric proteins further include a signal cleavage site at the N-terminus containing between 15-25 aa. Alternatively, or in addition, recombinant HA homotrimeric proteins further include a transmembrane domain positioned between amino acids 525-535 of HA depending on the influenza A virus subtype. In a preferred embodiment, the HA protein is derived from one or more strains of an Influenza A virus. Recombinant HA homotrimeric proteins of the invention retain the native signal sequence to enable secretion. Moreover, recombinant HA homotrimeric proteins of the invention contain a same signal sequence, which is not derived from HA. Futhermore, signal sequences used with recombinant HA homotrimeric proteins of the invention include those signal sequences known in the art that allow efficient secretion of proteins, such as the signal sequence of the immunoglobulin light kappa chain. Alternatively, recombinant HA homotrimeric proteins, or the HA0 precursors thereof, may have the native signal sequences in the expression constructs used by Immune Technology Corp. (http://www.immune-tech.com/). Signal sequences are retained or manipultated to allow efficient secretion from, for instance, art-recognized cell lines maintained in vitro, e.g. 293 HEK cells.
Recombinant HA homotrimeric proteins may retain a native HA1/HA2 protease cleavage site, which is critical for viral pathogenicity. In one aspect of the invention, recombinant HA homotrimeric proteins contain a substituted HA1/HA2 protease cleavage site. For example, the recombinant HA protein encoded by SEQ ID NO: 737 does not have a native cleavage site, but rather a cleavage site substituted from another HA protein. Furthermore, these proteins optionally retain sialic acid-containing receptor binding sites within the HA1 subunit.
According to the methods of the invention, human antibodies obtained from blood, serum, plasma, or cerebral spinal fluid, are contacted to recombinant and soluble HA homotrimers of the invention in vitro, wherein the recombinant and soluble HA homotrimers act as targets for human antibody binding to confirm specificity of the isolated human antibody for an Influenza HA homotrimer in its native conformation. In general, the methods include obtaining serum or plasma samples from subjects or patients that have been infected with or vaccinated against an infectious agent. These serum or plasma samples are then screened to identify those that contain antibodies specific for a particular polypeptide associated with the infectious agent, such as, e.g. a polypeptide specifically expressed on the surface of cells infected with the infectious agent, but not uninfected cells. In particular embodiments, the serum or plasma samples are screened by contacting the samples with a cell that has been transfected with an expression vector that expresses the polypeptide expressed on the surface of infected cells. In particular embodiments the serum or plasma samples are screened by contacting the samples with a recombinant protein which represents a particular protein of the infectious agent such as, e.g. hemagglutinin of the influenza A virus. In particular embodiments the serum or plasma samples are screened by contacting the samples with a purified form of the infectious agent such as, e.g. intact whole virions of the influenza A virus. In particular embodiments, the serum or plasma samples are screened by contacting the samples with a live form of the infectious agent such as, e.g. intact whole virions of the influenza A virus to determine the presence of serum antibodies that inhibit or neutralize infection of susceptible cells. Exemplary susceptible cells are eukaryotic or mammalian cells, such as MDCK cells.
Once a subject or patient is identified as having serum or plasma containing an antibody specific for the infectious agent polypeptide or virus of interest, mononuclear and/or B cells obtained from the same subject or patient are used to identify a cell or clone thereof that produces the antibody, using any of the methods described herein or available in the art. Once a B cell that produces the antibody is identified, cDNAs encoding the variable regions or fragments thereof of the antibody may be cloned using standard RT-PCR vectors and primers specific for conserved antibody sequences, and subcloned into expression vectors used for the recombinant production of monoclonal antibodies specific for the infectious agent polypeptide of interest.
More specifically, B cells are collected from a particular donor, i.e. a subject or patient is identified as having serum or plasma containing an antibody specific for HA, cultured, and antibody is secreted from these B cells into the culture medium. The culture medium is separated from these B cells, the B cells are lysed, and then frozen for storage. The culture medium is then screened for antibody binding to various HA targets and/or inhibition/neutralization of infection in vitro. When a culture well is identified as having an antibody of the desired specificity, reverse-transcriptase polymerase chain reaction (RT-PCR) is applied to the B-cell lysate to amplify the antibody variable regions and subsequently clone, express, and test for binding and function of the recombinant antibody,
Human antibodies, such as the MAbs listed in Table 10, which bind the recombinant and soluble HA homotrimer and/or bind whole virions, and optionally inhibit or neutralize infection of live virus are recombinantly reproduced and formulated into a pharmaceutical composition for administration to a subject at risk of contacting an Influenza virus. Furthermore, recombinant and soluble HA homotrimers are derived from multiple strains of Influenza viruses, including multiple strains of influenza A virus. Exemplary human antibodies specifically bind Influenza A, and may be selected for an inability to bind influenza B and C virus strains.
The invention further provides a novel process whereby full-length HA is expressed in mammalian cell lines, which allows for the identification of human antibodies that bind this cell-expressed HA. The huMHA antibodies have been shown to bind conformational determinants on the HA-transfected cells, as well as native HA, which can be isolated, or contacted to huMHA antibodies when presented either on Influenza infected cells or on Influenza A virus. Alternatively, or in addition, huMHA antibodies bind native HA, recombinant homotrimeric HA, purified virus, infected cells, linear peptide, synthetic HA peptide, HA transfected mammalian cells, and HA expressed on the surface of genetically altered bacteriophage virus, which are used for gene fragment display assays. Thus, this invention has allowed for the identification and production of human monoclonal antibodies that exhibit novel specificity for a very broad range of Influenza A virus strains. These antibodies may be used prophylactically to prevent Influenza A infection, diagnostically to identify Influenza A infection and therapeutically to treat Influenza A infection. Moreover, the epitopes to which huMHA antibodies of the invention bind are used as vaccines to prevent influenza A infection.
The huMHA antibodies of the invention has one or more of the following characteristics: a) binds to an epitope in an HA1 subunit of an Influenza hemagglutinin (HA) protein; b) binds to an epitope in the HA2 subunit of Influenza hemagglutinin (HA) protein; c) binds to an epitope in the extracellular domain of an Influenza hemagglutinin (HA) protein, consisting of an HA1 subunit and an HA2 subunit; d) binds to an epitope of a recombinant homotrimeric Influenza HA0 protein; e) binds to an epitope of an Influenza HA protein expressed on an infected cell; binds to an epitope of an Influenza HA protein expressed on a modified cell; g) binds to an Influenza virus; or h) inhibits virus infection of susceptible eukaryotic cells. The huMHA antibodies of the invention eliminate Influenza infected cells through immune effector mechanisms such as ADCC and/or CDC and promote direct viral clearance by binding to Influenza virions.
Exemplary Influenza A strains used for screening human plasma samples, B Cell Culture supernatants (BCC SN), and monoclonal transfection supernatants (MN are shown in Table 8 below). Live strains were used for the neutralization assays described herein. Inactivated strains were used for the virus binding assays described herein. Recombinant homotrimeric HA protein was used in the trimeric HA binding assay.











TABLE 8









Trimeric





Virus
HA


Virus
Subtype
Neutralization
binding
binding







A/California/4/09
H1


+


A/Solomon Islands/3/06
H1
+
+
+


A/South Carolina/1/18
H1


+


A/Japan/305/57
H2

+
+


A/Wisconsin/67/05
H3
+
+
+


A/s wine/Ontario/01911-
H4


+


2/99


A/Vietnam/1203/04
H5


+


A/Indonesia/5/05
H5


+


A/Egypt/3300-
H5


+


NAMRU3/08


A/common magpie/Hong
H5


+


Kong/5052/07


A/Anhui/1/05
H5


+


A/chicken/Vietnam/
H5


+


NCVD-016/08


A/Hong Kong/156/97
H5


+


A/northern shoveler/
H6


+


California/HKWF115/07


A/Netherlands/219/03
H7


+


A/duck/Yangzhou/02/05
H8


+


A/Hong Kong/2108/03
H9


+


A/Hong Kong/1073/99
H9


+









Exemplary HA sequences include those sequences listed on Table 9 below.











TABLE 9






GenBank






Accession


SEQ ID


Type
No.
Subtype
HA Sequence from Strain
NO:







A
ACP41105
H1
A/California/04/2009(H1N1)
SEQ ID






NO: 727


A
ABU99109
H1
A/Solomon Islands/3/
SEQ ID





2006 (H1N1)
NO: 728


A
AFI17241
H1
A/South Carolina/1/18
SEQ ID





(H1N1)
NO: 729


A
AAA43185
H2
A/Japan/305/1957 (H2N2)
SEQ ID






NO: 730


A
ACF54576
H3
A/Wisconsin/67/2005 (H3N2)
SEQ ID






NO: 731


A
AAG17427
H4
A/Swine/Ontario/01911-2/
SEQ ID





99 (H4N6)
NO: 732


A
AF028709
H5
A/Hong Kong/156/97 (H5N1)
SEQ ID






NO: 733


A
AAT73274
H5
A/VietNam/1203/
SEQ ID





2004 (H5N1)
NO: 734


A
ABW06108
H5
A/Indonesia/5/2005 (H5N1)
SEQ ID






NO: 735


A
ACI06185
H5
A/Egypt/3300-NAMRU3/
SEQ ID





2008 (H5N1)
NO: 736


A
ACJ26242
H5
A/common magpic/Hong
SEQ ID





Kong/5052/2007 (H5N1)
NO: 737


A
ABD28180
H5
A/Anhui/1/2005(H5N1)
SEQ ID






NO: 738


A
ACO07033
H5
A/chicken/Vietnam/NCVD-
SEQ ID





016/2008(H5N1)
NO: 739


A
ACE81692
H6
A/northern shoveler/
SEQ ID





California/HKWF115/2007
NO: 740





(H6N1)


A
AAR02640
H7
A/Netherlands/219/03 (H7N7)
SEQ ID






NO: 741


A
ABK32094
H8
A/duck/Yangzhou/02/
SEQ ID





2005 (H8N4)
NO: 742


A
ABB58945
H15
A/HK/2108/2003 (H9N2)
SEQ ID






NO: 743


A
NC_004908
H9
A/Hong Kong/1073/99
SEQ ID





(H9N2)
NO: 744









In one embodiment, the huMHA antibodies of the invention bind to an HA that wholly or partially includes the amino acid residues from position 1 to position 525 of Influenza hemagglutinin when numbered in accordance with SEQ ID NO: 727-744. Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as the mAbs listed in Table 10.









TABLE 10






BCC well ID
Theraclone ID








3251_K17
TCN-504



3280_D18
TCN-521



3212_I12
TCN-522



5248_A17
TCN-523



5237_B21
TCN-524



5084_C17
TCN-526



5086_C06
TCN-527



5087_P17
TCN-528



5297_H01
TCN-529



5248_H10a
TCN-530



5091_H13
TCN-531



5262_H18
TCN-532



5256_A17
TCN-533



5249_B02
TCN-534



5246_P19
TCN-535



5095_N01
TCN-536



3194_D21
TCN-537



3206_O17
TCN-538



5056_A08
TCN-539



5060_F05
TCN-540



5062_M11
TCN-541



5079_A16
TCN-542



5081_G23
TCN-543



5082_A19
TCN-544



5082_I15
TCN-545



5089_L08
TCN-546



5092_F11
TCN-547



5092_P01
TCN-548



5092_P04
TCN-549



5096_F06
TCN-550



5243_D01
TCN-551



5249_I23
TCN-552



5261_C18
TCN-553



5277_M05
TCN-554



5246_L16
TCN-555



5089_K12
TCN-556



5081_A04
TCN-557



5248_H10b
TCN-558



5097_G08
TCN-559



5084_P10
TCN-560









The antibodies of the invention are able to neutralize Influenza A. Monoclonal antibodies can be produced by known procedures, e.g., as described by R. Kennet et al. in “Monoclonal Antibodies and Functional Cell Lines; Progress and Applications”. Plenum Press (New York), 1984. Further materials and methods applied are based on known procedures, e.g., such as described in J. Virol. 67:6642-6647, 1993.
These antibodies can be used as prophylactic or therapeutic agents upon appropriate formulation, or as a diagnostic tool.
A “neutralizing antibody” is one that can neutralize the ability of that pathogen to initiate and/or perpetuate an infection in a host and/or in target cells in vitro. The invention provides a neutralizing monoclonal human antibody, wherein the antibody recognizes an antigen from an Influenza virus, which is preferably derived from the HA protein. Preferably an antibody according to the invention is a novel monoclonal antibody referred to herein as TCN-522 (corresponding to BCC plate and well location 3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), and TCN-504 (3251_K17). These antibodies were initially isolated from human samples and are produced by the B cell cultures referred to as 3212_I12, 3280_D18, 5248_A17, 5237_B21, 5084_C17, 5086_C06, 5087_P17, 5297_H01, 5248_H10a, 5091_H13, 5262_H18, 5256_A17, 5249_B02, 5246_P19, 5095_N01, 3194_D21, 3206_O17, 5056_A08, 5060_F05, 5062_M11, 5079_A16, 5081_G23, 5082_A19, 5082_I15, 5089_L08, 5092_F11, 5092_P01, 5092_P04, 5096_F06, 5243_D01, 5249_I23, 5261_C18, 5277_M05, 5246_L16, 5089_K12, 5081_A04, 5248_H10b, 5097_G08, 5084_P10, and 3251_K17. These antibodies have broad neutralizing activity or broad binding activity for Influenza A in vitro.
The CDRs of the antibody heavy chains are referred to as CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of the antibody light chains are referred to as CDRL1, CDRL2 and CDRL3, respectively. The position of the CDR amino acids is defined according to the IMGT numbering system as: CDR1—IMGT positions 27 to 38, CDR2—IMGT positions 56 to 65 and CDR3—IMGT positions 105 to 117. (Lefranc, M P. et al. 2003 IMGT unique numbering for immunoglobulin and T cell receptor variable regions and Ig superfamily V-like domains. Dev Comp Immunol. 27(1):55-77; Lefranc, M P. 1997. Unique database numbering system for immunogenetic analysis. Immunology Today, 18:509; Lefranc, M P. 1999. The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains. The Immunologist, 7:132-136.)
The sequences of the antibodies were determined, including the sequences of the variable regions of the Gamma heavy and Kappa or Lambda light chains of the antibodies designated. In addition, the sequence of each of the polynucleotides and polypeptides encoding the antibody sequences was determined for TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), and TCN-504 (3251_K17).
Shown below are the polypeptide and polynucleotide sequences of the heavy and light chains, with the signal peptides at the N-terminus (or 5′ end) and the constant regions at the C-terminus (or 3′ end) of the variable regions, which are shown in bolded text.








TCN-504 (3251_K17) heavy chain variable region nucleotide sequence:









(SEQ ID NO: 745)









CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCGGAGAGCCTGTCCCTCACTTGCGCTGTCTCT



 


GGTGTCTCCATCAGCAATATTGATTTCTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTAGAATGGATT


 


GGCAATATCTATTATACGGGGATCACCTTCTACAACCCGTCCCTCAGCAGTCGAGTCGCCATATCCATTGACACC


 


TCCAAGAACCAGTTCTCCCTGACTCTGACTTCTGTGACCGCCGCAGACACGGCTATGTATTACTGTGCGAGACAT


 


TACGGTGACTCCGAGGCAATAAACGATGCCTTTGACATCTGGGGCCAAGGGACAATGCTCACCGTCTCGAGC


 


TCN-504 (3251_K17) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 746)









QVQLQESGPGLVKPSETLSLTCAVSGVSISNIDFYWGWIRQPPGKGLEWIGNIYYTGITFYNPSLSSRV



 


AISIDTSKNQFSLTLTSVTAADTAMYYCARHYGDSEAINDAFDIWGQGTMLTVSS


 


TCN-504 (3251_K17) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 747)









NIDFYWG



 


CDR 2:








(SEQ ID NO: 748)









NIYYTGITFYNPSLSS



 


CDR 3:








(SEQ ID NO: 749)









HYGDSEAINDAFDI



 


TCN-504 (3251_K17) gamma heavy chain Chothia CDRs


CDR 1:








(SEQ ID NO: 750)









GVSISN



 


CDR 2:








(SEQ ID NO: 751)









NIYYTGITF



 


CDR 3:








(SEQ ID NO: 749)









HYGDSEAINDAFDI



 


TCN-504 (3251_K17) light chain variable region nucleotide sequence:








(SEQ ID NO: 752)









GAGATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTGGCAATAGTTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGT


 


GCATCCACCAGGGCCACTGGTATCCCACCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATC


 


AGCAGCCTGCAGACTGAAGATTTTGCAGTTTATTACTGTCAACAATATATTAACTGGCGTCCGCTCAGTTTTGGC


 


GGAGGGACCAAGGTGGAGATCAAA


 


TCN-504 (3251_K17) light chain variable region amino acid sequence (KabatCDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 753)









EIVMTQSPATLSVSPGERATLSCRASQSVGNSLAWYQQRPGQAPRLLIYGASTRATGIPPRFSGSGSGT



 


EFTLTISSLQTEDFAVYYCQQYINWRPLSFGGGTKVEIK


 


TCN-504 (3251_K17) light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 754)









RASQSVGNSLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 756)









QQYINWRPLS



 


TCN-504 (3251_K17) light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 754)









RASQSVGNSLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 756)









QQYINWRPLS



 


TCN-521 (3280_D18) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 758)









GAAGTGCAGTTGGTGCAGTCTGGAGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCGCCTGTGTAGTCTCT



 


GGGTTCACCGTCACCAGCAATTATATAACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT


 


ATTTATAGTCATGGTCGCGCATATTATTCAGCCTCCGTGAATGGCCGATTCACCATCTCCAGACACACTTCCAAG


 


AACACAGTTTATCTTGAAATGAACAGCCTGAGACCTGAGGACACGGCCGTCTATTACTGTGCGGGCGGGGGCCTA


 


GTCGGTGGCTACGACGAATATTTCTTTGACTATTGGGGCCAGGGAACCCTGGCCACCGTCTCCTCA


 


TCN-521 (3280_D18) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 759)









EVQLVQSGGGLVQPGGSLRLACVVSGFTVTSNYITWVRQAPGKGLEWVSVIYSHGRAYYSASVNGRF



 


TISRHTSKNTVYLEMNSLRPEDTAVYYCAGGGLVGGYDEYFFDYWGQGTLATVSS


 


TCN-521 (3280_D18) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 760)









SNYIT



 


CDR 2:








(SEQ ID NO: 761)









VIYSHGRAYYSASVNG



 


CDR 3:








(SEQ ID NO: 762)









GGLVGGYDEYFFDY



TCN-521 (3280_D18) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 763)









GFTVTS



 


CDR 2:








(SEQ ID NO: 764)









VIYSHGRAY



 


CDR 3:








(SEQ ID NO: 762)









GGLVGGYDEYFFDY



 


TCN-521 (3280_D18) light chain variable region nucleotide sequence:








(SEQ ID NO: 765)









GAAACTGTCTTGACGCAATCTCCAGGCACCTTGTCTTTGACTCCAGGGGAAAGAGCCACCCTCTCCTGCAGAGTC



 


GGTCAGAGTGTTAGCGGCAGCCACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT


 


GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCGGTGGCAGTGTGTCTGGGACAGACTTCACTCTCACC


 


ATCAGCAGACTGGAGCCTGAAGATTCTGCAGTTTATTACTGTCAGCAGTATGGTGACTCACGATACACTTTTGGC


 


CAGGGGACCAAGCTGGAGATCAAA


 


TCN-521 (3280_D18) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 766)









ETVLTQSPGTLSLTPGERATLSCRVGQSVSGSHLAWYQQKPGQAPRLLIYGASSRATGIPDRFGGSVSG



 


TDFTLTISRLEPEDSAVYYCQQYGDSRYTFGQGTKLEIK


 


TCN-521 (3280_D18) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 767)









RVGQSVSGSHLA



 


CDR 2:








(SEQ ID NO: 768)









GASSRAT



 


CDR 3:








(SEQ ID NO: 769)









QQYGDSRYT



 


TCN-521 (3280_D18) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 767)









RVGQSVSGSHLA



 


CDR 2:








(SEQ ID NO: 768)









GASSRAT



 


CDR 3:








(SEQ ID NO: 769)









QQYGDSRYT



 


TCN-522 (3212_I12) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 770)









CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTTTTGAAACCTTCGGAGACCCTGTCCCTCACCTGCACTGTGTCT



 


GGGGGGTCCCTCACTGATTACTCTTGGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATCGGTGAC


 


ACCCTTCATAATGGCTACACCAACTACAACCCGTCCCTCAGGGGTCGAGTTTCCATCTCAATAGACACGTCCAAG


 


AACCAGGTCTCACTCAGGCTGACCTCTGTGACCGCCGCGGACACGGCTCTTTATTACTGTGCGAGAGGCTCAGGT


 


GGATATGGTGGCTTCGATTATTTTGGCAAGCTCCGGACATGGGACTTCTGGGGCCAGGGAACGCTGGTCACCGTC


 


TCCTCA


 


TCN-522 (3212_I12) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 771)









QVQLQQWGAGLLKPSETLSLTCTVSGGSLTDYSWNWIRQPPGKGLEWIGDTLHNGYTNYNPSLRGR



 


VSISIDTSKNQVSLRLTSVTAADTALYYCARGSGGYGGFDYFGKLRTWDFWGQGTLVTVSS


 


TCN-522 (3212_I12) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 772)









DYSWN



 


CDR 2:








(SEQ ID NO: 773)









DTLHNGYTNYNPSLRG



 


CDR 3:








(SEQ ID NO: 774)









GSGGYGGFDYFGKLRTWDF



 


TCN-522 (3212_I12) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 775)









GGSLTD



 


CDR 2:








(SEQ ID NO: 776)









DTLHNGYTN



 


CDR 3:








(SEQ ID NO: 774)









GSGGYGGFDYFGKLRTWDF



TCN-522 (3212_I12) light chain variable region nucleotide sequence:








(SEQ ID NO: 777)









GACATTCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAAAAACCAGGGAACGCCCCTAAGCGCCTGATCTTTGGT


 


GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACAATC


 


AGCAGCCTGCAGCCTGAGGACTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGTACACTTTTGGCCAG


 


GGGACCAAGCTGGAGATCAAG


 


TCN-522 (3212_I12) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 778)









DIQLTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGNAPKRLIFGASSLQSGVPSRFSGSGSGT



 


EFTLTISSLQPEDFATYYCLQHNSYPYTFGQGTKLEIK


 


TCN-522 (3212_I12) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 779)









RASQGIRNDLG



 


CDR 2:








(SEQ ID NO: 780)









GASSLQS



 


CDR 3:








(SEQ ID NO: 781)









LQHNSYPYT



 


TCN-522 (3212_I12)Light chain Chothia CDRs


CDR 1:








(SEQ ID NO: 779)









RASQGIRNDLG



 


CDR 2:








(SEQ ID NO: 780)









GASSLQS



 


CDR 3:








(SEQ ID NO: 781)









LQHNSYPYT



 


TCN-523 (5248_A17) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 782)









CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT



 


GGAGGCAGCTTCAGCAACTATGCCTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ACCATCCCTCTACTTGGTACAACAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTT CCGCGGACCAATTC


 


ACGAGCACAGCCTACATGGAGCTGGGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTACGAGACGGAAA


 


ATGACTACGGCTTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA


 


TCN-523 (5248_A17) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 783)









QVQLVQSGAEVKKPGSSVKVSCKASGGSFSNYAFSWVRQAPGQGLEWMGGTIPLLGTTNYAQKFQ



 


GRVTISADQFTSTAYMELGSLRSEDTAVYYCTRRKMTTAFDSWGQGTLVTVSS


 


TCN-523 (5248_A17) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 784)









NYAFS



 


CDR 2:








(SEQ ID NO: 785)









GTIPLLGTTNYAQKFQG



 


CDR 3:








(SEQ ID NO: 786)









RKMTTAFDS



 


TCN-523 (5248_A17)gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 787)









GGSFSN



 


CDR 2:








(SEQ ID NO: 788)









GTIPLLGTTN



 


CDR 3:








(SEQ ID NO: 786)









RKMTTAFDS



 


TCN-523 (5248_A17)light chain variable region nucleotide sequence:








(SEQ ID NO: 789)









CAGCCTGTTCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACCTGCACCCTGAGC



 


AGCGCCTACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGCTTTGTGATGCGAGTG


 


GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTTCTCAGTCTTGGGCTCAGGCCTGAAT


 


CGGTACCTGACCATCAAGAACATCCAGGAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGG


 


AGCAACTTCGTGTCCCCTTACGTATTCGGCGGAGGGACCAAGCTGACCGTTCTA


 


TCN-523 (5248_A17)light chain variable region amino acid sequence (Kabat CDRs



in bold, Chothia CDRs underlined)








(SEQ ID NO: 790)









QPVLTQPPSASASLGASVTLTCTLSSAYSNYKVDWYQQRPGKGPRFVMRVGTGGIVGSKGDGIPDRF



 


SVLGSGLNRYLTIKNIQEEDESDYHCGADHGSGSNFVSPYVFGGGTKLTVL


 


TCN-523 (5248_A17)Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 791)









TLSSAYSNYKVD



 


CDR 2:








(SEQ ID NO: 792)









VGTGGIVGSKGD



 


CDR 3:








(SEQ ID NO: 793)









GADHGSGSNFVSPYV



 


TCN-523 (5248_A17)Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 791)









TLSSAYSNYKVD



 


CDR 2:








(SEQ ID NO: 792)









VGTGGIVGSKGD



 


CDR 3:








(SEQ ID NO: 793)









GADHGSGSNFVSPYV



 


TCN-563 (5237_B21) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 794)









CAGGTGCAGCTGGCGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTCGGTGAAAGTCTCATGCACGGCTTCT



 


GGAGGCATCTTCAGGAAGAATGCAATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG


 


ATCATCGCAGTCTTTAACACAGCAAATTACGCGCAGAAGTTTCAGGGCAGAGTCAAAATTACCGCAGACGAATCC


 


GGGAATACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTCACCCA


 


AAATATTTCTATGGTTCGGGGAGTTATCCGGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-563 (5237_B21) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 795)









QVQLAQSGAEVKRPGSSVKVSCTASGGIFRKNAISWVRQAPGQGLEWMGGIIAVFNTANYAQKFQG



 


RVKITADESGNTAYMELSSLRSDDTAVYYCASHPKYFYGSGSYPDFWGQGTLVTVSS


 


TCN-563 (5237_B21)gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 796)









KNAIS



 


CDR 2:








(SEQ ID NO: 797)









GIIAVFNTANYAQKFQG



 


CDR 3:








(SEQ ID NO: 798)









HPKYFYGSGSYPDF



 


TCN-563 (5237_B21)gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 799)









GGIFRK



 


CDR 2:








(SEQ ID NO: 800)









GIIAVFNTAN



 


CDR 3:








(SEQ ID NO: 798)









HPKYFYGSGSYPDF



 


TCN-563 (5237_B21)light chain variable region nucleotide sequence:








(SEQ ID NO: 801)









CAATCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAAGC



 


AGCAGTGATGTTGGTGCTTCTAACTCTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCGTTATT


 


TATGATGTCACTGAGCGACCCTCAGGGGTCCCTCATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACCGTCTCTGGGCTCCAGCCTGAGGACGAGGCTGATTATTTCTGCTGCGCATATGGAGGCAAATATCTTGTGGTC


 


TTCGGCGGAGGGACCAAGGTGACCGTCCTC


 


TCN-563 (5237_B21)light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 802)









QSALTQPRSVSGSPGQSVTISCTGSSSDVGASNSVSWYQQHPGKAPKLVIYDVTERPSGVPHRFSGSKS



 


GNTASLTVSGLQPEDEADYFCCAYGGKYLVVFGGGTKVTVL


 


TCN-563 (5237_B21)light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 803)









TGSSSDVGASNSVS



 


CDR 2:








(SEQ ID NO: 804)









DVTERPS



 


CDR 3:








(SEQ ID NO: 805)









CAYGGKYLVV



 


TCN-563 (5237_B21)light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 803)









TGSSSDVGASNSVS



 


CDR 2:








(SEQ ID NO: 804)









DVTERPS



 


CDR 3:








(SEQ ID NO: 805)









CAYGGKYLVV



 


TCN-526 (5084_C17)heavy chain variable region nucleotide sequence:








(SEQ ID NO: 806)









GAGGTGCTGATGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCGTGAGACTCTCCTGTGTAGCCTCT



 


GGATTCAGTTTCAGTAGTCATTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC


 


ATAGAGGACGATGGAGGTGACAAGTACTATGTGGACTCTGTGAAGGGCCGATTCATTATCTCCAGAGACAACGCC


 


AAGAATTCAGTGTATCTGCAAATGAACAGCCTAAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGTTCG


 


GGGAGCTCTGATAGAAGTGATTATGACCCCCACTACTACTACTACTTGGACGTCTGGGGCAAAGGGGCCACGGTC


 


ACCGTCTCCTCA


 


TCN-526 (5084_C17) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 807)









EVLMVESGGGLVQPGGSVRLSCVASGFSFSSHWMTWVRQAPGKGLEWVANIEDDGGDKYYVDSVK



 


GRFIISRDNAKNSVYLQMNSLRAEDTAVYFCARGSGSSDRSDYDPHYYYYLDVWGKGATVTVSS


 


TCN-526 (5084_C17) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 808)









SHWMT



 


CDR 2:








(SEQ ID NO: 809)









NIEDDGGDKYYVDSVKG



 


CDR 3:








(SEQ ID NO: 810)









GSGSSDRSDYDPHYYYYLDV



 


TCN-526 (5084_C17) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 811)









GFSFSS



 


CDR 2:








(SEQ ID NO: 812)









NIEDDGGDKY



 


CDR 3:








(SEQ ID NO: 810)









GSGSSDRSDYDPHYYYYLDV



 


TCN-526 (5084_C17) light chain variable region nucleotide sequence:








(SEQ ID NO: 813)









GACATCCAGCTGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AGTCAGAGCATTAGTAGGTATTTAAATTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGCTGTTTGCT


 


GCTTCTACTTTGCTAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC


 


AGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACGGAATCACAGTCCCTCGTGGACGTTCGGCCAA


 


GGGACCAGGGTGGAAATCAAA


 


TCN-526 (5084_C17) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 814)









DIQLTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLLFAASTLLDGVPSRFSGSGSGT



 


DFTLTISSLQPEDFATYYCQRNHSPSWTFGQGTRVEIK


 


TCN-526 (5084_C17) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 815)









RASQSISRYLN



 


CDR 2:








(SEQ ID NO: 816)









AASTLLD



 


CDR 3:








(SEQ ID NO: 817)









QRNHSPSWT



 


TCN-526 (5084_C17) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 815)









RASQSISRYLN



 


CDR 2:








(SEQ ID NO: 816)









AASTLLD



 


CDR 3:








(SEQ ID NO: 817)









QRNHSPSWT



 


TCN-527 (5086_C06) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 818)









CAGGTGCAGCTGCAAGAGTCGGGCCCGGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAACTGCGCTGTCTCT



 


GGAGGCTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGCTAT


 


ATCTCTTACAATGGGAGGCCCAAGTACAACCCCTCCCTCACGAGTCGAGTCACCATATCCGTCGACACGTCCAAG


 


GACCAGTTCTCCCTGGAGCTGCGCTCTGTGACCGCTGCGGACACGGCCCTTTATTACTGTGCGAGAGAAACGCGG


 


TTCGGGGAGTTATTATCTCCCTATGATGCTTTTGAAATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA


 


TCN-527 (5086_C06) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 819)









QVQLQESGPGLVKPSETLSLNCAVSGGSISNYYWSWIRQPPGKGLEWIGYISYNGRPKYNPSLTSRVTI



 


SVDTSKDQFSLELRSVTAADTALYYCARETRFGELLSPYDAFEIWGQGTMVTVSS


 


TCN-527 (5086_C06) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 820)









NYYWS



 


CDR 2:








(SEQ ID NO: 821)









YISYNGRPKYNPSLTS



 


CDR 3:








(SEQ ID NO: 822)









ETRFGELLSPYDAFEI



 


TCN-527 (5086_C06) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 824)









GGSISN



 


CDR 2:








(SEQ ID NO: 823)









YISYNGRPK



 


CDR 3:








(SEQ ID NO: 822)









ETRFGELLSPYDAFEI



 


TCN-527 (5086_C06) light chain variable region nucleotide sequence:








(SEQ ID NO: 825)









GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATGACTTGCCGGGCA



 


AGTCAGAACATTAGAAGCTATTTAAATTGGTATCAGCAGAGACCAGGGACAGCCCCTAAACTCCTGATCTATGCT


 


GCATCCACTTTACACAGTGGGGTCCCATCAAGGTTCAGTGGCGGTGGGTCTGGGACAGATTTCACTCTCACCATC


 


AATAATCTGCAACCTGAAGATTTTGCATCTTACTACTGTCAACAGAGTTACGATAACCCTCAGACGTTCGGCCAA


 


GGGACCAAGGTGGAAATCAAA


 


TCN-527 (5086_C06) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 826)









DIQMTQSPSSLSASVGDRVTMTCRASQNIRSYLNWYQQRPGTAPKLLIYAASTLHSGVPSRFSGGGSG



 


TDFTLTINNLQPEDFASYYCQQSYDNPQTFGQGTKVEIK


 


TCN-527 (5086_C06) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 827)









RASQNIRSYLN



 


CDR 2:








(SEQ ID NO: 828)









AASTLHS



 


CDR 3:








(SEQ ID NO: 829)









QQSYDNPQT



 


TCN-527 (5086_C06) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 827)









RASQNIRSYLN



 


CDR 2:








(SEQ ID NO: 828)









AASTLHS



 


CDR 3:








(SEQ ID NO: 829)









QQSYDNPQT



 


TCN-528 (5087_P17) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 830)









CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGATACACCTTCACCAATTATGACATCAACTGGATTCGACAGGCCCCTGGTCAAGGACTTGAGTGGATGGGCTGG


 


ATAAATCCCAACAGTGGAACCACGGGCTCTGCACAGAGGTTCCAGGGCAGAGTCACCATAACCGTGGACACCTCC


 


ATAACCACAGTCTACATGGAACTGAGCAGCCTGAGATCTGACGACACGGCCATTTACTACTGCGCGAGAGGCCGT


 


GAGCTCCTCCGGCTTCAACATTTTTTGACTGACTCCCAGTCCGAGAGGAGGGACTGCTTCGACCCCTGGGGCCAG


 


GGAACCCTGGTCACCGTCTCCTCA


 


TCN-528 (5087_P17) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 831)









QVQLVQSGSEVKKPGASVKVSCKASGYTFTNYDINWIRQAPGQGLEWMGWINPNSGTTGSAQRFQG



 


RVTITVDTSITTVYMELSSLRSDDTAIYYCARGRELLRLQHFLTDSQSERRDCFDPWGQGTLVTVSS


 


TCN-528 (5087_P17) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 832)









NYDIN



 


CDR 2:








(SEQ ID NO: 833)









WINPNSGTTGSAQRFQG



 


CDR 3:








(SEQ ID NO: 834)









GRELLRLQHFLTDSQSERRDCFDP



 


TCN-528 (5087_P17) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 835)









GYTFTN



 


CDR 2:








(SEQ ID NO: 836)









WINPNSGTTG



 


CDR 3:








(SEQ ID NO: 834)









GRELLRLQHFLTDSQSERRDCFDP



 


TCN-528 (5087_P17) light chain variable region nucleotide sequence:








(SEQ ID NO: 837)









GATATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AATCAAGACATTGGCATTTATTTAAATTGGTATCAACAGAATCCAGGGAAAGTCCCTAAACTCCTGCTCCATGGT


 


GCGTCCAGTTTGCAGGGCGGGGTCCCATCAAGGTTCAGTGCCAGTGGATCTGGGACAGATTTCACTCTCACCATT


 


CACAGTCTACAACCTGAAGATTTAGCAACCTACTACTGTCAACAGAGTCGCCGTCTACCGTACACTTTTGGCCAG


 


GGGACCAGGGTGGAACTCAAA


 


TCN-528 (5087_P17) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 838)









DIQMTQSPSSLSASVGDRVTITCRANQDIGIYLNWYQQNPGKVPKLLLHGASSLQGGVPSRFSASGSG



 


TDFTLTIHSLQPEDLATYYCQQSRRLPYTFGQGTRVELK


 


TCN-528 (5087_P17) Light chain Kabat CDRs:



CDR 1:








(SEQ ID NO: 839)









RANQDIGIYLN



 


CDR 2:








(SEQ ID NO: 840)









GASSLQG



 


CDR 3:








(SEQ ID NO: 841)









QQSRRLPYT



 


TCN-528 (5087_P17) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 839)









RANQDIGIYLN



 


CDR 2:








(SEQ ID NO: 840)









GASSLQG



 


CDR 3:








(SEQ ID NO: 841)









QQSRRLPYT



 


TCN-529 (5297_H01) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 842)









CAGATCACCTTGAGGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCT



 


GGGTTTTCACTCAGCACTAATGGAGTGAATGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT


 


GCACTCATTTACTGGGATGATGATAAGCGCTACAGTCCGTCTCTGAAGAGAAGGCTCACCATCACCAAGGACACC


 


TCCAAAAACCAAGTGGTCCTTACACTGACCAACATGGACCCTGTAGATACAGCCACATATTACTGTGCACACAGA


 


CCCGACTTCTATGGTGACTTCGAGTACTGGGGCCCGGGAACCCTGGTCACCGTCTCCTCA


 


TCN-529 (5297_H01) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 843)









QITLRESGPTLVKPTQTLTLTCTFSGFSLSTNGVNVGWIRQPPGKALEWLALIYWDDDKRYSPSLKRR



 


LTITKDTSKNQVVLTLTNMDPVDTATYYCAHRPDFYGDFEYWGPGTLVTVSS


 


TCN-529 (5297_H01) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 844)









TNGVNVG



 


CDR 2:








(SEQ ID NO: 845)









LIYWDDDKRYSPSLKR



 


CDR 3:








(SEQ ID NO: 846)









RPDFYGDFEY



 


TCN-529 (5297_H01)gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 847)









GFSLSTNG



 


CDR 2:








(SEQ ID NO: 848)









LIYWDDDKR



 


CDR 3:








(SEQ ID NO: 846)









RPDFYGDFEY



 


TCN-529 (5297_H01) light chain variable region nucleotide sequence:








(SEQ ID NO: 849)









CAGTCTGCACTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCCGGACAGTCGATCACCATCTCCTGCACTGGAAGC



 


AGCAGTGACATTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAGGCCCCCAAACTCATGATT


 


TACGATGTCAATAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACTATCTCTGGGCTCCAGACTGACGACGAGGCTGATTATTACTGCGGCTCATATACAGGCAGTCCTCATTATGTC


 


TTCGGAACTGGGACCAAGGTCACCGTCCTA


 


TCN-529 (5297_H01) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 850)









QSALTQPASVSGSPGQSITISCTGSSSDIGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVSNRFSGSKS



 


GNTASLTISGLQTDDEADYYCGSYTGSPHYVFGTGTKVTVL


 


TCN-529 (5297_H01) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 851)









TGSSSDIGGYNYVS



 


CDR 2:








(SEQ ID NO: 852)









DVNNRPS



 


CDR 3:








(SEQ ID NO: 853)









GSYTGSPHYV



 


TCN-529 (5297_H01)Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 851)









TGSSSDIGGYNYVS



 


CDR 2:








(SEQ ID NO: 852)









DVNNRPS



 


CDR 3:








(SEQ ID NO: 853)









GSYTGSPHYV



 


TCN-530 (5248_H10a) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 854)









CAGGTCCAACTGGTGCAATCTGGGGCTGAGGTGAGGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT



 


GGAGGCCCCTTCATGAGTTATGCTATCGGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCAACCCTGTGTTTGGTAGACCGCACTACGCACAGAAGTTCCAGGGCAGAGTCACCATCGCCACGGACGACTCC


 


ACGAAGACATCGTACATGGAACTGAGTAGCCTGACGTCTGAGGACACGGGCATGTATTACTGTGCGAGTAGGTAT


 


AGTAGGTCGTCCCCAGGGACCTTTGAGTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-530 (5248_H10a) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 855)









QVQLVQSGAEVRKPGSSVKVSCKASGGPFMSYAIGWVRQAPGQGLEWMGGINPVFGRPHYAQKFQ



 


GRVTIATDDSTKTSYMELSSLTSEDTGMYYCASRYSRSSPGTFESWGQGTLVTVSS


 


TCN-530 (5248_H10a) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 856)









SYAIG



 


CDR 2:








(SEQ ID NO: 857)









GINPVFGRPHYAQKFQG



 


CDR 3:








(SEQ ID NO: 858)









RYSRSSPGTFES



 


TCN-530 (5248_H10a) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 859)









GGPFMS



 


CDR 2:








(SEQ ID NO: 860)









GINPVFGRPH



 


CDR 3:








(SEQ ID NO: 858)









RYSRSSPGTFES



 


TCN-530 (5248_H10a) light chain variable region nucleotide sequence:








(SEQ ID NO: 861)









GAAATAGTGATGACGCAGTTTCCAGCCACCCTGTCTGTGTCTCCCGGGGAACGAGTCACCCTCTCCTGTAGGGCC



 


AGTCAGAGTGTTAGCAACAATTTAGCCTGGTACCAGCAAAAACCTGGCCAGCCTCCCAGGCTCCTCATCTATGAT


 


GCATCTACCAGGGCCACGGGTGTCCCAGCCAAGTTCAGTGGCACTGGGTCTGGCACAGAGTTCACTCTCAGCATC


 


AGCAGCCTGCAGTCCGAAGATTTTGCAGTTTATTACTGTCAGCAGTATCACAACTGGCCTCCCTCGTACAGTTTT


 


GGCCTGGGGACCAAGCTGGAGATCAAA


 


TCN-530 (5248_H10a) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 862)









EIVMTQFPATLSVSPGERVTLSCRASQSVSNNLAWYQQKPGQPPRLLIYDASTRATGVPAKFSGTGSGT



 


EFTLSISSLQSEDFAVYYCQQYHNWPPSYSFGLGTKLEIK


 


TCN-530 (5248_H10a) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 863)









RASQSVSNNLA



 


CDR 2:








(SEQ ID NO: 864)









DASTRAT



 


CDR 3:








(SEQ ID NO: 865)









QQYHNWPPSYS



 


TCN-530 (5248_H10a) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 863)









RASQSVSNNLA



 


CDR 2:








(SEQ ID NO: 864)









DASTRAT



 


CDR 3:








(SEQ ID NO: 865)









QQYHNWPPSYS



 


TCN-531 (5091_H13) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 866)









GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTACAGCCAGGGCGGTCCCTGAAACTCTCCTGCACAGGTTCT



 


GGATTCACCTTTGGTGATTATGGTGTGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTC


 


ATTAGAACCAGACCTTGGGGTGGGACAGCAGATACCGCCGCGTCTGTGAAAGGCAGATTCACTATTTCAAGAGAT


 


GATTCCAAAAGTCTCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTTGTAGA


 


GATGCCCCTCCAAATGTGGAAGTGGCTTCTATGACCAACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTC


 


ACCGTCTCCTCA


 


TCN-531 (5091_H13) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 867)









EVQLVESGGDLVQPGRSLKLSCTGSGFTFGDYGVTWVRQAPGKGLEWVGFIRTRPWGGTADTAASV



 


KGRFTISRDDSKSLAYLQMNSLKTEDTAVYYCCRDAPPNVEVASMTNWYFDLWGRGTLVTVSS


 


TCN-531 (5091_H13) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 868)









DYGVT



 


CDR 2:








(SEQ ID NO: 869)









FIRTRPWGGTADTAASVKG



 


CDR 3:








(SEQ ID NO: 870)









DAPPNVEVASMTNWYFDL



 


TCN-531 (5091_H13) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 871)









GFTFGD



 


CDR 2:








(SEQ ID NO: 872)









FIRTRPWGGTAD



 


CDR 3:








(SEQ ID NO: 870)









DAPPNVEVASMTNWYFDL



 


TCN-531 (5091_H13) light chain variable region nucleotide sequence:








(SEQ ID NO: 873)









GACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCG



 


AGTCAGGGCATTCTCAATTGTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGTTCCTAACCTCCTGATGTATGCT


 


GCATCCACATTGCAGTCAGGGGTCCCATCTCGGTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATC


 


AGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAACGTATGGCGGTGTCTCTACTTTCGGCGGAGGG


 


ACCAAGGTGGAGATCAGA


 


TCN-531 (5091_H13) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 874)









DIQLTQSPSSLSASVGDRVTITCRASQGILNCLAWYQQKPGKVPNLLMYAASTLQSGVPSRFSGSGFG



 


TDFTLTISSLQPEDVATYYCQTYGGVSTFGGGTKVEIR


 


TCN-531 (5091_H13) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 875)









RASQGILNCLA



 


CDR 2:








(SEQ ID NO: 876)









AASTLQS



 


CDR 3:








(SEQ ID NO: 877)









QTYGGVST



 


TCN-531 (5091_H13) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 875)









RASQGILNCLA



 


CDR 2:








(SEQ ID NO: 876)









AASTLQS



 


CDR 3:








(SEQ ID NO: 877)









QTYGGVST



 


TCN-532 (5262_H18) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 878)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCTTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCGTCAGCAGTGAGACTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTAGAGTGGATT


 


GGATATATCTATTACATTGGGAACACCGACTACAGGCCCTCCCTCAAGAGTCGAGTCACCATATCACTGGACACG


 


TCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGAGGC


 


GCTTATTATGATAGTAGTGGTTACCCGGCTTTTTATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA


 


TCN-532 (5262_H18) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 879)









QVQLQESGPGLVKPSETLSLTCTVSGGSVSSETYYWSWIRQPPGKGLEWIGYIYYIGNTDYRPSLKSR



 


VTISLDTSKNQFSLKLSSVTAADTAVYYCARGAYYDSSGYPAFYIWGQGTMVTVSS


 


TCN-532 (5262_H18) gamma heavy chain Kabat CDRs:


 


CDR 1:








(SEQ ID NO: 880)









SETYYWS



 


CDR 2:








(SEQ ID NO: 881)









YIYYIGNTDYRPSLKS



 


CDR 3:








(SEQ ID NO: 882)









GAYYDSSGYPAFYI



 


TCN-532 (5262_H18) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 883)









GGSVSSET



 


CDR 2:








(SEQ ID NO: 884)









YIYYIGNTD



 


CDR 3:








(SEQ ID NO: 882)









GAYYDSSGYPAFYI



 


TCN-532 (5262_H18) light chain variable region nucleotide sequence:








(SEQ ID NO: 885)









CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGC



 


AGCTCCAACATCGGGTCAGATTATGATGTGCACTGGTACAAGCAACTTCCAGGAACAGCCCCCAAACTCCTCATC


 


TTTGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTG


 


GCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAATCCTATGACAGCAGCCTGAGTGGTTTT


 


CATGTCTTCGGAAGTGGGACCAAGGTCACCGTCCTA


 


TCN-532 (5262_H18) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 886)









QSVLTQPPSVSGAPGQRVTISCTGSSSNIGSDYDVHWYKQLPGTAPKLLIFGNSNRPSGVPDRFSGSKS



 


GTSASLAITGLQAEDEADYYCQSYDSSLSGFHVFGSGTKVTVL


 


TCN-532 (5262_H18) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 887)









TGSSSNIGSDYDVH



 


CDR 2:








(SEQ ID NO: 888)









GNSNRPS



 


CDR 3:








(SEQ ID NO: 889)









QSYDSSLSGFHV



 


TCN-532 (5262_H18) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 887)









TGSSSNIGSDYDVH



 


CDR 2:








(SEQ ID NO: 888)









GNSNRPS



 


CDR 3:








(SEQ ID NO: 889)









QSYDSSLSGFHV



 


TCN-533 (5256_A17) heavy chain variable region nucleotide sequence:









(SEQ ID NO: 890)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGACGTGAAGAAGCCTGGGTCCTCGGTGACGGTCTCCTGCAAGGCTTCT



 


GGAGGCAGCTTCAGCAACTATGGAATCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGGGA


 


ATCATCCCTCTCATTAATGCACCGAACTACGCACCGAAGTTCCAGGGCAGAGTGACGATTACCGCGGACATGTTC


 


TCGAATATAGTCTCCTTGCAGTTGACCAGCCTGAGAACTGACGACACGGCCGTGTATTATTGTGCGAGACGAAAA


 


ATGACTACGGCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA


 


TCN-533 (5256_A17) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 891)









QVQLVQSGADVKKPGSSVTVSCKASGGSFSNYGINWVRQAPGQGLEWMGGIIPLINAPNYAPKFQG



 


RVTITADMFSNIVSLQLTSLRTDDTAVYYCARRKMTTAIDYWGQGTLVTVSS


 


TCN-533 (5256_A17) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 892)









NYGIN



 


CDR 2:








(SEQ ID NO: 893)









NAPNYAPKFQG



 


CDR 3:








(SEQ ID NO: 894)









RKMTTAIDY



 


TCN-533 (5256_A17) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 787)









GGSFSN



 


CDR 2:








(SEQ ID NO: 895)









GIIPLINAPN



 


CDR 3:








(SEQ ID NO: 894)









RKMTTAIDY



 


TCN-533 (5256_A17) light chain variable region nucleotide sequence:








(SEQ ID NO: 896)









CAGCCTGTGTTGAGTCAGCCACCTTCTGCATCGGCCTCCCTGGGAGCCTCCGTCACACTCACCTGCAC



 


CCTGAGTAGCGGCTTCGATAATTATCAAGTGGCCTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGCT


 


TTGTGATGCGGGTGGGCAATGGTGGGAATGTGGCTTCCAAGGGGGATGGCATTCCTGATCGTTTCTCA


 


GTCTCGGGCTCAGGCCTGAATCGGTACCTGACCATCAAGAACATCCAGGAAGACGATGAGAGTGACTA


 


TTATTGTGGGGCAGACCATGGCAGTGGGAACAACTTCGTGTCCCCTTATGTGTTTGGCGGAGGGACCA


 


AGCTGACCGTTCTA


 


TCN-533 (5256_A17) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 897)









QPVLSQPPSASASLGASVTLTCTLSSGFDNYQVAWYQQRPGKGPRFVMRVGNGGNVASKG



 


DGIPDRFSVSGSGLNRYLTIKNIQEDDESDYYCGADHGSGNNFVSPYVFGGGTKLTVL


 


TCN-533 (5256_A17) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 898)









TLSSGFDNYQVA



 


CDR 2:








(SEQ ID NO: 899)









VGNGGNVASKGD



 


CDR 3:








(SEQ ID NO: 900)








GADHGSGNNFVSPYV


 


TCN-533 (5256_A17) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 898)









TLSSGFDNYQVA



 


CDR 2:








(SEQ ID NO: 899)









VGNGGNVASKGD



 


CDR 3:








(SEQ ID NO: 900)









GADHGSGNNFVSPYV



 


TCN-534 (5249_B02) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 901)









CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCAGGGTCCTCGGTGAAGGTCTCCTGCAGGGAATCT



 


GGAGGCACCTTCAACGGCTACACTATCACCTGGGTGCGACAGGCCCCTGGGCAAGGCCTTGAGTGGATGGGAGGG


 


ATCATCCCTATGATGGGGACAGTCAACTACGCACAGAAGTTGCAGGGCAGAGTCACCATTACCACGGACTATTTC


 


ACGAAAACAGCCTACATGGATCTGAACAATTTAAGATCTGAAGACACGGCCATGTATTATTGTGTGAAAATCAGA


 


TATACTGGGCAGCAGCTGCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA


 


TCN-534 (5249_B02) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 902)









QVQLVQSGAEVKKPGSSVKVSCRESGGTFNGYTITWVRQAPGQGLEWMGGIIPMMGTVNYAQKLQ



 


GRVTITTDYFTKTAYMDLNNLRSEDTAMYYCVKIRYTGQQLLWGQGTLVTVSS


 


TCN-534 (5249_B02) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 903)









GYTIT



 


CDR 2:








(SEQ ID NO: 904)









GIIPMMGTVNYAQKLQG



 


CDR 3:








(SEQ ID NO: 905)









IRYTGQQLL



 


TCN-534 (5249_B02) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 906)









GGTFNG



 


CDR 2:








(SEQ ID NO: 907)









GIIPMMGTVN



 


CDR 3:








(SEQ ID NO: 905)









IRYTGQQLL



 


TCN-534 (5249_B02) light chain variable region nucleotide sequence:








(SEQ ID NO: 908)









GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCGGCATCTATAGGAGACAGAGTCACCATCACTTGCCGGGCC



 


AGTCAGAGTATTGCAAGTTGGTTGGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAG


 


GCAGTTAATTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC


 


AGCAGCCTGCAGCCCGATGATTTTGCAACTTATTTCTGCCAACATTATGGTACTATTTCTCAGACCTTCGGCGGA


 


GGGACCAAGGTGGAGATCAAA


 


TCN-534 (5249_B02) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 909)









DIQMTQSPSTLSASIGDRVTITCRASQSIASWLAWYQQKPGKAPKLLIYEAVNLESGVPSRFSGSGSGT



 


DFTLTISSLQPDDFATYFCQHYGTISQTFGGGTKVEIK


 


TCN-534 (5249_B02) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 910)









RASQSIASWLA



 


CDR 2:








(SEQ ID NO: 911)









EAVNLES



 


CDR 3:








(SEQ ID NO: 912)









QHYGTISQT



 


TCN-534 (5249_B02) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 910)









RASQSIASWLA



 


CDR 2:








(SEQ ID NO: 911)









EAVNLES



 


CDR 3:








(SEQ ID NO: 912)








QHYGTISQT


 


TCN-535 (5246_P19) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 913)









CAGGTCCAGCTGGTGCAATCTGGGAGTGAGGTGAAGAAGCCTGGGACCTCGGTGAAGGTCTCCTGCACGGCCTCT



 


GGAAGTGTCTTCACCAATTATGGAATTAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCATCCCTCTCTTTGGCGCAGCCAAGTACGCACAGAAATTCCAGGGCAGAGTCACCATCACAGCGGACGAATCC


 


ACGAAGACAGTCTACATGGAGCTGAGCAGGCTGACATCTAAAGACACGGCCATATATTTCTGTGCGAAGGCCCCC


 


CGTGTCTACGAGTACTACTTTGATCAGTGGGGCCAGGGAACCCCAGTCACCGTCTCCTCA


 


TCN-535 (5246_P19) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 914)









QVQLVQSGSEVKKPGTSVKVSCTASGSVFTNYGISWVRQAPGQGLEWMGGIIPLFGAAKYAQKFQG



 


RVTITADESTKTVYMELSRLTSKDTAIYFCAKAPRVYEYYFDQWGQGTPVTVSS


 


TCN-535 (5246_P19) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 915)









NYGIS



 


CDR 2:








(SEQ ID NO: 916)









GIIPLFGAAKYAQKFQG



 


CDR 3:








(SEQ ID NO: 917)









APRVYEYYFDQ



 


TCN-535 (5246_P19) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 918)









GSVFTN



 


CDR 2:








(SEQ ID NO: 919)









GIIPLFGAAK



 


CDR 3:








(SEQ ID NO: 917)









APRVYEYYFDQ



 


TCN-535 (5246_P19) light chain variable region nucleotide sequence:








(SEQ ID NO: 920)









GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTAGCAGCAGTCAATTAGCCTGGTACCAGCAAAAACCTGGCCAGGCTCCCAGGCTCATCATCTAT


 


GGTGCGTCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGAAGTGGGTCTGGGACAGACTTCACTCTCACC


 


ATCGGCAGACTGGAGCCTGAAGATTTTGCAGTGTTTTTCTGTCAGCAGTATAGTACCTCACCTCCGACGTTCGGC


 


CAAGGGACCAAGGTGGATTTCAAA


 


TCN-535 (5246_P19) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 921)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSSQLAWYQQKPGQAPRLIIYGASTRATGIPDRFSGSGSGT



 


DFTLTIGRLEPEDFAVFFCQQYSTSPPTFGQGTKVIDEK


 


TCN-535 (5246_P19) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 922)









RASQSVSSSQLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 923)









QQYSTSPPT



 


TCN-535 (5246_P19) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 922)









RASQSVSSSQLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 923)









QQYSTSPPT



 


TCN-536 (5095_N01) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 924)









CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT



 


GGTGGGTCCTTCAGTGTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAGGGGGCTGGAGTGGATT


 


GGGGAAATCAGTCATGGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTGGACACG


 


ACCAAGAACCAGTTCTCCCTGAGACTGAGCTCTGTGACCGCCGCGGACACGGCCGTCTATTACTGTGCGAGAGGG


 


ACAGACCCTGACACGGAAGTATATTGTCGTGTTGGTAACTGCGCGGCCTTTGACTACTGGGGCCAGGGAAGCCTG


 


GTCACCGTCTCCTCA


 


TCN-536 (5095_N01) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 925)









QVQLQQWGAGLLKPSETLSLTCAVYGGSFSVSGYYWSWIRQPPGRGLEWIGEISHGGSTNYNPSLKS



 


RVTISVDTTKNQFSLRLSSVTAADTAVYYCARGTDPDTEVYCRVGNCAAFDYWGQGSLVTVSS


 


TCN-536 (5095_N01) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 926)









VSGYYWS



 


CDR 2:








(SEQ ID NO: 927)









EISHGGSTNYNPSLKS



 


CDR 3:








(SEQ ID NO: 928)









GTDPDTEVYCRVGNCAAFDY



 


TCN-536 (5095_N01) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 929)









GGSFSVSG



 


CDR 2:








(SEQ ID NO: 930)









EISHGGSTN



 


CDR 3:








(SEQ ID NO: 928)









GTDPDTEVYCRVGNCAAFDY



 


TCN-536 (5095_N01) light chain variable region nucleotide sequence:








(SEQ ID NO: 931)









GAAATTATATTGGCGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAGAGAGCCACCCTCTCCTGCAGGGCC



 


AGCCAGTTTGTTAGCACCAGATCCCTGGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGACTCCTCATCTAT


 


GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACGCTCACC


 


ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTATGGTTACTCACCTAGGTACGCTTTT


 


GGCCAGGGGTCCAAGGTTGAGATCAAA


 


TCN-536 (5095_N01) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 932)









EIILAQSPGTLSLSPGERATLSCRASQFVSTRSLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGT



 


DFTLTISRLEPEDFAVYYCQHYGYSPRYAFGQGSKVEIK


 


TCN-536 (5095_N01) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 933)









RASQFVSTRSLA



 


CDR 2:








(SEQ ID NO: 768)









GASSRAT



 


CDR 3:








(SEQ ID NO: 934)









QHYGYSPRYA



 


TCN-536 (5095_N01) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 933)









RASQFVSTRSLA



 


CDR 2:








(SEQ ID NO: 768)









GASSRAT



 


CDR 3:








(SEQ ID NO: 934)









QHYGYSPRYA



 


TCN-537 (3194_D21) heavy chain variable region nucleotide sequence:









(SEQ ID NO: 935)









CAGGTGCAGCTCCAACAGTGGGGCTCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT



 


GGTGGGTCCTTCAGAGATGACTACTGGACCTGGATTCGCCAGCCCCCAGGCAAGGGGCTGGAGTGGATTGGGGAA


 


ATCAATCATAGTGGAAGAACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCCTG


 


AAACAGTTCTCCTTGAAGGTGATTTCTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAGAGGGACGAGC


 


CATGTTTCCCGGTATTTTGATTGGTTACCACCCACCAACTGGTTCGACCCCTGGGGCCAGGGAACCCAGGTCACC


 


GTCTCGAGC


 


TCN-537 (3194_D21) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 936)









QVQLQQWGSGLLKPSETLSLTCAVYGGSFRDDYWTWIRQPPGKGLEWIGEINHSGRTNYNPSLKSRV



 


TISVDTSLKQFSLKVISVTAADTAVYYCARGTSHVSRYFDWLPPTNWFDPWGQGTQVTVSS


 


TCN-537 (3194_D21) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 937)









DDYWT



 


CDR 2:








(SEQ ID NO: 938)









EINHSGRTNYNPSLKS



 


CDR 3:








(SEQ ID NO: 939)









GTSHVSRYFDWLPPTNWFDP



 


TCN-537 (3194_D21) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 940)









GGSFRD



 


CDR 2:








(SEQ ID NO: 941)









EINHSGRTN



 


CDR 3:








(SEQ ID NO: 939)









GTSHVSRYFDWLPPTNWFDP



 


TCN-537 (3194_D21) light chain variable region nucleotide sequence:








(SEQ ID NO: 942)








GACATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC


 


AGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCGTCATGTAT


 


GGTGCAGCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGCCAGACTTCACTCTCACC


 


ATCAGCAGACTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAGCAGTATGGTAACTCACCGATCACCTTCGGC


 


CAAGGGACACGACTGGAGATCAAA


 


TCN-537 (3194_D21) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 943)









DIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLVMYGAATRATGIPDRFSGSGS



 


GPDFTLTISRLEPEDFAMYYCQQYGNSPITFGQGTRLEIK


 


TCN-537 (3194_D21) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 944)









RASQSVSSSYLA



 


CDR 2:








(SEQ ID NO: 945)









GAATRAT



 


CDR 3:








(SEQ ID NO: 946)









QQYGNSPIT



 


TCN-537 (3194_D21) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 944)









RASQSVSSSYLA



 


CDR 2:








(SEQ ID NO: 945)









GAATRAT



 


CDR 3:








(SEQ ID NO: 946)









QQYGNSPIT



 


TCN-538 (3206_O17) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 947)









CAGATCACCTTGAAGGAGTCTGGTCCTACACTGGTGAAACCCACACAGACCCTCACACTGACCTGCGTCTTCTCT



 


GGGTTCTCACTCAGCATTACTGGAGTGCGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT


 


GCACTCATTTCTTGGGATGATGAAAAGCACTACAGCCCATCTCTGCAGAGTAGGCTCACCATCACCAAGGACACC


 


TCCAAAAACCAGGTGGTCCTTACAATGACCAACCTGGACCCTGTCGACACAGCCACATATTACTGTGCACGGTCA


 


ACCGACAGGGGCCACGTCTTACGATATTTTGGCTGGATGTTACCGGGTGATGCATTTGATGTCTGGGGCCAAGGG


 


ACAATGGTCACCGTCTCGAGC


 


TCN-538 (3206_O17) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 948)









QITLKESGPTLVKPTQTLTLTCVFSGFSLSITGVRVGWIRQPPGKALEWLALISWDDEKHYSPSLQSRL



 


TITKDTSKNQVVLTMTNLDPVDTATYYCARSTDRGHVLRYFGWMLPGDAFDVWGQGTMVTVSS


 


TCN-538 (3206_O17) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 949)









ITGVRVG



 


CDR 2:








(SEQ ID NO: 950)









LISWDDEKHYSPSLQS



 


CDR 3:








(SEQ ID NO: 951)









STDRGHVLRYFGWMLPGDAFDV



 


TCN-538 (3206_O17) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 952)









GFSLSITG



 


CDR 2:








(SEQ ID NO: 953)









LISWDDEKH



 


CDR 3:








(SEQ ID NO: 951)









STDRGHVLRYFGWMLPGDAFDV



 


TCN-538 (3206_O17) light chain variable region nucleotide sequence:








(SEQ ID NO: 954)









GACATCGTGATGACCCAGTCTCCAGACTTCCTGCCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCC



 


AGCCAGAGAGTTTTATACAGCTCCAACAATAAAAACTACTTAGCTTGGTACCAGCTGAAACCAGGGCAGCCTCCT


 


AAGTTGATCATTTATTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA


 


GAATTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATAGTCGT


 


CCGTACACTTTTGGCCAGGGGACCAAGCTCGAGATCAAA


 


TCN-538 (3206_O17) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 955)









DIVMTQSPDFLPVSLGERATINCKSSQRVLYSSNNKNYLAWYQLKPGQPPKLIIYWASTRESGVPDRFS



 


GSGSGTEFTLTISSLQAEDVAVYYCQQYYSRPYTFGQGTKLEIK


 


TCN-538 (3206_O17) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 956)









KSSQRVLYSSNNKNYLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 958)









QQYYSRPYT



 


TCN-538 (3206_O17) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 956)









KSSQRVLYSSNNKNYLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 958)









QQYYSRPYT



 


TCN-539 (5056_A08) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 959)









CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GAAATCACCTTCATTACCTATGCTATGCACTGGGTCCGCCAGGCCCCAGGCAAGGGGCTGGAGTGGGTGGCACTT


 


ATATCAGATGATGGAAGCAATAAATTCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC


 


AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGCTTATTACTGTGCGAGAGAAGGG


 


GTTTACTTTGATTCGGGGACTTATAGGGGCTACTTTGACTACTGGGGCCAGGAAACCCTGGTCACCGTCTCGAGC


 


TCN-539 (5056_A08) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 960)









QVQLVESGGGVVQPGRSLRLSCAASEITFITYAMHWVRQAPGKGLEWVALISDDGSNKFYADSVKG



 


RFTISRDNSKNTLYLQMNSLRAEDTAAYYCAREGVYFDSGTYRGYFDYWGQETLVTVSS


 


TCN-539 (5056_A08) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 961)









TYAMH



 


CDR 2:








(SEQ ID NO: 962)









LISDDGSNKFYADSVKG



 


CDR 3:








(SEQ ID NO: 963)









EGVYFDSGTYRGYFDY



 


TCN-539 (5056_A08) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 964)









EITFIT



 


CDR 2:








(SEQ ID NO: 965)









LISDDGSNKF



 


CDR 3:








(SEQ ID NO: 963)









EGVYFDSGTYRGYFDY



 


TCN-539 (5056_A08) light chain variable region nucleotide sequence:








(SEQ ID NO: 966)









GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT


 


GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC


 


AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCCACTGGCCTCCGATCACCTTCGGC


 


CAAGGGACACGACTGGAGATCAAA


 


TCN-539 (5056_A08) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 967)









EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD



 


FTLTISSLEPEDFAVYYCQQRSHWPPITFGQGTRLEIK


 


TCN-539 (5056_A08) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 968)









RASQSVSSYLA



 


CDR 2:








(SEQ ID NO: 969)









DASNRAT



 


CDR 3:








(SEQ ID NO: 970)









QQRSHWPPIT



 


TCN-539 (5056_A08) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 968)









RASQSVSSYLA



 


CDR 2:








(SEQ ID NO: 969)









DASNRAT



 


CDR 3:








(SEQ ID NO: 970)









QQRSHWPPIT



 


TCN-540 (5060_F05) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 971)









CAGGTGCAGCTGGTACAATCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GGATTCACCTTCAGTAGCTACGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCTATT


 


ATATCATACGACGGAAATGATCAATACTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCTCC


 


AAAGTGTATCTCCAAATGCACAGGCTGAGACCTGAGGACACGGCTGTTTATTACTGTGCGAAAGAATTTGAAACT


 


AGTGGTTATTTTCATGGGAGTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCGAGC


 


TCN-540 (5060_F05) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 972)









QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAIISYDGNDQYYTDSVKG



 


RFTISRDSSKVYLQMHRLRPEDTAVYYCAKEFETSGYFHGSFDYWGQGILVTVSS


 


TCN-540 (5060_F05) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 973)









SYAMH



 


CDR 2:








(SEQ ID NO: 974)









IISYDGNDQYYTDSVKG



 


CDR 3:








(SEQ ID NO: 975)









EFETSGYFHGSFDY



 


TCN-540 (5060_F05) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 976)









GFTFSS



 


CDR 2:








(SEQ ID NO: 977)









IISYDGNDQY



 


CDR 3:








(SEQ ID NO: 975)









EFETSGYFHGSFDY



 


TCN-540 (5060_F05) light chain variable region nucleotide sequence:








(SEQ ID NO: 978)









CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACC



 


AGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCTTGATT


 


TATGAGGTCACTAATTGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGACTATTACTGCAGCTCATATGCGGGCAGCAGCACTTGGGTG


 


TTCGGCGGAGGGACCAGGGTGACCGTTCTA


 


TCN-540 (5060_F05) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 979)









QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLLIYEVTNWPSGVSNRFSGSK



 


SGNTASLTISGLQAEDEADYYCSSYAGSSTWVFGGGTRVTVL


 


TCN-540 (5060_F05) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 980)









TGTSSDVGGYNYVS



 


CDR 2:








(SEQ ID NO: 981)









EVTNWPS



 


CDR 3:








(SEQ ID NO: 982)









SSYAGSSTWV



 


TCN-540 (5060_F05) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 980)









TGTSSDVGGYNYVS



 


CDR 2:








(SEQ ID NO: 981)









EVTNWPS



 


CDR 3:








(SEQ ID NO: 982)









SSYAGSSTWV



 


TCN-541 (5062_M11) heavy chain variable region nucleotide sequence:









(SEQ ID NO: 983)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCATCAATAGTTACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGCTAT


 


ATCTATCACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATTTCGGTAGACACGTCCAAG


 


AACCAGTTCTCCCTGCAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGACTCCGGACG


 


GACTACGGTGACCCCGACTCGGTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCG


 


AGC


 


TCN-541 (5062_M11) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 984)









QVQLQESGPGLVKPSETLSLTCTVSGGSINSYYWNWIRQPPGKGLEWIGYIYHSGSTNYNPSLKSRVTI



 


SVDTSKNQFSLQLSSVTAADTAVYYCARLRTDYGDPDSVYYYGMDVWGQGTTVTVSS


 


TCN-541 (5062_M11) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 985)









SYYWN



 


CDR 2:








(SEQ ID NO: 986)









YIYHSGSTNYNPSLKS



 


CDR 3:








(SEQ ID NO: 987)









LRTDYGDPDSVYYYGMDV



 


TCN-541 (5062_M11) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 988)









GGSINS



 


CDR 2:








(SEQ ID NO: 989)









YIYHSGSTN



 


CDR 3:








(SEQ ID NO: 987)









LRTDYGDPDSVYYYGMDV



 


TCN-541 (5062_M11) light chain variable region nucleotide sequence:








(SEQ ID NO: 990)









TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGAT



 


GCATTGCCAAAGCAAAATGCTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGCTGATATATAAAGAC


 


AGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGCTCAGGGACAACAGTCACGTTGACCATCAGT


 


GGAGTCCAGGCAGAGGACGAGGCTGACTATTACTGTCAATCAGCAGACAGCAGTGGTACTTCTTGGGTGTTCGGC


 


GGAGGGACCAAACTGACCGTTCTA


 


TCN-541 (5062_M11) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 991)









SYELTQPPSVSVSPGQTARITCSGDALPKQNAYWYQQKPGQAPVLLIYKDSERPSGIPERFSGSSSGTT



 


VTLTISGVQAEDEADYYCQSADSSGTSWVFGGGTKLTVL


 


TCN-541 (5062_M11) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 994)









SGDALPKQNAY



 


CDR 2:








(SEQ ID NO: 995)









KDSERPS



 


CDR 3:








(SEQ ID NO: 996)









QSADSSGTSWV



 


TCN-541 (5062_M11) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 994)









SGDALPKQNAY



 


CDR 2:








(SEQ ID NO: 995)









KDSERPS



 


CDR 3:








(SEQ ID NO: 996)









QSADSSGTSWV



 


TCN-542 (5079_A16) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 992)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCATCAGCAGTGGTAATTACTACTGGAACTGGGTCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT


 


GGGTACATCTATTACAGAGGGAGCACCTTCTACAACCCGTCCCTCAAGAGTCGAGTGACCATATCAATAGACACG


 


TCTAAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACGGCCGCGGACACGGCCGTGTATTACTGTGCGAAGGAT


 


ACAAGGTCGAGCCTAGACAATTACCAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC


 


TCN-542 (5079_A16) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 993)









QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGNYYWNWVRQHPGKGLEWIGYIYYRGSTFYNPSLKSR



 


VTISIDTSKNQFSLRLSSVTAADTAVYYCAKDTRSSLDNYQYGMDVWGQGTTVTVSS


 


TCN-542 (5079_A16) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 997)









SGNYYWN



 


CDR 2:








(SEQ ID NO: 998)









YIYYRGSTFYNPSLKS



 


CDR 3:








(SEQ ID NO: 999)









DTRSSLDNYQYGMDV



 


TCN-542 (5079_A16) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1000)








GGSISSGN


 


CDR 2:








(SEQ ID NO: 1001)









YIYYRGSTF



 


CDR 3:








(SEQ ID NO: 999)









DTRSSLDNYQYGMDV



 


TCN-542 (5079_A16) light chain variable region nucleotide sequence:








(SEQ ID NO: 1002)









CAGACTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGCTTCCAGC



 


ACTGGAGCAGTCACCAGTAGTTACTTTCCAAACTGGTTCCAGCAGAAACCTGGACAAGCGCCCAGGCCACTGATT


 


TATAGTACAACTATCAGACACTCCTGGACCCCGGCCCGATTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTG


 


ACACTGTCAGGTGTGCAGCCTGAGGACGAGGCTGACTATTACTGCCTGCTCTACTCTGGTGGTGATCCAGTGGCT


 


TTCGGCGGAGGGACCAAACTGACCGTTCTA


 


TCN-542 (5079_A16) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1003)









QTVVTQEPSLTVSPGGTVTLTCASSTGAVTSSYFPNWFQQKPGQAPRPLIYSTTIRHSWTPARFSGSLL



 


GGKAALTLSGVQPEDEADYYCLLYSGGDPVAFGGGTKLTVL


 


TCN-542 (5079_A16) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1004)








ASSTGAVTSSYFPN


 


CDR 2:








(SEQ ID NO: 1005)









STTIRHS



 


CDR 3:








(SEQ ID NO: 1006)









LLYSGGDPVA



 


TCN-542 (5079_A16) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1004)









ASSTGAVTSSYFPN



 


CDR 2:








(SEQ ID NO: 1005)









STTIRHS



 


CDR 3:








(SEQ ID NO: 1006)









LLYSGGDPVA



 


TCN-543 (5081_G23) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1007)









CAGGTTCATCTGGTGCAGTCTGGAGCTGAGGTGAGGAAGCCTGGGGACTCAGTGAAGGTCTCCTGTAAGACTTCT



 


GGTTACACCTTTTCCACCTATCCTGTCGCCTGGGTGCGACAGGTCCCCGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAGCACTTACAATGGAAACACAAACTTTGCACAGAACTTCCAGGGCAGAGTCACCCTGACCACAGACACAACC


 


ACGAACACAGCCTACATGGAAGTGAGGAGCCTGAAATTTGACGACACGGCCGTCTATTACTGTGCGAGAGTGGAA


 


GGCTCGTACAGGGATTTTTGGAATAATCAAAACAGATTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCG


 


AGC


 


TCN-543 (5081_G23) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1008)









QVHLVQSGAEVRKPGDSVKVSCKTSGYTFSTYPVAWVRQVPGQGLEWMGWISTYNGNTNFAQNFQ



 


GRVTLTTDTTTNTAYMEVRSLKFDDTAVYYCARVEGSYRDFWNNQNRFDPWGQGTLVTVSS


 


TCN-543 (5081_G23) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1009)









TYPVA



 


CDR 2:








(SEQ ID NO: 1010)









WISTYNGNTNFAQNFQG



 


CDR 3:








(SEQ ID NO: 1011)









VEGSYRDFWNNQNRFDP



 


TCN-543 (5081_G23) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1012)









GYTFST



 


CDR 2:








(SEQ ID NO: 1013)









WISTYNGNTN



 


CDR 3:








(SEQ ID NO: 1011)









VEGSYRDFWNNQNRFDP



 


TCN-543 (5081_G23) light chain variable region nucleotide sequence:








(SEQ ID NO: 1014)









TCCTATGTACTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTTCCTGTGGGGGAAGC



 


AACATTGGAGGGAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGAT


 


AGCGGCCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACACGGCCACCCTGACCATCAGC


 


AGGGTCGAAGCCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGATAATTTCGGGGGAGTCTTCGGAACTGGG


 


ACCAAGGTCACCGTTCTA


 


TCN-543 (5081_G23) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1015)









SYVLTQPPSVSVAPGQTARISCGGSNIGGKSVHWYQQKPGQAPVLVVYDDSGRPSGIPERFSGSNSGD



 


TATLTISRVEAGDEADYFCQVWDNFGGVFGTGTKVTVL


 


TCN-543 (5081_G23) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1016)









GGSNIGGKSVH



 


CDR 2:








(SEQ ID NO: 1017)









DDSGRPS



 


CDR 3:








(SEQ ID NO: 1018)









QVWDNFGGV



 


TCN-543 (5081_G23) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1016)









GGSNIGGKSVH



 


CDR 2:








(SEQ ID NO: 1017)









DDSGRPS



 


CDR 3:








(SEQ ID NO: 1018)









QVWDNFGGV



 


TCN-544 (5082_A19) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1019)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGGCTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


CGTGGCTCCATCGGTCATTACTTCTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGTTAT


 


ATCTCTTACAGTGGGAGCACCAAGTACAACCCCTCCCTCAGGAGTCGAGTCACCATATCAGTAGACACGTCCAAG


 


AACCAGTTCTCCCTGAATCTGAACTCTGTCACCGCTACGGACACGGCCCTATATTACTGTGCGAGAGAGGATTAC


 


GATATTTTGACTGGGGCGGGACCCGGTATGGAGGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC


 


TCN-544 (5082_A19) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1020)









QVQLQESGPGLVKPSETLSLTCTVSRGSIGHYFWSWIRQPPGKGLEWIGYISYSGSTKYNPSLRSRVTIS



 


VDTSKNQFSLNLNSVTATDTALYYCAREDYDILTGAGPGMEVWGQGTTVTVSS


 


TCN-544 (5082_A19) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1021)









HYFWS



 


CDR 2:








(SEQ ID NO: 1022)









YISYSGSTKYNPSLRS



 


CDR 3:








(SEQ ID NO: 1023)









EDYDILTGAGPGMEV



 


TCN-544 (5082_A19) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1024)









RGSIGH



 


CDR 2:








(SEQ ID NO: 1025)









YISYSGSTK



 


CDR 3:








(SEQ ID NO: 1023)









EDYDILTGAGPGMEV



 


TCN-544 (5082_A19) light chain variable region nucleotide sequence:








(SEQ ID NO: 1026)









CAGTCTATGTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGGAGC



 


AGCTCCAACATCGGAAGTAATACTGTCAACTGGTTCAAACATCTCCCAGGAACGGCCCCCAAACTCCTCATCTAC


 


AGAAATGATCTGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC


 


ATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAACATGGGATGACAGCCTGAATGGTTTTTAT


 


GTCTTCGGAACTGGGACCAAAGTCACCGTTCTA


 


TCN-544 (5082_A19) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1027)









QSMLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWFKHLPGTAPKLLIYRNDLRPSGVPDRFSGSKSGT



 


SASLAISGLQSEDEADYYCATWDDSLNGFYVFGTGTKVTVL


 


TCN-544 (5082_A19) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1028)









SGSSSNIGSNTVN



 


CDR 2:








(SEQ ID NO: 1029)









RNDLRPS



 


CDR 3:








(SEQ ID NO: 1030)









ATWDDSLNGFYV



 


TCN-544 (5082_A19) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1028)









SGSSSNIGSNTVN



 


CDR 2:








(SEQ ID NO: 1029)









RNDLRPS



 


CDR 3:








(SEQ ID NO: 1030)









ATWDDSLNGFYV



 


TCN-545 (5082_I15) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1031)









CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCTCCTGCGCTGTCTTT



 


GGTGGGTCCTTCAGTGATTACTACTGGACCTGGATACGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGCGAA


 


ATCAAACATAGTGGAAGAACCAACTACAACCCGTCCCTTGAGAGTCGAGTCACCATATCAGTGGACACGTCCAAG


 


AACCAGTTTTCCCTGAAACTGAGTTCTGTGACCGCCGCGGACACGGCTATATATTATTGTGCGAGAGGGACAGAC


 


CCTGACACGGAGGGATATTGTCGTAGTGGTAGCTGCTCGGCCTTTGACTTCTGGGGCCAGGGAACCCTGGTCACC


 


GTCTCGAGC


 


TCN-545 (5082_I15) gamma heavy chain variable region amino acid sequence:



(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1032)









QVQLQQWGAGLLKPSETLSLSCAVFGGSFSDYYWTWIRQPPGKGLEWIGEIKHSGRTNYNPSLESRV



 


TISVDTSKNQFSLKLSSVTAADTAIYYCARGTDPDTEGYCRSGSCSAFDFWGQGTLVTVSS


 


TCN-545 (5082_I15) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1033)









DYYWT



 


CDR 2:








(SEQ ID NO: 1034)









EIKHSGRTNYNPSLES



 


CDR 3:








(SEQ ID NO: 1035)









GTDPDTEGYCRSGSCSAFDF



 


TCN-545 (5082_I15) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1036)









GGSFSD



 


CDR 2:








(SEQ ID NO: 1037)









EIKHSGRTN



 


CDR 3:








(SEQ ID NO: 1035)









GTDPDTEGYCRSGSCSAFDF



 


TCN-545 (5082_I15) light chain variable region nucleotide sequence:








(SEQ ID NO: 1038)









GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCACTTTGTGAACTACAGGTCCTTAGCCTGGTACCAGCAGACACCTGGCCAGGTTCCCAGGCTCCTCATCTAT


 


GGTGCGTCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC


 


ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTTCTGTCAGCAGTATGGTGGCTCACCTAGGTACACTTTT


 


GGCCAGGGGACCAGGCTGGAGATCAAA


 


TCN-545 (5082_I15) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1039)









EIVLTQSPGTLSLSPGERATLSCRASHFVNYRSLAWYQQTPGQVPRLLIYGASTRATGIPDRFSGSGSGT



 


DFTLTISRLEPEDFAVYFCQQYGGSPRYTFGQGTRLEIK


 


TCN-545 (5082_I15) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1040)









RASHFVNYRSLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 1041)









QQYGGSPRYT



 


TCN-545 (5082_I15) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1040)









RASHFVNYRSLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 1041)









QQYGGSPRYT



 


TCN-546 (5089_L08) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1042)









CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGGTGTCTAT



 


GGTGGGTCCCTCAGTGATTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGAGAA


 


ATCAATCATAGTGGAGGCACCAACTACAATCCGTCCCTCAAGAGACGAGTCACCATATCAGTAGACACGTCAAAG


 


AAGCAATTCTCCCTGAAGATGAACTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGGGACAGAC


 


CCTGACACGGAAGTATATTGTCGTGCTGGTAACTGCGCGGCCTTTGACTTCTGGGGCCAGGGAACCCTGGTCACC


 


GTCTCGAGC


 


TCN-546 (5089_L08) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1043)









QVQLQQWGAGLLKPSETLSLTCGVYGGSLSDYYWSWIRQPPGKGLEWIGEINHSGGTNYNPSLKRRV



 


TISVDTSKKQFSLKMNSVTAADTAVYYCARGTDPDTEVYCRAGNCAAFDFWGQGTLVTVSS


 


TCN-546 (5089_L08) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1044)









DYYWS



 


CDR 2:








(SEQ ID NO: 1045)









EINHSGGTNYNPSLKR



 


CDR 3:








(SEQ ID NO: 1046)









GTDPDTEVYCRAGNCAAFDF



 


TCN-546 (5089_L08) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1047)









GGSLSD



 


CDR 2:








(SEQ ID NO: 1048)









EINHSGGTN



 


CDR 3:








(SEQ ID NO: 1046)









GTDPDTEVYCRAGNCAAFDF



 


TCN-546 (5089_L08) light chain variable region nucleotide sequence:








(SEQ ID NO: 1049)









GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAGAGAGCCACCCTCTCCTGCCGGGCC



 


AGTCACTTTGTTATAGGCAGGGCTGTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAC


 


GGTGCATCCAGCAGGGCCACTGGCATCCCGGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC


 


ATCAGCAGACTGGAGACTGAAGATTTTGCTGTGTTTTACTGTCAGCACTATGGTAGCTCACCTAGGTACGCTTTT


 


GGCCAGGGGACCAAGCTGGAGATCAAA


 


TCN-546 (5089_L08) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1050)









EIVLTQSPGTLSLSPGERATLSCRASHFVIGRAVAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT



 


DFTLTISRLETEDFAVFYCQHYGSSPRYAFGQGTKLEIK


 


TCN-546 (5089_L08) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1051)









RASHFVIGRAVA



 


CDR 2:








(SEQ ID NO: 768)









GASSRAT



 


CDR 3:








(SEQ ID NO: 1052)









QHYGSSPRYAF



 


TCN-546 (5089_L08) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1051)









RASHFVIGRAVA



 


CDR 2:








(SEQ ID NO: 768)









GASSRAT



 


CDR 3:








(SEQ ID NO: 1052)









QHYGSSPRYAF



 


TCN-547 (5092_F11) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1053)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGACTCCATTAGTAGTGTTGATCACTACTGGAGCTGGATCCGCCAACACCCAGTGAAGGGCCTGGAGTGGATT


 


GGGTTCATGTATTACAGTGCGAGCACCTATTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAACGGACACG


 


TCTAAGAACCAGTTCTCCCTGAGGCTGAGTTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGAGAGGC


 


ACTTGTGCTGGTGACTGCTCCCTTCACTACTACTACTACGGTTTGGACGTCTGGGGCCAAGGGAGGACGGTCACC


 


GTCTCGAGC


 


TCN-547 (5092_F11) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1054)









QVQLQESGPGLVKPSQTLSLTCTVSGDSISSVDHYWSWIRQHPVKGLEWIGFMYYSASTYYNPSLKSR



 


VTISTDTSKNQFSLRLSSVTAADTAVYYCARGTCAGDCSLHYYYYGLDVWGQGRTVTVSS


 


TCN-547 (5092_F11) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1055)









SVDHYWS



 


CDR 2:








(SEQ ID NO: 1056)









FMYYSASTYYNPSLKS



 


CDR 3:








(SEQ ID NO: 1057)









GTCAGDCSLHYYYYGLDV



 


TCN-547 (5092_F11) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1058)









GDSISSVD



 


CDR 2:








(SEQ ID NO: 1059)









FMYYSASTY



 


CDR 3:








(SEQ ID NO: 1057)









GTCAGDCSLHYYYYGLDV



 


TCN-547 (5092_F11) light chain variable region nucleotide sequence:








(SEQ ID NO: 1060)









GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCACAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCT


 


GTATCCATTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGGCAGATTTCACTCTCACCATC


 


AGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTTCCCCGCTCACTTTCGGCGGA


 


GGGACCAAGGTGGAGATCAAA


 


TCN-547 (5092_F11) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1061)









DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQHKPGKAPKVLMYAVSILQSGVPSRFSGSGSGA



 


DFTLTISSLQPEDFATYYCQQSYSSPLTFGGGTKVEIK


 


TCN-547 (5092_F11) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1062)









RASQSISSYLN



 


CDR 2:








(SEQ ID NO: 1063)









AVSILQS



 


CDR 3:








(SEQ ID NO: 1064)









QQSYSSPLT



 


TCN-547 (5092_F11) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1062)









RASQSISSYLN



 


CDR 2:








(SEQ ID NO: 1063)









AVSILQS



 


CDR 3:








(SEQ ID NO: 1064)









QQSYSSPLT



 


TCN-548 (5092_P01) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1065)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


AGTGGCCCCATGAGTGATTATTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGCAT


 


GTCTCTGTCTCTCACGGAGGGAGGACCAAATCCAATCCCTCCGTCATGAGTCGAGTCACCATTTCAGTAGAAACG


 


TCCAAGAACCAATTCTCCCTGAAACTGACCTCCGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGATTA


 


AATTACTATGATAGAAGTGGTTATCATTCGCCTGACGGCCCCTCGAACAACTGGTTCGACCCCTGGGGCCAGGGA


 


ACCCTGGTCACCGTCTCGAGC


 


TCN-548 (5092_P01) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1066)









QVQLQESGPGLVKPSETLSLTCTVSSGPMSDYYWSWIRQPPGKGLEWIGHVSVSHGGRTKSNPSVMS



 


RVTISVETSKNQFSLKLTSVTAADTAVYYCARLNYYDRSGYHSPDGPSNNWFDPWGQGTLVTVSS


 


TCN-548 (5092_P01) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1044)









DYYWS



 


CDR 2:








(SEQ ID NO: 1067)









HVSVSHGGRTKSNPSVMS



 


CDR 3:








(SEQ ID NO: 1068)









LNYYDRSGYHSPDGPSNNWFDP



 


TCN-548 (5092_P01) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1069)









SGPMSD



 


CDR 2:








(SEQ ID NO: 1070)









HVSVSHGGRTK



 


CDR 3:








(SEQ ID NO: 1068)









LNYYDRSGYHSPDGPSNNWFDP



 


TCN-548 (5092_P01) light chain variable region nucleotide sequence:








(SEQ ID NO: 1071)









GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCC



 


AGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT


 


AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGAATCAGCGGCAGCGGGTCTGGGGCA


 


GATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTATTTTGCTACT


 


CCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA


 


TCN-548 (5092_P01) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1072)









DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRI



 


SGSGSGADFTLTISSLQAEDVAVYYCQQYFATPRTFGQGTKVEIK


 


TCN-548 (5092_P01) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1073)









KSSQSVLYSSNNKNYLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 1074)









QQYFATPRT



 


TCN-548 (5092_P01) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1073)









KSSQSVLYSSNNKNYLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 1074)









QQYFATPRT



 


TCN-549 (5092_P04) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1075)









CAGGTACAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAACCCTAACAGTGGTGACACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC


 


ATCACCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATTCC


 


CCCTATAGCAGCAGCTGGTCCTTCTTTGACTACTGGGGCCAGGGACCCCTGGTCACCGTCTCGAGC


 


TCN-549 (5092_P04) gamma heavy chain variable region amino acid sequence:



(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1076)









QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGDTNYAQKF



 


QGRVTMTRDTSVITAYMELSSLRSDDTAVYYCARDSPYSSSWSFFDYCWGQGPLVTVSS


 


TCN-549 (5092_P04) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1077)









GYYMH



 


CDR 2:








(SEQ ID NO: 1078)









WINPNSGDTNYAQKFQG



 


CDR 3:








(SEQ ID NO: 1079)









DSPYSSSWSFFDY



 


TCN-549 (5092_P04) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1080)









GYTFTG



 


CDR 2:








(SEQ ID NO: 1081)









WINPNSGDTN



 


CDR 3:








(SEQ ID NO: 1079)









DSPYSSSWSFFDY



 


TCN-549 (5092_P04) light chain variable region nucleotide sequence:








(SEQ ID NO: 1082)









GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCC



 


AGCCAGAGTGTTTTATACAGCTCCAACAATAAGAGCCACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT


 


AAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA


 


GATTTCACCCTCATCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAATATTATTTTTCT


 


CCCCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA


 


TCN-549 (5092_P04) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1083)









DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKSHLAWYQQKPGQPPKLLIYWASTRESGVPDRF



 


SGSGSGTDFTLIISSLQAEDVAVYYCQQYYFSPLTFGGGTKVEIK


 


TCN-549 (5092_P04) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1084)









KSSQSVLYSSNNKSHLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 1085)









QQYYFSPLT



 


TCN-549 (5092_P04) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1084)









KSSQSVLYSSNNKSHLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 1085)









QQYYFSPLT



 


TCN-550 (5096_F06) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1086)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGCCTCCATCAATAGTCACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT


 


GTCTATTACAGTGGGAGCACCACCTACAACCCCTCCCTCAAGAGTCGAGTCACCTTATCAGTAGATACGTCCAAG


 


AACCAGTTCTCCCTGAACCTGAGCTCTGTGACCGCCGCAGACACGGCCTTCTATTACTGTGCGAGACATCCCTAC


 


GATGTTTTGACTGGTTCCGGGGACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-550 (5096_F06) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1087)









QVQLQESGPGLVKPSETLSLTCTVSGASINSHYWSWIRQPPGKGLEWIGYVYYSGSTTYNPSLKSRVT



 


LSVDTSKNQFSLNLSSVTAADTAFYYCARHPYDVLTGSGDWFDPWGQGTLVTVSS


 


TCN-550 (5096_F06) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1088)









SHYWS



 


CDR 2:








(SEQ ID NO: 1089)









YVYYSGSTTYNPSLKS



 


CDR 3:








(SEQ ID NO: 1090)









HPYDVLTGSGDWFDP



 


TCN-550 (5096_F06) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1091)









GASINSH



 


CDR 2:








(SEQ ID NO: 1092)









YVYYSGSTT



 


CDR 3:








(SEQ ID NO: 1090)









HPYDVLTGSGDWFDP



 


TCN-550 (5096_F06) light chain variable region nucleotide sequence:








(SEQ ID NO: 1093)









TCCTATGTTCTGACTCAGGCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAAT



 


GCCATTGGAAGTAAAAAAGTTCACTGGTACCAGCACAAGGCAGGCCAGGCCCCTGTACTCGTCGTCTATGATGAT


 


ACAGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTTGGAGCACGGCCACCCTGACCATCAAC


 


AGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATTTTACCATTGATCATGTGGTCTTCGGC


 


GGAGGGACCAAGCTGACCGTTCTA


 


TCN-550 (5096_F06) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1094)









SYVLTQAPSVSVAPGQTARITCGGNAIGSKKVHWYQHKAGQAPVLVVYDDTDRPSGIPERFSGSNSW



 


STATLTINRVEAGDEADYYCQVWDFTIDHVVFGGGTKLTVL


 


TCN-550 (5096_F06) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1095)









GGNAIGSKKVH



 


CDR 2:








(SEQ ID NO: 1096)









DDTDRPS



 


CDR 3:








(SEQ ID NO: 1097)









QVWDFTIDHVV



 


TCN-550 (5096_F06) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1095)









GGNAIGSKKVH



 


CDR 2:








(SEQ ID NO: 1096)









DDTDRPS



 


CDR 3:








(SEQ ID NO: 1097)









QVWDFTIDHVV



 


TCN-551 (5243_D01) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1098)









GAGGTGCAACTGGTTCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCT



 


GGCTACAGCTTTAGCAACTACTGGATCGGCTGGGTGCGCCACATGCCCGGGAAAGGCCTGGAATGGATGGGGATC


 


ATTTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATGTCAGCCGACAAGTCC


 


AGCAGCACCGTCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATTTATTATTGTGCGAGACGGGGC


 


GGACATAGTTTTGGATATGGGTCGGGGGGGGACACGCACAGTGAATTCGACTCCTGGGGCCAGGGAACCCTGGTC


 


ACCGTCTCGAGC


 


TCN-551 (5243_D01) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1099)









EVQLVQSGSEVKKPGESLKISCKGSGYSFSNYWIGWVRHMPGKGLEWMGIIYPGDSDTRYSPSFQGQ



 


VTMSADKSSSTVYLQWSSLKASDTAIYYCARRGGHSFGYGSGGDTHSEFDSWGQGTLVTVSS


 


TCN-551 (5243_D01) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1100)









NYWIG



 


CDR 2:








(SEQ ID NO: 1101)









IIYPGDSDTRYSPSFQG



 


CDR 3:








(SEQ ID NO: 1102)









RGGHSFGYGSGGDTHSEFDS



 


TCN-551 (5243_D01) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1103)









GYSFSN



 


CDR 2:








(SEQ ID NO: 1104)









IIYPGDSDTR



 


CDR 3:








(SEQ ID NO: 1102)









RGGHSFGYGSGGDTHSEFDS



 


TCN-551 (5243_D01) light chain variable region nucleotide sequence:








(SEQ ID NO: 1105)









CAGTCTGTATTGACGCAGTCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGC



 


GACTCCAACATTGGTGATTATTTTGTATGTTGGTACCAGCACCTCCCAGGAAAACCCCCCCAACTCCTCATCTAT


 


GAAAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGC


 


ATCACCGGAATCCAGACCGGGGACGAGGCCGATTACTACTGCGCAACTTGGGATGGCAGCCTGAGTGCTTGGGTG


 


TTCGGCGGAGGGACCAAGCTGACCGTTCTA


 


TCN-551 (5243_D01) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1106)









QSVLTQSPSVSAAPGQKVTISCSGSDSNIGDYFVCWYQHLPGKPPQLLIYENNKRPSGIPDRFSGSKSGT



 


SATLGITGIQTGDEADYYCATWDGSLSAWVFGGGTKLTVL


 


TCN-551 (5243_D01) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1107)









SGSDSNIGDYFVC



 


CDR 2:








(SEQ ID NO: 1108)









ENNKRPS



 


CDR 3:








(SEQ ID NO: 1109)









ATWDGSLSAWV



 


TCN-551 (5243_D01) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1107)









SGSDSNIGDYFVC



 


CDR 2:








(SEQ ID NO: 1108)









ENNKRPS



 


CDR 3:








(SEQ ID NO: 1109)









ATWDGSLSAWV



 


TCN-552 (5249_I23) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1110)









CAGGTCCAAGTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAGGGTCTCCTGCCAGGCTTCT



 


GGAGGCACCTTCATGAATTATGCTATCATTTGGGTGCGACGGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG


 


ATCATCCCTGTCTTTCCTACACCAAACTACGCACAGATGTTCCAGGGCAGAGTCACGATTTCCACGGACGAATCC


 


AGGAGCACATCCTTCTTGGAACTGACCAACCTGAGATATGAGGACACGGCCGTTTATTACTGTGCGAGGCGAATT


 


TATCACGGTGGTAACTCCGGCTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-552 (5249_I23) gamma heavy chain variable region amino acid sequence:



(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1111)









QVQVVQSGAEVKKPGSSVRVSCQASGGTFMNYAIIWVRRAPGQGLEWMGGIIPVFPTPNYAQMFQG



 


RVTISTDESRSTSFLELTNLRYEDTAVYYCARRIYHGGNSGFDFWGQGTLVTVSS


 


TCN-552 (5249_I23) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1112)









NYAII



 


CDR 2:








(SEQ ID NO: 1113)









GIIPVFPTPNYAQMFQG



 


CDR 3:








(SEQ ID NO: 1114)









RIYHGGNSGFDF



 


TCN-552 (5249_I23) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1115)









GGTFMN



 


CDR 2:








(SEQ ID NO: 1116)









GIIPVFPTPN



 


CDR 3:








(SEQ ID NO: 1114)









RIYHGGNSGFDF



 


TCN-552 (5249_I23) light chain variable region nucleotide sequence:








(SEQ ID NO: 1117)









GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTGGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT


 


TCATCCAACAGGGCCCCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC


 


AGCAGCCTCGCGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAAGTGGCCTCCCATGTACAGTTTT


 


GGCCATGGGACCAAGCTGGAGATCAAA


 


TCN-552 (5249_I23) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1118)









EIVLTQSPATLSLSPGERATLSCRASQSVGNYLAWYQQKPGQAPRLLIYDSSNRAPGIPARFSGSGSGT



 


DFTLTISSLAPEDFAVYYCQQRSKWPPMYSFGHGTKLEIK


 


TCN-552 (5249_I23) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1119)









RASQSVGNYLA



 


CDR 2:








(SEQ ID NO: 1120)









DSSNRAP



 


CDR 3:








(SEQ ID NO: 1121)









QQRSKWPPMYS



 


TCN-552 (5249_I23) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1119)









RASQSVGNYLA



 


CDR 2:








(SEQ ID NO: 1120)









DSSNRAP



 


CDR 3:








(SEQ ID NO: 1121)









QQRSKWPPMYS



 


TCN-553 (5261_C18) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1122)









CAGGTCCAGGTGGTGCAGTCTGGGACTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCCAGACTTCT



 


GGAGGCAGGTTCATGAGTTATGCTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC


 


ATCGTCCCTGTCTTCGGAACAGCAAACTACGCTCAGAAGTTCCAGGGCAGAGTCACGATCACCACGGACGATTCC


 


ACGCGCACAGCCTATATGGAGTTGAGCAGCCTGAGAAGTGAGGACACGGCCGTTTATTACTGTGGGTTCCGATAC


 


GGCTCTGGTTACGGGTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-553 (5261_C18) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1123)









QVQVVQSGTEVKKPGSSVKVSCQTSGGRFMSYAITWVRQAPGQGLEWMGGIVPVFGTANYAQKFQ



 


GRVTITTDDSTRTAYMELSSLRSEDTAVYYCGFRYGSGYGFDSWGQGTLVTVSS


 


TCN-553 (5261_C18) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1124)









SYAIT



 


CDR 2:








(SEQ ID NO: 1125)









GIVPVFGTANYAQKFQG



 


CDR 3:








(SEQ ID NO: 1126)









RYGSGYGFDS



 


TCN-553 (5261_C18) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1127)









GGRFMS



 


CDR 2:








(SEQ ID NO: 1128)









GIVPVFGTAN



 


CDR 3:








(SEQ ID NO: 1126)









RYGSGYGFDS



 


TCN-553 (5261_C18) light chain variable region nucleotide sequence:








(SEQ ID NO: 1129)









GAAATTGTATTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTAGTAGCAGCTACTTAGCCTGGTATCAGAAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT


 


GGTGCTTCCACTAGGGCCACTGGCATCCCGGACCGGTTCACTGGCAGTGGGTCTGGGACAGACTTCACTCTCAGC


 


ATCAGTAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTTTGGTACCTCAGTCTTCACTTTCGGC


 


GGAGGGACCAAGGTTGAGATCAAA


 


TCN-553 (5261_C18) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1130)









EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQKKPGQAPRLLIYGASTRATGIPDRFTGSGSGT



 


DFTLSISRLEPEDFAVYYCQHFGTSVFTFGGGTKVEIK


 


TCN-553 (5261_C18) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 944)









RASQSVSSSYLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 1131)









QHFGTSVFT



 


TCN-553 (5261_C18) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 944)









RASQSVSSSYLA



 


CDR 2:








(SEQ ID NO: 755)









GASTRAT



 


CDR 3:








(SEQ ID NO: 1131)









QHFGTSVFT



 


TCN-554 (5277_M05) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1132)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGATCTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGATACACCTTCACCGACTACTATATTCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAACCCTGAAAGTGGTGACACAAAGTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC


 


ATCACCACAGCCTACATGGAGCTGGGTAGGCTGAGATCCGACGACACGGCCGTGTATTACTGTGCGAGAGATGTA


 


AGTACGACCTGGAGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-554 (5277_M05) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1133)









QVQLVQSGADLKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWINPESGDTKYAQKFQ



 


GRVTMTRDTSITTAYMELGRLRSDDTAVYYCARDVSTTWSWFAPWGQGTLVTVSS


 


TCN-554 (5277_M05) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1134)









DYYIH



 


CDR 2:








(SEQ ID NO: 1135)









WINPESGDTKYAQKFQG



 


CDR 3:








(SEQ ID NO: 1136)









DVSTTWSWFAP



 


TCN-554 (5277_M05) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1137)









GYTFTD



 


CDR 2:








(SEQ ID NO: 1138)









WINPESGDTK



 


CDR 3:








(SEQ ID NO: 1136)









DVSTTWSWFAP



 


TCN-554 (5277_M05) light chain variable region nucleotide sequence:








(SEQ ID NO: 1139)









GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCAGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGGTCC



 


AGCCAGAGTATTTTCCACAACTCCAACAATGAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT


 


AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA


 


GATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCGGTTTATTTCTGTCAGCAATATTATAATGCT


 


CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA


 


TCN-554 (5277_M05) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1140)









DIVMTQSPDSLAVSLGERATINCRSSQSIFHNSNNENYLAWYQQKPGQPPKLLIYWASTRESGVPDRF



 


SGSGSGTDFTLTISSLQAEDVAVYFCQQYYNAPLTFGGGTKVEIK


 


TCN-554 (5277_M05) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1141)









RSSQSIFHNSNNENYLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 1142)









QQYYNAPLT



 


TCN-554 (5277_M05) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1141)









RSSQSIFHNSNNENYLA



 


CDR 2:








(SEQ ID NO: 957)









WASTRES



 


CDR 3:








(SEQ ID NO: 1142)









QQYYNAPLT



 


TCN-555 (5246_L16) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1143)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTCGGTGAAGGTCTCATGCACGGCTTCT



 


GGAGGCATCTTCAGGAAGAATGCAATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG


 


ATCATCGCAGTCTTTAACACAGCAAATTACGCGCAGAAGTTCCAGAACAGAGTCAAAATTACCGCAGACGAGTCA


 


GGCAATACGGCCTACATGGAGCTGAGCAGCCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGTCACCCA


 


AAATATTTCTATGGTTCGGGGAGTTATCCGGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-555 (5246_L16) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1144)









QVQLVQSGAEVKRPGSSVKVSCTASGGIFRKNAISWVRQAPGQGLEWMGGIIAVFNTANYAQKFQN



 


RVKITADESGNTAYMELSSLTSDDTAVYYCASHPKYFYGSGSYPDFWGQGTLVTVSS


 


TCN-555 (5246_L16) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 796)









KNAIS



 


CDR 2:








(SEQ ID NO: 797)









GIIAVFNTANYAQKFQN



 


CDR 3:








(SEQ ID NO: 798)









HPKYFYGSGSYPDF



 


TCN-555 (5246_L16) gamma heavy chain Chothia CDRs:



CDR 1:








(SEQ ID NO: 799)









GGIFRK



 


CDR 2:








(SEQ ID NO: 800)









GIIAVFNTAN



 


CDR 3:








(SEQ ID NO: 798)









HPKYFYGSGSYPDF



 


TCN-555 (5246_L16) light chain variable region nucleotide sequence:








(SEQ ID NO: 1145)









CAATCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAATCACCATCTCCTGTACTGGTGGC



 


AGCAGTGATATTGGTGCTTCTAACTCTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCGTTATT


 


TTTGATGTCACTGAGCGACCCTCAGGGGTCCCGCATCGGTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACCGTCTCTGGGCTCCAGCCTGACGACGAGGCTGATTATTTCTGCTGCGCATATGGAGGCAAATATCTTGTGGTC


 


TTCGGCGGAGGGACCAAGGTGACCGTTCTA


 


TCN-555 (5246_L16) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1146)









QSALTQPRSVSGSPGQSITISCTGGSSDIGASNSVSWYQQHPGKAPKLVIFDVTERPSGVPHRFSGSKSG



 


NTASLTVSGLQPDDEADYFCCAYGGKYLVVFGGGTKVTVL


 


TCN-555 (5246_L16) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1147)









TGGSSDIGASNSVS



 


CDR 2:








(SEQ ID NO: 804)









DVTERPS



 


CDR 3:








(SEQ ID NO: 805)









CAYGGKYLVV



 


TCN-555 (5246_L16) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1147)









TGGSSDIGASNSVS



 


CDR 2:








(SEQ ID NO: 804)









DVTERPS



 


CDR 3:








(SEQ ID NO: 805)









CAYGGKYLVV



 


TCN-556 (5089_K12) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1148)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAACCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGATACACCTTCATCGGCTATGATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAACGCTAAAAGAGGTGGCACAAACTATGCACAAAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCT


 


ATCAGCACAGCCTACATGGAGCTGAACAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGGGGTG


 


GGGTCACGAACTACGATTTTTGGAGTTCTCAACCCGGAATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC


 


TCGAGC


 


TCN-556 (5089_K12) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1149)








QVQLVQSGAEVKKPGASVKVSCKASGYTFIGYDMHWVRQAPGQGLEWMGWINAKRGGTNYAQKF


 


QGRVTMTRDTSISTAYMELNSLRSDDTAVYYCARGVGSRTTIFGVLNPEFDYWGQGTLVTVSS


 


TCN-556 (5089_K12) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1150)









GYDMH



 


CDR 2:








(SEQ ID NO: 1151)









WINAKRGGTNYAQKFQG



 


CDR 3:








(SEQ ID NO: 1152)









GVGSRTTIFGVLNPEFDY



 


TCN-556 (5089_K12) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1153)









GYTFIG



 


CDR 2:








(SEQ ID NO: 1154)









WINAKRGGTN



 


CDR 3:








(SEQ ID NO: 1152)









GVGSRTTIFGVLNPEFDY



 


TCN-556 (5089_K12) light chain variable region nucleotide sequence:








(SEQ ID NO: 1155)









CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGATCC



 


AGCAGTGACGTTGGTGGTTATGACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCCTGATT


 


TATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCGGGCAACTACAATCATGTC


 


TTCGGACCTGGGACCAAGGTCACCGTTCTA


 


TCN-556 (5089_K12) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1156)









QSALTQPPSASGSPGQSVTISCTGSSSDVGGYDYVSWYQQHPGKAPKLLIYEVTKRPSGVPDRFSGSKS



 


GNTASLTVSGLQAEDEADYYCSSYAGNYNHVFGPGTKVTVL


 


TCN-556 (5089_K12) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1157)









TGSSSDVGGYDYVS



 


CDR 2:








(SEQ ID NO: 1158)









EVTKRPS



 


CDR 3:








(SEQ ID NO: 1159)









SSYAGNYNHV



 


TCN-556 (5089_K12) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1157)









TGSSSDVGGYDYVS



 


CDR 2:








(SEQ ID NO: 1158)









EVTKRPS



 


CDR 3:








(SEQ ID NO: 1159)









SSYAGNYNHV



 


TCN-557 (5081_A04) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1160)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGACACACCTTCACCGGCTACTACATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAACCCTGACAGTGGTGCCACCAGTTCTGCACAGAACTTTCAGGGCAGGGTCACCATGACCGGGGACACGTCC


 


TCTAGCACAGCCTACATGGAGCTGAGTAGGCTGAGTTTTGACGACACGGCCGTCTATTACTGTGCGAGAGTACTG


 


TTTACCAGTCCTTTTGACTTCTGGGGTGAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-557 (5081_A04) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1161)









QVQLVQSGAEVKKPGASVKVSCKASGHTFTGYYIHWVRQAPGQGLEWMGWINPDSGATSSAQNFQ



 


GRVTMTGDTSSSTAYMELSRLSFDDTAVYYCARVLFTSPFDFWGEGTLVTVSS


 


TCN-557 (5081_A04) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1162)









GYYIH



 


CDR 2:








(SEQ ID NO: 1163)









WINPDSGATSSAQNFQG



 


CDR 3:








(SEQ ID NO: 1164)









VLFTSPFDF



 


TCN-557 (5081_A04) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1165)









GHTFTG



 


CDR 2:








(SEQ ID NO: 1166)









WINPDSGATS



 


CDR 3:








(SEQ ID NO: 1164)









VLFTSPFDF



 


TCN-557 (5081_A04) light chain variable region nucleotide sequence:








(SEQ ID NO: 1167)









CAGGCTGTGGTGACTCAGGAGCCCTCACTGGCTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGC



 


ACTGGAGCTGTCACCAGGGGTCATTATCCCTATTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGGCACTCATT


 


TATGATAGTGCAGGCAACAGACACTCCTGGACTCCCGCCCGATTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCC


 


CTGACCCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGTCTGGGTG


 


TTCGGCGGAGGGACGAAGCTGACCGTTCTA


 


TCN-557 (5081_A04) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1168)









QAVVTQEPSLAVSPGGTVTLTCGSSTGAVTRGHYPYWFQQKPGQAPRALIYDSAGNRHSWTPARFSG



 


SLLGGKAALTLSGAQPEDEAEYYCLLSYSGVWVFGGGTKLTVL


 


TCN-557 (5081_A04) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1169)









GSSTGAVTRGHYPY



 


CDR 2:








(SEQ ID NO: 1170)









DSAGNRHS



 


CDR 3:








(SEQ ID NO: 1171)









LLSYSGVWV



 


TCN-557 (5081_A04) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1169)









GSSTGAVTRGHYPY



 


CDR 2:








(SEQ ID NO: 1170)









DSAGNRHS



 


CDR 3:








(SEQ ID NO: 1171)









LLSYSGVWV



 


TCN-558 (5248_H10b) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1172)









CAGGTCCAGCTGGTGCAATCTGGGAGTGAGGTGAAGAAGCCTGGGACCTCGGTGAAGGTCTCCTGCACGGCCTCT



 


GGAAGTGTCTTCACCAATTATGGAATTAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCATCCCTCTCTTTGGCGCAGCCAAGTACGCACAGAAATTCCAGGGCAGAGTCACCATCACAGCGGACGAATCC


 


ACGAAGACAGTCTACATGGAGCTGAGCAGGCTGACATCTAAAGACACGGCCATATATTTCTGTGCGAAGGCCCCC


 


CGTGTCTACGAGTACTACTTTGATCAGTGGGGCCAGGGAACCCCAGTCACCGTCTCCTCA


 


TCN-558 (5248_H10b) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 914)









QVQLVQSGSEVKKPGTSVKVSCTASGSVFTNYGISWVRQAPGQGLEWMGGIIPLFGAAKYAQKFQG



 


RVTITADESTKTVYMELSRLTSKDTAIYFCAKAPRVYEYYFDQWGQGTPVTVSS


 


TCN-558 (5248_H10b) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 915)









NYGIS



 


CDR 2:








(SEQ ID NO: 916)









GIIPLFGAAKYAQKFQG



 


CDR 3:








(SEQ ID NO: 917)









APRVYEYYFDQ



 


TCN-558 (5248_H10b) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 918)









GSVFTN



 


CDR 2:








(SEQ ID NO: 919)









GIIPLFGAAK



 


CDR 3:








(SEQ ID NO: 917)









APRVYEYYFDQ



 


TCN-558 (5248_H10b) light chain variable region nucleotide sequence:








(SEQ ID NO: 1173)









GAAATAGTGATGACGCAGTTTCCAGCCACCCTGTCTGTGTCTCCCGGGGAACGAGTCACCCTCTCCTGTAGGGCC



 


AGTCAGAGTGTTAGCAACAATTTAGCCTGGTACCAGCAAAAACCTGGCCAGCCTCCCAGGCTCCTCATCTATGAT


 


GCATCTACCAGGGCCACGGGTGTCCCAGCCAAGTTCAGTGGCACTGGGTCTGGCACAGAGTTCACTCTCAGCATC


 


AGCAGCCTGCAGTCCGAAGATTTTGCAGTTTATTACTGTCAGCAGTATCACAACTGGCCTCCCTCGTACAGTTTT


 


GGCCTGGGGACCAAGCTGGAGATCAAA


 


TCN-558 (5248_H10b) light chain variable region amino acid sequence (Kabat CDRs



in bold, Chothia CDRs underlined)








(SEQ ID NO: 862)









EIVMTQFPATLSVSPGERVTLSCRASQSVSNNLAWYQQKPGQPPRLLIYDASTRATGVPAKFSGTGSGT



 


EFTLSISSLQSEDFAVYYCQQYHNWPPSYSFGLGTKLEIK


 


TCN-558 (5248_H10b) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 863)









RASQSVSNNLA



 


CDR 2:








(SEQ ID NO: 864)









DASTRAT



 


CDR 3:








(SEQ ID NO: 865)









QQYHNWPPSYS



 


TCN-558 (5248_H10b) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 863)









RASQSVSNNLA



 


CDR 2:








(SEQ ID NO: 864)









DASTRAT



 


CDR 3:








(SEQ ID NO: 865)









QQYHNWPPSYS



 


TCN-559 (5097_G08) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1174)









CAAGAGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


AGAAAGTCCTTCATTGGCTACTATGTACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAGCCCTGACAGTGATGCCACAAAGTACGCACAGAAGTTTCAGGGCTCCGTCATCATGACCAGGGACACGTCC


 


GTCAGCACAGTGTACATGGAGCTGAGTAGGCTGACATCTGACGACACGGCCCTTTATTACTGTCTCCTTTTTCGA


 


GGTGGAAACTCCCTCTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC


 


TCN-559 (5097_G08) gamma heavy chain variable region amino acid sequence:


(Kabat. CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1175)









QEQLVQSGAEVKKPGASVKVSCKASRKSFIGYYVHWVRQAPGQGLEWMGWISPDSDATKYAQKFQ



 


GSVIMTRDTSVSTVYMELSRLTSDDTALYYCLLFRGGNSLSWGQGTLVTVSS


 


TCN-559 (5097_G08) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1176)









GYYVH



 


CDR 2:








(SEQ ID NO: 1177)









WISPDSDATKYAQKFQG



 


CDR 3:








(SEQ ID NO: 1178)









FRGGNSLS



 


TCN-559 (5097_G08) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1179)









RKSFIG



 


CDR 2:








(SEQ ID NO: 1180)









WISPDSDATK



 


CDR 3:








(SEQ ID NO: 1178)









FRGGNSLS



 


TCN-559 (5097_G08) light chain variable region nucleotide sequence:








(SEQ ID NO: 1181)









CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACCCTCACCTGTGGCTCCAGC



 


ACTGGACCTGTCACCAGTGGTCATTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACATTGATT


 


TCTGCTACATCCAACACACACTCCTGGACACCTGCCCGCTTCTCAGGCTCCCTCCTTGGGGGCAGAGCTGCCCTG


 


ACCCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGACTATTATTGCTTTCTCTCCTACAGTGGTGCTTGGGTGTTC


 


GGCGGAGGGACCACGCTGACCGTTCTA


 


TCN-559 (5097_G08) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1182)









QAVVTQEPSLTVSPGGTVTLTCGSSTGPVTSGHYPYWFQQKPGQAPRTLISATSNTHSWTPARFSGSL



 


LGGRAALTLSGAQPEDEADYYCFLSYSGAWVFGGGTTLTVL


 


TCN-559 (5097_G08) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1183)









GSSTGPVTSGHYPY



 


CDR 2:








(SEQ ID NO: 1184)









ATSNTHS



 


CDR 3:








(SEQ ID NO: 1185)









FLSYSGAWV



 


TCN-559 (5097_G08) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1183)









GSSTGPVTSGHYPY



 


CDR 2:








(SEQ ID NO: 1184)









ATSNTHS



 


CDR 3:








(SEQ ID NO: 1185)









FLSYSGAWV



 


TCN-560 (5084_P10) heavy chain variable region nucleotide sequence:








(SEQ ID NO: 1186)









GAGGTGCAGCTGGTGGAATCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GGATTTATCTTTAGAAATTACTGGATGAGCTGGGTCCGGCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC


 


ATAAAACAAGATGGAAGAGAGAAGTACTATGTGGACTCTCTGAGGGGCCGAGTCAACATCTCCAGAGACAACGCC


 


GAGAACTCATTGTATCTGCACATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTTCTGTGCGAGAGCTCGG


 


ATGGTGGTGGTTACTGGCGATGGTTTTGATGTCTGGGGCCATGGGACAATGGTCACCGTCTCGAGC


 


TCN-560 (5084_P10) gamma heavy chain variable region amino acid sequence:


(Kabat CDRs in bold, Chothia CDRs underlined)








(SEQ ID NO: 1187)









EVQLVESGGGLVQPGGSLRLSCAASGFIFRNYWMSWVRQAPGKGLEWVANIKQDGREKYYVDSLR



 


GRVNISRDNAENSLYLHMNSLRVEDTAVYFCARARMVVVTGDGFDVWGHGTMVTVSS


 


TCN-560 (5084_P10) gamma heavy chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1188)









NYWMS



 


CDR 2:








(SEQ ID NO: 1189)









NIKQDGREKYYVDSLRG



 


CDR 3:








(SEQ ID NO: 1190)









ARMVVVTGDGFDV



 


TCN-560 (5084_P10) gamma heavy chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1191)









GFIFRN



 


CDR 2:








(SEQ ID NO: 1192)









NIKQDGREKY



 


CDR 3:








(SEQ ID NO: 1190)









ARMVVVTGDGFDV



 


TCN-560 (5084_P10) light chain variable region nucleotide sequence:








(SEQ ID NO: 1193)









GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AGTCAGAATATTAAGAGGTATTTCAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT


 


GCATCCAATTTAGAAAATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC


 


AGCAGTCTGCAACCTGAGGATTTTGCGACTTATTACTGTCAGCAGAGTTTCAGTAAATCGTGGACATTCGGCCAA


 


GGGACCAACGTGGACATCAAA


 


TCN-560 (5084_P10) light chain variable region amino acid sequence (Kabat CDRs


in bold, Chothia CDRs underlined)








(SEQ ID NO: 1194)









DIQMTQSPSSLSASVGDRVTITCRASQNIKRYFNWYQQKPGKAPKLLIYAASNLENGVPSRFSGSGSGT



 


DFTLTISSLQPEDFATYYCQQSFSKSWTFGQGTNVDIK


 


TCN-560 (5084_P10) Light chain Kabat CDRs:


CDR 1:








(SEQ ID NO: 1195)









RASQNIKRYFN



 


CDR 2:








(SEQ ID NO: 1196)









AASNLEN



 


CDR 3:








(SEQ ID NO: 1197)









QQSFSKSWT



 


TCN-560 (5084_P10) Light chain Chothia CDRs:


CDR 1:








(SEQ ID NO: 1195)









RASQNIKRYFN



 


CDR 2:








(SEQ ID NO: 1196)









AASNLEN



 


CDR 3:








(SEQ ID NO: 1197)









QQSFSKSWT







The invention provides an isolated fully human monoclonal anti-HA antibody or fragment thereof, wherein said antibody includes a variable heavy chain (VH) region comprising CDR1 and CDR2, wherein the VH region is encoded by a human IGHV1 (or specifically, IGHV1-18, IGHV1-2, IGHV1-69, IGHV1-8), IGHV2 (or specifically, IGHV2-5) , IGHV3 (or specifically, IGHV3-30, IGHV3-33, IGHV3-49, IGHV3-53, 66, IGHV3-7), IGHV4 (or specifically, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61), or IGHV5 (or specifically, IGHV5-51) VH germline sequence or an allele thereof, or a nucleic acid sequence that is homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 VH germline gene sequence or an allele thereof. In one aspect, the nucleic acid sequence that is homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 VH germline sequence is at least 75% homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 VH germline sequence or an allele thereof. Exemplary alleles include, but are not limited to, IGHV1-18*01, IGHV1-2*02, IGHV1-2*04, IGHV1-69*01, IGHV1-69*05, IGHV1-69*06, IGHV1-69*12, IGHV1-8*01, IGHV2-5*10, IGHV3-30-3*01, IGHV3-30*03, IGHV3-30*18, IGHV3-33*05, IGHV3-49*04, IGHV3-53*01, IGHV3-66*03, IGHV3-7*01, IGHV4-31*03, IGHV4-31*06, IGHV4-34*01, IGHV4-34*02, IGHV4-34*03, IGHV4-34*12, IGHV4-39*01, IGHV4-59*01, IGHV4-59*03, IGHV4-61*01, IGHV4-61*08, and IGHV5-51*01. Exemplary sequences for each allele are provided below.








IGHV1-18*01 nucleotide sequence









(SEQ ID NO: 1198)









CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCC


 


ACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV1-2*02 nucleotide sequence








(SEQ ID NO: 1199)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC


 


ATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV1-2*04 nucleotide sequence








(SEQ ID NO: 1200)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCTGGGTCACCATGACCAGGGACACGTCC


 


ATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA


 


IGHV1-69*01 nucleotide sequence








(SEQ ID NO: 1201)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT



 


GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCC


 


ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV1-69*05 nucleotide sequence








(SEQ ID NO: 1202)









CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT



 


GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCACGGACGAATCC


 


ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA


 


IGHV1-69*06 nucleotide sequence








(SEQ ID NO: 1203)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT



 


GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCC


 


ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV1-69*12 nucleotide sequence








(SEQ ID NO: 1204)









CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT



 


GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCC


 


ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV1-8*01 nucleotide sequence








(SEQ ID NO: 1205)









CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGG


 


ATGAACCCTAACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCC


 


ATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGG


 


IGHV2-5*10 nucleotide sequence








(SEQ ID NO: 1206)









CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCT



 


GGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT


 


GCACTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACC


 


TCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGG


 


IGHV3-30-3*01 nucleotide sequence








(SEQ ID NO: 1207)









CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT


 


ATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC


 


AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGA


 


IGHV3-30*03 nucleotide sequence








(SEQ ID NO: 1208)









CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT


 


ATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC


 


AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGA


 


IGHV3-30*18 nucleotide sequence








(SEQ ID NO: 1209)









CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT


 


ATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC


 


AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGA


 


IGHV3-33*05 nucleotide sequence








(SEQ ID NO: 1210)









CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCT



 


GGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT


 


ATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC


 


AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGA


 


IGHV3-49*04 nucleotide sequence








(SEQ ID NO: 1211)









GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTCCTGTACAGCTTCT



 


GGATTCACCTTTGGTGATTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTC


 


ATTAGAAGCAAAGCTTATGGTGGGACAACAGAATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGAT


 


GATTCCAAAAGCATCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACTAGA


 


GA


 


IGHV3-53*01 nucleotide sequence








(SEQ ID NO: 1212)









GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GGGTTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT


 


ATTTATAGCGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAG


 


AACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV3-66*03 nucleotide sequence








(SEQ ID NO: 1213)









CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGTTACAGTTTCACCACCTATGGTATGAATTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


TTCAACACCTACACTGGGAACCCAACATATGCCCAGGGCTTCACAGGACGGTTTGTCTTCTCCATGGACACCTCT


 


GCCAGCACAGCATACCTGCAGATCAGCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATA


 


IGHV3-7*01 nucleotide sequence








(SEQ ID NO: 1214)









GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT



 


GGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC


 


ATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC


 


AAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGA


 


IGHV4-31*03 nucleotide sequence








(SEQ ID NO: 1215)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCATCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT


 


GGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACG


 


TCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV4-31*06 nucleotide sequence








(SEQ ID NO: 1216)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCATCAGCAGTGGTAGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT


 


GGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACG


 


TCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTG


 


IGHV4-34*01 nucleotide sequence








(SEQ ID NO: 1217)









CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATT


 


GGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACG


 


TCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACA


 


IGHV4-34*02 nucleotide sequence








(SEQ ID NO: 1218)









CAGGTGCAGCTACAACAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT



 


GGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA


 


ATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG


 


AACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGG


 


IGHV4-34*03 nucleotide sequence








(SEQ ID NO: 1219)









CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT



 


GGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA


 


ATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG


 


AACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTG


 


IGHV4-34*12 nucleotide sequence








(SEQ ID NO: 1220)









CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT



 


GGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA


 


ATCATTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG


 


AACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGA


 


IGHV4-39*01 nucleotide sequence








(SEQ ID NO: 1221)









CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATT


 


GGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACG


 


TCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACA


 


IGHV4-59*01 nucleotide sequence








(SEQ ID NO: 1222)









GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTAAAGACTGGAGGGGTCTCTGAGACTCTCCTGTGCAGCCTC



 


TGGATTCACCTTCAGTAGCTCTGCTATGCACTGGGTCCACCAGGCTCCAGGAAAGGGTTTGGAGTGGGTCTCAGT


 


TATTAGTACAAGTGGTGATACCGTACTCTACACAGACTCTGTGAAGGGCTGATTCACCATCTCTAGAGACAATGC


 


CCAGAATTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGACGACATGGCTGTGTATTACTGTGTGAAAGA


 


IGHV4-59*03 nucleotide sequence








(SEQ ID NO: 1223)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT


 


ATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG


 


AACCAATTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCG


 


IGHV4-61*01 nucleotide sequence








(SEQ ID NO: 1224)









CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT



 


GGTGGCTCCGTCAGCAGTGGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATT


 


GGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACG


 


TCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGA


 


IGHV4-61*08 nucleotide sequence








(SEQ ID NO: 1225)









CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT



 


GGTTACAGTTTCACCACCTATGGTATGAATTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG


 


TTCAACACCTACACTGGGAACCCAACATATGCCCAGGGCTTCACAGGACGGTTTGTCTTCTCCATGGACACCTCT


 


GCCAGCACAGCATACCTGCAGATCAGCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATA


 


IGHV5-51*01 nucleotide sequence








(SEQ ID NO: 1226)









GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCT



 


GGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATC


 


ATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCC


 


ATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACA






In certain embodiments of the invention, the antibody further includes a variable light chain (VL) region encoded by a human IGKV1 (or specifically, IGKV1-17, IGKV1-27, IGKV1-39, IGKV1D-39, IGKV1-5), IGKV2 (or specifically, IGKV2-30), IGKV3 (or specifically, IGKV3-11, IGKV3-15, IGKV3-20), IGKV4 (or specifically, IGKV4-1, IGKV4-1), IGLV1 (or specifically, IGLV1-40, IGLV1-44, IGLV1-55), IGLV2 (or specifically, IGLV2-11, IGLV2-14, IGLV2-8), IGLV3 (or specifically, IGLV3-21 or IGLV3-25), IGLV7 (or specifically, IGLV7-43 or IGLV7-46), or IGLV9 (or specifically, IGLV9-49) or an allele thereof. VL germline gene sequence IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 or an allele thereof, or a nucleotide acid sequence that is homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 VL germline gene sequence or an allele thereof. Furthermore, the nucleic acid sequence that is homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 VL germline sequence or an allele thereof is at least 65% homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 VL germline sequence or an allele thereof.








IGKV1-17*01 nucleotide sequence









(SEQ ID NO: 1227)









GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCT


 


GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATC


 


AGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTCC


 


IGKV1-27*01 nucleotide sequence








(SEQ ID NO: 1228)









GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCG



 


AGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCT


 


GCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC


 


AGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCC


 


IGKV1-39*01 nucleotide sequence








(SEQ ID NO: 1229)









GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT


 


GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC


 


AGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCC


 


IGKV1D-39*01 nucleotide sequence








(SEQ ID NO: 1230)









GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA



 


AGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT


 


GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC


 


AGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCC


 


IGKV1-5*03 nucleotide sequence








(SEQ ID NO: 1231)









GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCC



 


AGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAG


 


GCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATC


 


AGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTCC


 


IGKV2-30*02 nucleotide sequence








(SEQ ID NO: 1232)









GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCT



 


AGTCAAAGCCTCGTACACAGTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGG


 


CGCCTAATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGAT


 


TTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCT


 


CC


 


IGKV3-11*01 nucleotide sequence








(SEQ ID NO: 1233)









GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT


 


GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC


 


AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCC


 


IGKV3-15*01 nucleotide sequence








(SEQ ID NO: 1234)









GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT


 


GCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATC


 


AGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCC


 


IGKV3-20*01 nucleotide sequence








(SEQ ID NO: 1235)









GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC



 


AGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT


 


GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC


 


ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCC


 


IGKV4-1*01 nucleotide sequence








(SEQ ID NO: 1236)









GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCC



 


AGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT


 


AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA


 


GATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACT


 


CCTCC


 


IGLV1-40*01 nucleotide sequence








(SEQ ID NO: 1237)









CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGC



 


AGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATC


 


TATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTG


 


GCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTTC


 


IGLV1-44*01 nucleotide sequence








(SEQ ID NO: 1238)









CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGC



 


AGCTCCAACATCGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT


 


AGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC


 


ATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTCC


 


IGLV1-51*02 nucleotide sequence








(SEQ ID NO: 1239)









CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGC



 


AGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTAT


 


GAAAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGC


 


ATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGAGTGCTGG


 


IGLV2-11*01 nucleotide sequence








(SEQ ID NO: 1240)









CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACC



 


AGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT


 


TATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTTC


 


IGLV2-14*01 nucleotide sequence








(SEQ ID NO: 1241)









CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACC



 


AGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT


 


TATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACTCTC


 


IGLV2-8*01 nucleotide sequence








(SEQ ID NO: 1242)









CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACC



 


AGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT


 


TATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG


 


ACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGGCAGCAACAATTTC


 


IGLV3-21*02 nucleotide sequence








(SEQ ID NO: 1243)









TCCTATGAGCTGACACAGCTACCCTCGGTGTCAGTGTCCCCAGGACAGACAGCCAGGATCACCTGCTCTGGAGAT



 


GTACTGGGGGAAAATTATGCTGACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGAGTTGGTGATATACGAAGAT


 


AGTGAGCGGTACCCTGGAATCCCTGAACGATTCTCTGGGTCCACCTCAGGGAACACGACCACCCTGACCATCAGC


 


AGGGTCCTGACCGAAGACGAGGCTGACTATTACTGTTTGTCTGGGGATGAGGACAATCC


 


IGLV3-25*03 nucleotide sequence








(SEQ ID NO: 1244)









TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGAT



 


GCATTGCCAAAGCAATATGCTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTGATATATAAAGAC


 


AGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGCTCAGGGACAACAGTCACGTTGACCATCAGT


 


GGAGTCCAGGCAGAAGACGAGGCTGACTATTACTGTCAATCAGCAGACAGCAGTGGT


 


IGLV7-43*01 nucleotide sequence








(SEQ ID NO: 1245)









CAGACTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGCTTCCAGC



 


ACTGGAGCAGTCACCAGTGGTTACTATCCAAACTGGTTCCAGCAGAAACCTGGACAAGCACCCAGGGCACTGATT


 


TATAGTACAAGCAACAAACACTCCTGGACCCCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTG


 


ACACTGTCAGGTGTGCAGCCTGAGGACGAGGCTGAGTATTACTGCCTGCTCTACTATGGTGGTGCTCAG


 


IGLV7-46*01 nucleotide sequence








(SEQ ID NO: 1246)









CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGC



 


ACTGGAGCTGTCACCAGTGGTCATTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATT


 


TATGATACAAGCAACAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTG


 


ACCCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGCTCGG


 


IGLV7-46*02 nucleotide sequence








(SEQ ID NO: 1247)









CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGC



 


ACTGGAGCTGTCACCAGTGGTCATTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATT


 


TATGATACAAGCAACAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTG


 


ACCCTTTTGGGTGCGCAGCCTGAGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGCTCGG


 


IGLV9-49*01 nucleotide sequence








(SEQ ID NO: 1248)









CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACCTGCACCCTGAGC



 


AGCGGCTACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGGTTTGTGATGCGAGTG


 


GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTTCTCAGTCTTGGGCTCAGGCCTGAAT


 


CGGTACCTGACCATCAAGAACATCCAGGAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGG


 


AGCAACTTCGTGTAACC


 


IGLV9-49*03 nucleotide sequence








(SEQ ID NO: 1249)









CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACCTGCACCCTGAGC



 


AGCGGCTACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGATTTGTGATGCGAGTG


 


GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTTCTCAGTCTTGGGCTCAGGCCTGAAT


 


CGGTACCTGACCATCAAGAACATCCAGGAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGG


 


AGCAACTTCGTGTAACC






The heavy chain of an isolated monoclonal anti-hemagglutinin (HA) antibody (i.e., anti-hemagglutinin antibody of the invention) is derived from a germ line V (variable) gene such as, for example, the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene or an allele thereof.
The HA antibodies of the invention include a variable heavy chain (VH) region encoded by a human IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele thereof. A IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence is shown, e.g., in SEQ ID NOs: 457 to 485. The HA antibodies of the invention include a VH region that is encoded by a nucleic acid sequence that is at least 75% homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele thereof. Preferably, the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97% homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele thereof, and more preferably, at least 98%, 99% homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele thereof. The VH region of the HA antibody is at least 75% homologous to the amino acid sequence of the VH region encoded by the IGHV 1, IGHV2, IGHV3, IGHV4, or IGHV5 VH germline gene sequence or an allele thereof. Preferably, the amino acid sequence of VH region of the HA antibody is at least 75%, 80%, 85%, 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the 75%, 80%, 85%, 90%, 95%, 96%, 97% germline gene sequence or an allele thereof, and more preferably, at least 98%, 99% homologous to the sequence encoded by the 75%, 80%, 85%, 90%, 95%, 96%, 97% germline gene sequence or an allele thereof.
The HA antibodies of the invention also include a variable light chain (VL) region encoded by a human IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof. A human IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 VL germline gene sequence, or an allele thereof is shown, e.g., at SEQ ID NOs: 486 to 508. Alternatively, the HA antibodies include a IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 VL region that is encoded by a nucleic acid sequence that is at least 65% homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof. Preferably, the nucleic acid sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof, and more preferably, at least 98%, 99% homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof. The VL region of the HA antibody is at least 65% homologous to the amino acid sequence of the VL region encoded the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof. Preferably, the amino acid sequence of VL region of the HA antibody is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof, and more preferably, at least 98%, 99% homologous to the sequence encoded by the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof.
HA Antibodies III
The present invention relates to an immunogen capable of inducing antibodies against a target peptide of the stem region of hemagglutinn protein of an influenza virus. The immunogen is a peptide or a synthetic peptide. In particular, the immunogen of this invention comprises one or more epitopes or epitope units. Optionally, the immunogen further comprises a general immune stimulator. These immunogens of the present invention are capable of inducing antibodies against influenza A virus to prevent infection by the virus.
In one aspect the invention provides an immunogen having an epitope or epitope unit recognized by a protective monoclonal antibody having the specificity for the stem region of hemagglutinn protein of an influenza virus.
The antibody binds both the HA1 and HA2 peptide. In some embodiments the epitope is recognized by monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or a monoclonal antibody that competes with the binding of monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 to the HA protein. Preferably, the epitope is the F10 epitope.
In some embodiments the hemagglutinin protein is in the neutral pH conformation.
The immunogen is a peptide or a synthetic peptide.
In some aspects the immunogen is a conjugate having one or more peptides or peptide fragments that are spatially positioned relative to each other so that they together form a non-linear sequence which mimics the tertiary structure of an F10 epitope. Optionally, the one or more peptides or peptide fragments are linked to a backbone. The conjugate competes with the binding of monoclonal antibody F10 to the HA protein.
The e conformation of the epitope is defined by amino acid residues 18, 38, 39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53, and 56 of HA2 when the hemagglutinin in the neutral pH conformation.
In some embodiments the immunogen is a peptide having one or more of the following amino acid sequences.
[Xaa0]m-Xaa1-Xaa2-[Xaa0]p, wherein, preferably, Xaa1 is S, T, F H or Y and Xaa 2 is H, Y, M, L or Q. Most preferably, Xaa1 is Y. Most preferably, Xaa2 is H.
[Xaa0]m-Xaa1-Xaa2-[Xaa0]p, wherein, preferably, Xaa1 is H, Q, Y, S, D, N or T and Xaa2 of is Q, E, K, I , V, M, E, R or T. Most preferably, Xaa1 is H. Most preferably, Xaa2 is Q.
[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-[Xaa0]p, wherein, preferably, Xaa1 is I, V, M, or L; Xaa2 is D, N, H, Y, D, A,S or E, Xaa3 is G or A, and Xaa4 is W, R, or G. Most preferably, Xaa1 is V; Xaa2 is D, Xaa3 is G, and Xaa4 is W.
[Xaa0]m-Xaa1-[Xaa0]q Xaa2-Xaa3-[Xaa0]q Xaa4-[Xaa0]r Xaa5-[Xaa0]q-Xaa6 Xaa7-[Xaa0]q-Xaa8-[Xaa0]p, and [Xaa0]m-Xaa1-[Xaa0]q Xaa2-Xaa3-[Xaa0]q Xaa4-[Xaa0]r Xaa5-[Xaa0]q-Xaa6 Xaa7-[Xaa0]s-[Xaa8]t-[Xaa0]p, wherein, preferably Xaa1 is K, Q, R, N, L, G, F, H or Y; Xaa2 is S or T, Xaa3 is Q or P; Xaa4 is F, V, I, M, L, or T; Xaa5 is I, T, S, N, Q, D, or A; Xaa6 is I, V, M, or L; Xaa7 is N, S, T, or D and Xaa8 is I, F,V, A, or T. Most preferably, Xaa1 is K; Xaa2 is T, Xaa3 is Q; Xaa4 is I; Xaa5 is T; Xaa6 is V; Xaa 7 is N, and Xaa8 is I.
For all of the preceding sequences, m, and p are independently 0 or 1-100, preferably about 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20 or 1-10; q is 2, r is 3, s is 0 or 2, and t is 0 or 1, and Xaa0, is independently any amino acid. Preferably s is 2 and t is 1.
In some aspects of the inventions, one or more amino acids are D-amino acids.
Optionally, the immunogen further comprises an adjuvant or is conjugated to a carrier.
In various aspects the invention includes a composition containing the immunogen together with one or more pharmaceutically acceptable excipients, diluents, and/or adjuvants. In some embodiments the composition further comprises an anti-influenza antibody of antigen binding fragment thereof. Preferably, the antibody is monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 ora monoclonal antibody that competes with the binding of monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 to the HA protein. Also provided by the invention are nucleic acids encoding the immunogens of the invention and composition comprising the nucleic acids.
The invention further comprises a method preventing a disease or disorder caused by an influenza virus by administering to person at risk of suffering from said disease or disorder an immunogen composition described herein. Optionally, the method includes further administering an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor. The anti-viral drug is a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel. The M2 ion channel inhibitor is amantadine or, rimantadine. The neuraminidase inhibitor zanamivir, or oseltamivir phosphate.
In another aspect the method includes further administering one or more antibodies specific to a Group I influenza virus and or a Group II influenza virus. The antibody is administered at a dose sufficient to neutralize the influenza virus.
Administration is prior to or after exposure to influenza virus.
Also disclosed are methods of treating subjects and methods of screening and producing antibodies. For example, disclosed is a method of treating a subject suffering or at risk of influenza infection, the method comprising administering to the subject one or more of the disclosed antibodies, such as the disclosed HA stem antibodies. For example, disclosed is a method of treating a subject, the method comprising administering to the subject the stem region of influenza hemagglutinin in the neutral pH conformation in isolation from other components of influenza virus, wherein the subject produces an immune response to the stem region. For example, disclosed is a method of treating a subject, the method comprising administering to the subject the stem region of influenza hemagglutinin in the neutral pH conformation in isolation from the head region of hemagglutinin, wherein the subject produces an immune response to the stem region. For example, disclosed is a method of treating a subject, the method comprising administering to the subject influenza hemagglutinin in the neutral pH conformation in isolation from other components of influenza virus, wherein the head region of the hemagglutinin is modified to reduce the antigenicity of the head region, wherein the subject produces an immune response to the stem region. For example, disclosed is a method, the method comprising screening antibodies reactive to hemagglutinin for binding to hemagglutinin immobilized on a surface, thereby identifying antibodies of interest. For example, disclosed is a method comprising screening antibodies reactive to hemagglutinin for binding to the stem region of influenza hemagglutinin in the neutral pH conformation in isolation from the head region of hemagglutinin, thereby identifying antibodies of interest. For example, disclosed is a method comprising screening antibodies reactive to hemagglutinin for binding to influenza hemagglutinin in the neutral pH conformation in isolation from other components of influenza virus, wherein the head region of the hemagglutinin is modified to reduce the antigenicity of the head region, thereby identifying antibodies of interest.
In some forms, the head region of the hemagglutinin can be modified by removing or replacing glycosylation sites. In some forms, the head region of the hemagglutinin can be modified by adding glycosylation sites. In some forms, the head region of the hemagglutinin can be modified by removing all or a portion of the head region.
In some forms, the disclosed antibodies, disclosed hemagglutinins, and disclosed methods can produce an immune reaction in a subject. For example, in some forms, the subject can produce an immune response that prevents or reduces the severity of an influenza infection. In some forms, the immune response can be reactive to influenza viruses within a subtype. In some forms, the immune response can be reactive to influenza viruses in each subtype within a cluster. In some forms, the immune response can be reactive to influenza viruses in each cluster within a group. In some forms, the immune response can be reactive to all influenza viruses in each subtype within a group. In some forms, the immune response can be reactive to influenza viruses within group 1.
In some forms, the disclosed methods can further comprise screening the antibodies of interest for competing with antibody F10 for binding to hemagglutinin, thereby identifying F10-competing antibodies. In some forms, the hemagglutinin can be hemagglutinin from a group 2 influenza virus. In some forms, the hemagglutinin can be hemagglutinin from a group 1 influenza virus. In some forms, the disclosed methods can further comprising producing the identified antibodies. Also disclosed are antibodies produced by the disclosed methods. Also disclosed are antibodies identified by the disclosed methods.
The disclosed compositions and methods are based upon the discovery of monoclonal antibodies which neutralize the influenza virus, e.g. influenza A virus. The influenza A virus is a Group I influenza A virus such as a H1 cluster influenza virus. The H1 cluster influenza virus is an H1a cluster or an H1b cluster. The monoclonal antibody is fully human. In some forms, the monoclonal antibody can be a bivalent antibody, a monovalent antibody, a single chain antibody or fragment thereof. Specifically, such monoclonal can bind to an epitope on the stem region of the hemagglutinin protein (HA), such as HA1 or HA2 polypeptide. The epitope can be non-linear.
The epitope can comprise both the HA1 and HA2. The epitope can be non-linear. In some forms the epitope can comprise the amino acid position 18, 38, 40, 291 of the Ha1 polypeptide and the amino acid at position 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53 and 56 of the HA2 polypeptide.
The disclosed compositions and methods are further based upon the discovery of a protocol for generating broadly neutralizing human antibodies that target a highly conserved epitope in the stem region of HA. Using the trimeric H5 ectodomain expressed in baculovirus which produces shorter N-glycans and uncharged mannoses absorbed on a plastic surface, allowed for the dominant presentation of the stem epitope while masking the normally immunodominat globular head. Accordingly, also disclosed is a method of producing an isolated antibody that specifically binds a pathogenic enveloped virus by exposing a single chain or Fab expression library to a membrane fusion protein of the virus, identifying an antibody in the library that specifically binds said protein; and isolating the antibody from the library. The fusion protein can be immobilized on a solid surface, e.g. plastic. In some forms the fusion protein can have modified glycosylations compared to a wild type fusion protein. For example, the fusion can be produced in a non-mammalian cell, such as an insect cell. The fusion protein can be, for example, a trimeric hemagglutinin (HA) protein.
Also disclosed is a method of vaccinating a subject against pathogenic enveloped virus such as an influenza virus by administering to the subject, for example, a membrane fusion protein (e.g., a trimeric hemagglutinin (HA) protein coated) or embedded in a biologically compatible matrix. In some forms the fusion protein can have modified glycosylations compared to a wild type fusion protein.
Also disclosed is a composition comprising a monoclonal antibody as described herein and kits containing the composition in one or more containers and instructions for use. The invention further provides a method of screening a compound for binding to an F10 antibody by contacting said F10 antibody with a compound of interest and detecting a compound-antibody complex. Also included in the invention are the compound identified by the method and their use as immunogens.
High affinity, cross-subtype, broadly-neutralizing human anti-HA mAbs have been identified. Specifically, a human Ab phage display library and H5 hemagglutinin (HA) ectodomain was used to select ten neutralizing mAbs (nAbs) with a remarkably broad range among Group 1 influenza viruses, including the H5N1 “bird flu” and the H1N1 “Spanish flu” and “Swine flu” strains. These nAbs inhibit the post-attachment fusion process by recognizing a novel and highly conserved neutralizing epitope within the stem region at a point where key elements of the conformational change—the fusion peptide and the exposed surface of helix aA—are brought into close apposition. The crystal structure of one mAb (mAbFlO) bound to H5N1 HA reveals that only the heavy chain inserts into a highly conserved pocket in the HA stem region, inhibiting the conformational changes required for membrane fusion. It has been discovered that nAbs targeting this pocket can provide broad protection against both seasonal and pandemic influenza A infections. The crystal structure further revealed that the epitope to which the F10 mAb is defined by amino acid residues 18, 38, 39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53, and 56 of HA2. This epitope is referred to herein as the F10 epitope. Structural and sequence analysis of all 16 HA subtypes points to the existence of only two variants of this epitope, corresponding to the two phylogenetic groupings of HA (Groups 1 and 2). This discovery indicates that a small cocktail of nAbs derived from a subset of each group can provide broad protection against both seasonal and pandemic influenza.
Remarkably, nAbs were isolated that utilize the same VH germline gene, IGHV1-69*01, and encode a CDR3 loop containing a tyrosine at an equivalent position to Y102, from a non-immune library. This indicates that broad anti-HA cross-immunity pre-exists in the H5-naive population, possibly due to previous exposure to H1, and, for library donors born before 1968, H2 subtypes. The recurrent use of this germline VH segment, the commonality of the CDR3 tyrosine introduced through N insertion and/or germline D gene assembly, and the promiscuous use of VL genes by the discovered nAbs discovered indicate that the precursor frequency of rearranged VH segments that could recognize this epitope is significant. This indicates that with suitable exposure to the F10 epitope identified here, these broad-spectrum nAbs can be readily induced in vivo. These discoveries led to the disclosed simple solution to provide universal protection against virus subtypes in both groups.
Three unique anti-HA-1 scFvs were identified by sequencing analysis of the 58 HA-1 positive clones. These scFvs were designated as 38B and 1C. The VH and VL amino acid sequence of 2A is shown herein. Ten unique anti-HAO scFvs were identified by sequencing analysis of the 97 HAO positive clones. These scFvs were designated as 7, 8, 10, 17, 40, 66, 80, 88, 90, and 98. Six different VH and 10 different VL genes were revealed. Some scFvs shared the same VH gene. Five out of the six different VH genes belonged to the IGHV1-69 gene family. Three out of ten VL genes were kappa chain. 2A scFv is a moderate neutralizing antibody, 38B and 1C are non-neutralizing antibodies. Ten scFvs, 7, 8, 10, 17, 40, 66, 80, 88, 90, and 98 are potent neutralizing antibodies. The nucleic acid and amino acid sequence of the neutralizing influenza antibodies are provided below. Methods of making these antibodies are disclosed in PCT/US2009/054950 (Publication No. WO 2010/027818), the entire contents of which are incorporated herein by reference.
Antibody 2A: Variable Region Nucleic Acid Sequences







VH chain of 2A







(SEQ ID NO: 1305)







CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGTGACAATGCTA


 


TCAGCTGGGTGCGACAGGCCCCAGGACAAGGGCTTGAGTGGATGGGGGGC


 


ATCATTCCTATCTTTGGAAAACCAAACTACGCACAGAAGTTCCAGGGCAG


 


AGTCACGATTACTGCGGACGAATCCACGAGCACAGCCTACATGGACCTGA


 


GGAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAGATTCA


 


GACGCGTATTACTATGGTTCGGGGGGTATGGACGTCTGGGGCCAAGGCAC


 


CCTGGTCACCGTCTCCTCA


 


VL chain of 2A







(SEQ ID NO: 1306)







CTGCCTGTGCTGACTCAATCATCCTCTGCCTCTGCTTCCCTGGGATCCTC


 


GGTCAAGCTCACCTGCACTCTGAGCAGTGGGCATAGTAACTACATCATCG


 


CATGGCATCAACAGCAGCCAGGGAAGGCCCCTCGGTACTTGATGAAGGTT


 


AATAGTGATGGCAGCCACACCAAGGGGGACGGGATCCCTGATCGCTTCTC


 


AGGCTCCAGCTCTGGGGCTGACCGCTACCTCACCATCTCCAACCTCCAGT


 


CTGAGGATGAGGCTAGTTATTTCTGTGAGACCTGGGACACTAAGATTCAT


 


GTCTTCGGAACTGGGACCAAGGTCTCCGTCCTCAG






Antibody 2A: Variable Region Aamino Acid Sequences







VH chain of 2A







(SEQ ID NO: 1307)







QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNAISWVRQAPGQGLEWMGG


 


IIPIFGKPNYAQKFQGRVTITADESTSTAYMDLRSLRSEDTAVYYCARDS


 


DAYYYGSGGMDVWGQGTLVTVSS


 


VL chain of 2A







(SEQ ID NO: 1308)







LPVLTQSSSASASLGSSVKLTCTLSSGHSNYIIAWHQQQPGKAPRYLMKV


 


NSDGSHTKGDGIPDRFSGSSSGADRYLT ISNLQSEDEASYFCETWDTK


 


I HVFGTGTKVSVL






Antibody D7: Variable Region Nucleic Acid Sequences







VH chain of D7







(SEQ ID NO: 1309)







CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTCTCCTGCAAGGCTCCTGGAGGTATCTTCAACACCAATGCTT


 


TCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGG


 


GTCATCCCTTTGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAG


 


AGTCACCATTACCGCGGACGAATCCACGAACACAGCCTACATGGAGCTTA


 


CCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGT


 


GGTTACCATTTTAGGAGTCACTTTGACTCCTGGGGCCTGGGAACCCTGGT


 


CACCGTCTCCTCA


 


VL chain of D7







(SEQ ID NO: 1310)







AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGCGTCTCCGGGGAAGAC


 


GGTGACCATCTCCTGCACCGGCAGCAGTGGCAACATTGCCGCCAACTATG


 


TGCAGTGGTACCAACAACGTCCGGGCAGTGCCCCCACTACTGTGATCTAT


 


GAGGATGACCGAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCAT


 


CGACAGGTCCTCCAACTCTGCCTCCCTCACCATCTCAGGACTGAAGACTG


 


AGGACGAGGCTGACTACTACTGTCAGACTTATGATACCAACAATCATGCT


 


GTGTTCGGAGGAGGCACCCACCTGACCGTCCTC






Antibody H98: Variable Region Nucleic Acid Sequences







VH chain of H98







(SEQ ID NO: 1311)







CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTCTCCTGCAAGGCTCCTGGAGGTATCTTCAACACCAATGCTT


 


TCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGG


 


GTCATCCCTTTGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAG


 


AGTCACCATTACCGCGGACGAATCCACGAACACAGCCTACATGGAGCTTA


 


CCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGT


 


GGTTACCATTTTAGGAGTCACTTTGACTCCTGGGGCCTGGGAACCCTGGT


 


CACCGTCTCCTCA


 


VL chain of H98







(SEQ ID NO: 1312)







TCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGAAACACGGGCAGAG


 


GGTCACCATCTCTTGTTCTGGAGGCACCTCCAACATCGGACGTAATCATG


 


TTAACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTAT


 


AGTAATGAACAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAA


 


ATCTGGCACCTCCGCCTCCCTGGCCGTGAGTGGGCTCCAGTCTGAGGATG


 


AGGCTGATTATTACTGTGCATCATGGGATGACAACTTGAGTGGTTGGGTG


 


TTCGGCGGAGGGACCAAGCTGACCGTCCTA






Antibody D7 and H98: Variable Region Chain Amino Acid Sequences







VH chain of D7 and H98


(SEQ ID NO: 1313)







QVQLVQSGAEVKKPGSSVKVSCKAPGGIFNTNAFSWVRQAPGQGLEWVGG


 


VIPLFRTASYAQNVQGRVT I TADESTNTAYMELTSLRSADTAVYYCAR


 


SSGYH FRSHFDSWGLGTLVTVSS


 


VL chain of D7







(SEQ ID NO: 1314)







NFMLTQPHSVSASPGKTVTISCTGSSGNIAANYVQWYQQRPGSAPTTVIY


 


EDDRRPSGVPDRFSGSIDRSSNSASLTISGLKTEDEADYYCQTYDTNNHA


 


VFGGGTHLTVL


 


VL chain of H98







(SEQ ID NO: 1315)







SYELTQPPSASGKHGQRVTISCSGGTSNIGRNHVNWYQQLPGTAPKLLIY


 


SNEQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEADYYCASWDDNLSGWV


 


FGGGTKLTVL






Antibody D8: Variable Region Nucleic Acid Sequences







VH chain of D8







(SEQ ID NO: 1316)







CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCGCTTATGCTT


 


TCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC


 


ATCACCGGAATGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAG


 


AGTCACGATTACCGCGGACGAACTCACGAGCACAGCCTACATGGAGTTGA


 


GCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTG


 


TATTACTATGAGAGTAGTCTTGACTATTGGGGCCAGGGAACCCTGGTCAC


 


CGTCTCCTCAG


 


VL chain of D8







(SEQ ID NO: 1317)







CAGTCTGTGCTGACTCAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTC


 


AGTCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACT


 


CTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT


 


TATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGCCTC


 


CAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGG


 


ATGAGGCTGATTATTTCTGCTGCTCATATGCAGGCCACAGTGCTTATGTC


 


TTCGGAACTGGGACCAAGGTCACCGTCCTG






Antibody D80: Variable Region Nucleic Acid Sequences







VH chain of D80







(SEQ ID NO: 1318)







CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTCTCCTGCAGGGCTTCTGGAGGCACCTTCAGCGCTTATGCTT


 


TCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC


 


ATCACCGGAATGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAG


 


AGTCACGATTACCGCGGACGAACTCACGAGCACAGCCTACATGGAGTTGA


 


GCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTG


 


TATTACTATGAGAGTAGTCTTGACTATTGGGGCCAGGGAACCCTGGTCAC


 


CGTCTCCTCAG


 


VK chain of D80







(SEQ ID NO: 1319)







GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA


 


AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTCTTAGCAGCAAGTACT


 


TAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT


 


GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG


 


GTCTGGGACAGACTTCACCCTCACCATCAGTAGACTGGAGCCTGAAGATT


 


TTGCAGTGTATTCCTGTCAGCAGTATGATGGCGTACCTCGGACGTTCGGC


 


CAAGGGACCACGGTGGAAATCAAA






Antibody D8 and D80: Variable Region Chain Amino Acid Sequences







VH chain of D8 and D80







(SEQ ID NO: 1320)







QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYAFTWVRQAPGQGLEWMGG


 


ITGMFGTANYAQKFQGRVTITADELTSTAYMELSSLTSEDTALY


 


VL chain of D8







(SEQ ID NO: 1321)







YCARGLYYYESSLDYWGQGTLVTVSSQSVLTQPPSASGSPGQSVTISCTG


 


TSSDVGGYNSVSWYQQHPGKAPKLMIYEVTKRPSGVPDRFSASKSGNTAS


 


LTVSGLQAEDEADYFCCSYAGHSAYVFGTGTKVTVL


 


VK chain of D80







(SEQ ID NO: 1322)







EIVLTQSPGTLSLSPGERATLSCRASQSLSSKYLAWYQQKPGQAPRLLIY


 


GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYSCQQYDGVPRTFG


 


QGTTVEIK






Antibody F10: Variable Region Nucleic Acid Sequences







VH chain of F10







(SEQ ID NO: 1323)







CAGGTGCAGCTGGTGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTCTCCTGCACGTCCTCTAAGTCACCTTCAGTAGTTTTGGCTA


 


TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGG


 


ATCAGCCCTATGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAG


 


AGTCACCATTACCGCGGACCAGTCCACGAGGACAGCCTACATGGACCTGA


 


GGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCT


 


TCTTACATTTGTTCTGGTGGAACCTGCGTCTTTGACCATTGGGGCCAGGG


 


AACCCTGGTCACCGTCTCCTCA


 


VL chain of F10







(SEQ ID NO: 1324)







CAGCCTGGGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGAC


 


CGCCACACTCACCTGCACTGGGAACAGCAACAATGTTGGCAACCAAGGAG


 


CAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTCCTATCCTAC


 


AGGAATAATGACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAG


 


GTCAGGAAACACAGCCTCCCTGACCATTACTGGACTCCAGCCTGAGGACG


 


AGGCTGACTATTACTGCTCAACATGGGACAGCAGCCTCAGTGCTGTGGTA


 


TTCGGCGGAGGGACCAAGCTGACCGTCCTA






Antibody E90: Variable Region Nucleic Acid Sequences







VH chain of E90







(SEQ ID NO: 1325)







CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTC


 


GGTGAAGGTCTCCTGCACGTCCTCTGAAGTCACCTTCAGTAGTTTTGCTA


 


TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGG


 


ATCAGCCCTATGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAG


 


AGTCACCATTACCGCGGACCAGTCCACGAGGACAGCCTACATGGACCTGA


 


GGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCT


 


TCTTACATTTGTTCTGGTGGAACCTGCGTCTTTGACCATTGGGGCCAGGG


 


AACCCTGGTCACCGTCTCCTCA


 


VL chain of E90







(SEQ ID NO: 1326)







GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA


 


CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA


 


ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT


 


GCATCCAGTTTGCAAAGAGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC


 


TGGGACAGACTTCACTCTCACCATTAGCAGCCTGCAGCCTGAAGATTTTG


 


CAGTGTATTACTGTCAGCAGTATGATAGTTCACCGTACACTTTTGGCCAG


 


GGGACCAAGGTAGAGATCAAA






Antibody F10 and E90 Variable Region Amino Acid Sequences







VH chain of F10 and E90







(SEQ ID NO: 1327)







QVQLVQSGAEVKKPGS SVKVSCTSSEVTFSSFAISWVRQAPGQGLEWLG


 


GISPMFGTPNYAQKFQGRVTITADQSTRTAYMDLRSLRSEDTAVYYCARS


 


PSYICSGGTCVFDHWGQGTLVTVSS


 


VL chain of F10







(SEQ ID NO: 1328)







QPGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSY


 


RNNDRPSGISERFSASRSGNTASLTITGLQPEDEADYYCSTWDSSLSAVV


 


FGGGTKLTVL


 


VL chain of E90







(SEQ ID NO: 1329)







DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA


 


ASSLQRGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYDSSPYTFGQ


 


GTKVEIK






Antibody G17: Variable Region Nucleic Acid Sequences







VH chain of G17







(SEQ ID NO: 1330)







CAGGTGCAGCTGGTGCAATCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTC


 


AGTGAAGGTCTCCTGCAAGACTTCTGGAGTCACCTTCAGCAGCTATGCTA


 


TCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG


 


ATCATCGGTGTCTTTGGTGTACCAAAGTACGCGCAGAACTTCCAGGGCAG


 


AGTCACAATTACCGCGGACAAACCGACGAGTACAGTCTACATGGAGCTGA


 


ACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGCCC


 


GGGTACTACGTAGGAAAGAATGGTTTTGATGTCTGGGGCCAAGGGACAAT


 


GGTCACCGTCTCTTCA


 


VL chain of G17







(SEQ ID NO: 1331)







TCCTATGAGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGAC


 


CGCCATACTCACCTGCACTGGAGACAGCAACAATGTTGGCCACCAAGGTA


 


CAGCTTGGCTGCAACAACACCAGGGCCACCCTCCCAAACTCCTATCCTAC


 


AGGAATGGCAACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAG


 


GTCAGGAAATACAGCCTCCCTGACCATTATTGGACTCCAGCCTGAGGACG


 


AGGCTGACTACTACTGCTCAGTATGGGACAGCAGCCTCAGTGCCTGGGTG


 


TTCGGCGGAGGGACCAAGCTGACCGTCCTA






Antibody G17 Variable Region Amino Acid Sequences







VH chain of G17







(SEQ ID NO: 1332)







QVQLVQSGAEVKKPGASVKVSCKTSGVTFSSYAISWVRQAPGQGLEWMGG


 


IIGVFGVPKYAQNFQGRVTITADKPTSTVYMELNSLRAEDTAVYYCAREP


 


GYYVGKNGFDVWGQGTMVTVSS


 


VL chain of G17







(SEQ ID NO: 1333)







SYELTQPPSVSKGLRQTAILTCTGDSNNVGHQGTAWLQQHQGHPPKLLSY


 


RNGNRPSGISERFSASRSGNTASLTIIGLQPEDEADYYCSVWDSSLSAWV


 


FGGGTKLTVL






Antibody H40: Variable Region Nucleic Acid Sequences







VH chain of H40







(SEQ ID NO: 1334)







CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGGCCTC


 


AGTGAAGGTCTCATGTAAGGCTTCTGGATACACCTTCACCGGTTATTATA


 


TTCACTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGTTGG


 


ATCAACCCTATGACTGGTGGCACAAACTATGCACAGAAGTTTCAGGTCTG


 


GGTCACCATGACCCGGGACACGTCCATCAACACAGCCTACATGGAGGTGA


 


GCAGGCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGGGGGGCT


 


TCCGTATTACGATATTTTGACTGGCAGCCCGAGGCTCTTGATATCTGGGG


 


CCTCGGGACCACGGTCACCGTCTCCTCA


 


VL chain of H40







(SEQ ID NO: 1335)







CAGCCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGAC


 


GGCCAGCATTCCCTGTGGGGGGAACAACATTGGAGGCTACAGTGTACACT


 


GGTACCAACAAAAGCCGGGCCAGGCCCCCCTCTTGGTCATTTATGACGAT


 


AAAGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCGCCAACTCTGG


 


GAGCACGGCCACCCTGACAATCAGCAGGGTCGAAGCCGGGGATGAGGGCG


 


ACTACTACTGTCAGGTGTGGGATAGTGGTAATGATCGTCCGCTGTTCGGC


 


GGAGGGACCAAGCTGACCGTCCTA






Antibody H40: Variable Region Amino Acid Sequences







VH chain of H40







(SEQ ID NO: 1336)







QVQLVQSGAEVRKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGW


 


INPMTGGTNYAQKFQVWVTMTRDTSINTAYMEVSRLTSDDTAVYYCARGA


 


SVLRYFDWQPEALDIWGLGTTVTVSS


 


VL chain of H40







(SEQ ID NO: 1337)







QPVLTQPPSVSVAPGQTASIPCGGNNIGGYSVHWYQQKPGQAPLLVIYDD


 


KDRPSGIPERFSGANSGSTATLTISRVEAGDEGDYYCQVWDSGNDRPLFG


 


GGTKLTVL






Antibody A66 Variable Region Nucleic Acid Sequences







VH chain of A66







(SEQ ID NO: 1338)







CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTC


 


GGTGAAGGTTTCCTGCAAGGCTTCTGGAGGCCCCTTCAGCATGACTGCTT


 


TCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGG


 


ATCAGCCCTATCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAG


 


AGTCACGATTACCGCGGACGAATCCACGAACACAGCCAACATGGAGCTGA


 


CCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTT


 


TCCTCCTACCAACCGAATAATGATGCTTTTGCTATCTGGGGCCAAGGGAC


 


AATGGTCACCGTCTCTTCA


 


VK chain of A66







(SEQ ID NO: 1339)







GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA


 


AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAG


 


CCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT


 


GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC


 


TGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTG


 


CAGTCTATTTCTGTCAGCAGTATGGTAGCTCACCTCAATTCGGCCAAGGG


 


ACACGACTGGAGATTAAA






Antibody A66 Variable Region Amino Acid Sequences







VH chain of A66







(SEQ ID NO: 1340)







QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGG


 


ISPIFRTPKYAQKFQGRVTITADESTNTANMELTSLKSEDTAVYYCARTL


 


VK chain of A66







(SEQ ID NO: 1341)







SSYQPNNDAFAIWGQGTMVTVSSEIVLTQSPATLSLSPGERATLSCRASQ


 


SVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT I


 


SRLEPEDFAVYFCQQYGSSPQFGQGTRLEIK






Antibody E88 Variable Region Nucleic Acid Sequences







VH chain of E88







(SEQ ID NO: 1342)







CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTC


 


GGTGAAGGTTTCCTGCAAGGCTTCTGGAGGCCCCTTCAGCATGACTGCTT


 


TCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGG


 


ATCAGCCCTATCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAG


 


AGTCACGATTACCGCGGACGAATCCACGAACACAGCCAACATGGAGCTGA


 


CCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTT


 


TCCTCCTACCAACCGAATAATGATGCTTTTGCTATCTGGGGCCAAGGGAC


 


AATGGTCACCGTCTCTTCA


 


VL chain of E88







(SEQ ID NO: 1343)







CTGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAG


 


GGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTG


 


TAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT


 


AGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAG


 


GTCAGGCACCTCAGCCTCCCTGGCCATCATTGGACTCCGGCCTGAGGATG


 


AAGCTGATTATTACTGTCAGTCGTATGACAGCAGGCTCAGTGCTTCTCTC


 


TTCGGAACTGGGACCACGGTCACCGTCCTC






Antibody E88 Variable Region Amino Acid Sequences







VH chain of E88







(SEQ ID NO: 1344)







QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGG


 


ISPIFRTPKYAQKFQGRVTITADESTNTANMELTSLKSEDTAVYYCARTL


 


SSYQPNNDAFAIWGQGTMVTVSS


 


VL chain of E88







(SEQ ID NO: 1345)







LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIY


 


SNNQRPSGVPDRFSGSRSGTSASLAIIGLRPEDEADYYCQSYDSRLSASL


 


FGTGTTVTVL






The amino acid sequences of the heavy and light chain complementary determining regions of the neutralizing influenza antibodies are shown below in Table 18.















TABLE 18








SEQ

SEQ

SEQ






ID

ID

ID


ANTIBODY
CHAIN
CDR1
NO:
CDR2
NO:
CDR3
NO:























CONSENSUS
HEAVY
SYAFS
299
GIIPMFGTPNYAQKFQG
1263
SSGYYYGGGFDV
1284



 


D7/H98
HEAVY
TNAFS
302
GVIPLFRTASYAQNV
1264
SSGYHFGRSHFDS
1285






QN


 


D8/D80
HEAVY
AYAFT
305
GITGMFGTANYAQKF
1265
GLYYYESSLDY
1286






QG


 


F10/E90
HEAVY
SFAIS
600
GISPMFGTPNYAQKF
1266
SPSYICSGGTCVF
1287






QG

DH


 


G17
HEAVY
SYAIS
606
GIIGVFGVPKYAQKFQG
1267
EPGYYGKNGFDV
1288


 


H40
HEAVY
GYYIH
630
WINPMTGGTNYAQKF
1268
GASVLRYFDWQP
1289






QV

EALDI


 


A66
HEAVY
MTAFT
641
GISPIFRTPKYAQKFQG
1269
TLSSYQPNNDAF
1290








AI


 


E88
HEAVY
MTAFT
647
GISPIFRTPKYAQKFQG
1270
TLSSYQPNNDAF
1291








AI


 


2A
HEAVY
DNAIS
668
GIIPIFGKPNYAQKFQG
1271
DSDAYYYGSGG
1292








MDV


 


CONSENSUS
LIGHT
TGSSSMNIGN
757
SNSDRPS
1272
QSYDSLSAYV
1293




YVA


 


D7
LIGHT
TGSSSNIAAN
1252
EDDRRPS
1273
QTYDTNNHAV
1294




YVQ


 


D8
LIGHT
TGTSSDVGGY
1253
EVTKRPS
1274
CSYAGHSAYV
1295




NSVS


 


F10
LIGHT
TGNSNNVGN
1254
RNNDRPS
1275
STWDSSLSAVV
1296




QGAA


 


G17
LIGHT
TGNSNNVGH
1255
RNGNRPS
1276
SVWDSSLSAWV
1297




QGTA


 


H40
LIGHT
GGNNIGGYSVH
1256
DDKDRPS
1277
QVWDSGNDRPL
1298


 


A66
LIGHT
RASQSVSSYLA
1257
DASNRAT
1278
QQYGSSPQF
1299


 


D80
LIGHT
RASQSLSSKY
1258
GASSRAT
1279
QQYDGVPRT
1300




LA


 


E88
LIGHT
SGSSSNIGSNT
1259
SNNQRPS
1280
QSYDSRLSASL
1301




VN


 


E90
LIGHT
RASQSISSYLN
1260
AASSLQR
1281
QQYDSSPYT
1302


 


H98
LIGHT
SGGTSNIGRN
1261
SNEQRPS
1282
ASWDDNLSGWV
1303




HVN


 


2A
LIGHT
TLSSGHSNYIIA
1262
VNSDGSHTKGD
1283
ETWDTKIHV
1304









Antibodies

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).
The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
The following definitions are useful in understanding the present invention:
The term “antibody” (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.
An “isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.
The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (ξ), respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
The term “variable” refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a n-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid,residues from a “complemeniarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a “hypervariable loop” (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a “hypervariable loop”/CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. e al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the VH when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001)).
By “germline nucleic acid residue” is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. “Germline gene” is the DNA found in a germ cell (i.e., a cell destined to become an egg or in the sperm). A “germline mutation” refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body. A germline mutation is in contrast to a somatic mutation which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
The monoclonal antibodies herein include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The present invention provides variable domain antigen-binding sequences derived from human antibodies. Accordingly, chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g., an FR or C region sequence. In addition, chimeric antibodies of primary interest herein include those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass. Chimeric antibodies of interest herein also include those containing variable domain antigen-binding sequences related to those described herein or derived from a different species, such as a non-human primate (e.g., Old World Monkey, Ape, etc). Chimeric antibodies also include primatized and humanized antibodies.
Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
A “humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is traditionally performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
A “human antibody” is an antibody containing only sequences present in an antibody naturally produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.
An “intact” antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH 1, CH 2 and CH 3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The phrase “functional fragment or analog” of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FcεRI.
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′)2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH 1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The “Fc” fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
“Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies; vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
As used herein, an antibody that “internalizes” is one that is taken up by (i.e., enters) the cell upon binding to an antigen on a mammalian cell (e.g., a cell surface polypeptide or receptor). The internalizing antibody will of course include antibody fragments, human or chimeric antibody, and antibody conjugates. For certain therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or adequate to kill a cell or inhibit its growth, especially an infected cell. Depending on the potency of the antibody or antibody conjugate, in some instances, the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly potent in killing such that internalization of one molecule of the toxin conjugated to the antibody is sufficient to kill the infected cell.
As used herein, an antibody is said to be “immunospecific,” “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with the antigen, preferably with an affinity constant, Ka, of greater than or equal to about 104 M−1, or greater than or equal to about 105 M−1, greater than or equal to about 106 M−1, greater than or equal to about 107 M−1, or greater than or equal to 108 M−1. Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant KD, and in certain embodiments, HuM2e antibody specifically binds to M2e if it binds with a KD of less than or equal to 10−4 M, less than or equal to about 10−5 M, less than or equal to about 10−6 M, less than or equal to 10−7 M, or less than or equal to 10−8 M. Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)).
Binding properties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
An antibody having a “biological characteristic” of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies. For example, in certain embodiments, an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody and/or have a common effector function as the designated antibody.
The term “antagonist” antibody is used in the broadest sense, and includes an antibody that partially or fully blocks, inhibits, or neutralizes a biological activity of an epitope, polypeptide, or cell that it specifically binds. Methods for identifying antagonist antibodies may comprise contacting a polypeptide or cell specifically bound by a candidate antagonist antibody with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.
An “antibody that inhibits the growth of infected cells” or a “growth inhibitory” antibody is one that binds to and results in measurable growth inhibition of infected cells expressing or capable of expressing an M2e epitope bound by an antibody. Preferred growth inhibitory antibodies inhibit growth of infected cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being infected cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the infected cells to the antibody. Growth inhibition of infected cells in vivo can be determined in various ways known in the art. The antibody is growth inhibitory in vivo if administration of the antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction the percent of infected cells or total number of infected cells within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.
An antibody that “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Preferably the cell is an infected cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.
Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).
“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In certain embodiments, the FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FCγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
“Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.
“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
The terms “influenza A” and “Influenzavirus A” refer to a genus of the Orthomyxoviridae family of viruses. Influenzavirus A includes only one species: influenza A virus which cause influenza in birds, humans, pigs, and horses. Strains of all subtypes of influenza A virus have been isolated from wild birds, although disease is uncommon. Some isolates of influenza A virus cause severe disease both in domestic poultry and, rarely, in humans.
A “mammal” for purposes of treating n infection, refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
The term “therapeutically effective amount” refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. See preceding definition of “treating.”
“Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM polyethylene glycol (PEG), and PLURONICSTM.
The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.
A “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, either in vitro or in vivo. Examples of growth inhibitory agents include agents that block cell cycle progression, such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vinca alkaloids (vincristine, vinorelbine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERETM, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
“Label” as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a “labeled” antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.
The term “epitope tagged” as used herein refers to a chimeric polypeptide comprising a polypeptide fused to a “tag polypeptide.” The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide is also preferably fairly unique so that the antibody does not substantially, cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
A “small molecule” is defined herein to have a molecular weight below about 500 Daltons.
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers. Polynucleotides may include genomic sequences, extra-genomic and plasmid sequences, and smaller engineered gene segments that express, or may be adapted to express polypeptides.
An “isolated nucleic acid” is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
The term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen or Influenza A-infected cell.
An “isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
A “native sequence” polynucleotide is one that has the same nucleotide sequence as a polynucleotide derived from nature. A “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g., antibody) derived from nature (e.g., from any species). Such native sequence polynucleotides and polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
A polynucleotide “variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.
A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art.
Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.
In many instances, a polypeptide variant will contain one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
When comparing polynucleotide and polypeptide sequences, two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.
For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
In one approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
“Homology” refers to the percentage of residues in the polynucleotide or polypeptide sequence variant that are identical to the non-variant sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. In particular embodiments, polynucleotide and polypeptide variants have at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.
“Vector” includes shuttle and expression vectors. Typically, the plasmid construct will also include an origin of replication (e.g., the ColE1 origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively, of the plasmids in bacteria. An “expression vector” refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragment of the invention, in bacterial or eukaryotic cells. Suitable vectors are disclosed below.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
The present invention includes human monoclonal anti-influenza antibodies comprising a polypeptide of the present invention, as well as fragments and variants thereof. In one embodiment, the antibody is an antibody designated herein as TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, 3242_P05, TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_D17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN 558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), TCN-504 (3251_K17), SC06-141, SC06-255, SC06-257, SC06-260, SC06-261, SC06-262, SC06-268, SC06-272, SC06-296, SC06-301, SC06-307, SC06-310, SC06-314, SC06-323, SC06-325, SC06-327, SC06-328, SC06-329, SC06-331, SC06-332, SC06-334, SC06-336, SC06-339, SC06-342, SC06-343, SC06-344, CR6141, CR6255, CR6257, CR6260, CR6261, CR6262, CR6268, CR6272, CR6296, CR6301, CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329, CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, or CR6344. These antibodies preferentially bind to or specifically bind to influenza A infected cells as compared to uninfected control cells of the same cell type.
In particular embodiments, the antibodies of the present invention bind to the M2 or HA protein. In certain embodiments, the present invention provides human anti-influenza antibodies that bind to epitopes within M2e or HA that are only present in the native conformation, i.e., as expressed in cells. In particular embodiments, these antibodies fail to specifically bind to an isolated M2e polypeptide, e.g., the 23 amino acid residue M2e fragment or an isolated HA polypeptide. It is understood that these antibodies recognize non-linear (i.e. conformational) epitope(s) of the M2 or HA peptide or protein.
These specific conformational epitopes within the M2 or HA protein, and particularly within M2e, may be used as vaccines to prevent the development of influenza infection within a subject.
As will be understood by the skilled artisan, general description of antibodies herein and methods of preparing and using the same also apply to individual antibody polypeptide constituents and antibody fragments.
The antibodies of the present invention may be polyclonal or monoclonal antibodies. However, in preferred embodiments, they are monoclonal. In particular embodiments, antibodies of the present invention are fully human antibodies. Methods of producing polyclonal and monoclonal antibodies are known in the art and described generally, e.g., in U.S. Pat. No. 6,824,780. Typically, the antibodies of the present invention are produced recombinantly, using vectors and methods available in the art, as described further below. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
Human antibodies may also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852. Such animals may be genetically engineered to produce human antibodies comprising a polypeptide of the present invention.
In certain embodiments, antibodies of the present invention are chimeric antibodies that comprise sequences derived from both human and non-human sources. In particular embodiments, these chimeric antibodies are humanized or primatizedTM. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
In the context of the present invention, chimeric antibodies also include fully human antibodies wherein the human hypervariable region or one or more CDRs are retained, but one or more other regions of sequence have been replaced by corresponding sequences from a non-human animal.
The choice of non-human sequences, both light and heavy, to be used in making the chimeric antibodies is important to reduce antigenicity and human anti-non-human antibody responses when the antibody is intended for human therapeutic use. It is further important that chimeric antibodies retain high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, chimeric antibodies are prepared by a process of analysis of the parental sequences and various conceptual chimeric products using three-dimensional models of the parental human and non-human sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
As noted above, antibodies (or immunoglobulins) can be divided into five different classes, based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences). Antibodies, or fragments thereof, of the present invention may be any class, and may, therefore, have a gamma, mu, alpha, delta, or epsilon heavy chain. A gamma chain may be gamma 1, gamma 2, gamma 3, or gamma 4; and an alpha chain may be alpha 1 or alpha 2.
In a preferred embodiment, an antibody of the present invention, or fragment thereof, is an IgG. IgG is considered the most versatile immunoglobulin, because it is capable of carrying out all of the functions of immunoglobulin molecules. IgG is the major Ig in serum, and the only class of Ig that crosses the placenta. IgG also fixes complement, although the IgG4 subclass does not. Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is that the cell can now internalize the antigen better. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions. Antibodies of the present invention may be of any IgG subclass.
In another preferred embodiment, an antibody, or fragment thereof, of the present invention is an IgE. IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen. As a consequence of its binding to basophils an mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms. IgE also plays a role in parasitic helminth diseases. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite. IgE does not fix complement.
In various embodiments, antibodies of the present invention, and fragments thereof, comprise a variable light chain that is either kappa or lambda. The lamba chain may be any of subtype, including, e.g., lambda 1, lambda 2, lambda 3, and lambda 4.
As noted above, the present invention further provides antibody fragments comprising a polypeptide of the present invention. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. For example, the smaller size of the fragments allows for rapid clearance, and may lead to improved access to certain tissues, such as solid tumors. Examples of antibody fragments include: Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single-chain antibodies; and multispecific antibodies formed from antibody fragments.
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab′)2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions. Thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
In certain embodiments, antibodies of the present invention are bispecific or multi-specific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen. Alternatively, an anti-M2e arm may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies possess an M2e-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.
Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.
In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Recent progress has facilitated the direct recovery of Fab′-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991). A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
Antibodies of the present invention further include single chain antibodies.
In particular embodiments, antibodies of the present invention are internalizing antibodies.
Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the antibody, or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final antibody, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides of the present invention may be included in antibodies of the present invention.
A useful method for identification of certain residues or regions of an antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells in Science, 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with PSCA antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-antibody variants are screened for the desired activity.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of an antibody include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.
Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative and non-conservative substitutions are contemplated.
Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion.from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and an antigen or infected cell. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
The antibody of the invention is modified with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-infection activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).
To increase the serum half-life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
Antibodies of the present invention may also be modified to include an epitope tag or label, e.g., for use in purification or diagnostic applications. The invention also pertains to therapy with immunoconjugates comprising an antibody conjugated to an anti-cancer agent such as a cytotoxic agent or a growth inhibitory agent. Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.
Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.
In one preferred embodiment, an antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules. Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.
Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.
There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research 52: 127-131 (1992). The linking groups include disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.
Immunoconjugates may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
Another immunoconjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company). Another drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
Examples of other agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).
Enzymatically active toxins and fragments thereof that can be used include, e.g., diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232.
The present invention further includes an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
For selective destruction of infected cells, the antibody includes a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-PSCA antibodies. Examples include At211, I131, I125, Y90, Re186, Rc188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
The radio- or other label is incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc99m or I123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal,CRC Press 1989) describes other methods in detail.
Alternatively, a fusion protein comprising the antibody and cytotoxic agent is made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
The antibodies of the present invention are also used in antibody dependent enzyme mediated prodrug therapy (ADET) by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug (see, e.g., WO 88/07378 and U.S. Pat. No. 4,975,278).
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form. Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulthase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as “abzymes”, can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a infected cell population.
The enzymes of this invention can be covalently bound to the antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature, 312: 604-608 (1984).
Other modifications of the antibody are contemplated herein. For example, the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
The antibodies disclosed herein are also formulated as immunoliposomes. A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant that is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired a diameter. Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81(19)1484 (1989).
Antibodies of the present invention, or fragments thereof, may possess any of a variety of biological or functional characteristics. In certain embodiments, these antibodies are Influenza A specific or M2 protein specific antibodies, indicating that they specifically bind to or preferentially bind to Influenza A or the M2 protein thereof, respectively, as compared to a normal control cell. In particular embodiments, the antibodies are HuM2e antibodies, indicating that they specifically bind to a M2e protein, preferably to an epitope of the M2e domain that is only present when the M2 protein is expressed in cells or present on a virus, as compared to a normal control cell.
In particular embodiments, an antibody of the present invention is an antagonist antibody, which partially or fully blocks or inhibits a biological activity of a polypeptide or cell to which it specifically or preferentially binds. In other embodiments, an antibody of the present invention is a growth inhibitory antibody, which partially or fully blocks or inhibits the growth of an infected cell to which it binds. In another embodiment, an antibody of the present invention induces apoptosis. In yet another embodiment, an antibody of the present invention induces or promotes antibody-dependent cell-mediated cytotoxicity or complement dependent cytotoxicity.
Methods of Identifying and Producing Antibodies Specific for Influenza Virus
The present invention provides novel methods for the identification of human anti-influenza antibodies raised against the M2e protein, as exemplified in Example 4, and for the identification of human anti-influenza antibodies raised against the HA protein, as exemplified in Example 13. These methods may be readily adapted to identify antibodies specific for other polypeptides expressed on the cell surface by infectious agents, or even polypeptides expressed on the surface of an infectious agent itself.
In general, the methods include obtaining serum samples from patients that have been infected with or vaccinated against an infectious agent. These serum samples are then screened to identify those that contain antibodies specific for a particular polypeptide associated with the infectious agent, such as, e.g., a polypeptide or protein specifically expressed on the surface of cells infected with the infectious agent, but not uninfected cells. In particular embodiments, the serum samples are screened by contacting the samples with a cell that has been transfected with an expression vector that expresses the polypeptide expressed on the surface of infected cells.
Once a patient is identified as having serum containing an antibody specific for the infectious agent polypeptide of interest is identified, mononuclear and/or B cells obtained from the same patient are used to identify a cell or clone thereof that produces the antibody, using any of the methods described herein or available in the art. Once a B cell that produces the antibody is identified, cDNAs encoding the variable regions or fragments thereof of the antibody may be cloned using standard RT-PCR vectors and primers specific for conserved antibody sequences, and subcloned in to expression vectors used for the recombinant production of monoclonal antibodies specific for the infectious agent polypeptide of interest.
In one embodiment, the present invention provides a method of identifying an antibody that specifically binds influenza A-infected cells, comprising: contacting an Influenza A virus or a cell expressing the M2 protein with a biological sample obtained from a patient having been infected by Influenza A; determining an amount of antibody in the biological sample that binds to the cell; and comparing the amount determined with a control value, wherein if the value determined is at least two-fold greater than the control value, an antibody that specifically binds influenza A-infected cells is indicated.
In various embodiments, the cells expressing an M2 or HA protein are cells infected with an Influenza A virus or cells that have been transfected with a polynucleotide that expressed the M2 or HA protein. Alternatively, the cells may express a portion of the M2 protein that includes the M2e domain and enough additional M2 sequence that the protein remains associated with the cell and the M2e domain is presented on the cell surface in the same manner as when present within full length M2 protein. Methods of preparing an M2 or HA expression vector and transfecting an appropriate cell, including those described herein, may be readily accomplished, in view of the M2 and HA sequences being publicly available. See, for example, the Influenza Sequence Database (ISD) (flu.lanl.gov on the World Wide Web, described in Macken et al., 2001, “The value of a database in surveillance and vaccine selection” in Options for the Control of Influenza IV. A.D.M.E., Osterhaus & Hampson (Eds.), Elsevier Science, Amsterdam, pp. 103-106) and the Microbial Sequencing Center (MSC) at The Institute for Genomic Research (TIGR) (tigr.org/msc/infl_a_virus.shtml on the World Wide Web).
The M2e- or HA-expressing cells or virus described above are used to screen the biological sample obtained from a patient infected with influenza A for the presence of antibodies that preferentially bind to the cell expressing the M2 or HA polypeptide using standard biological techniques. For example, in certain embodiments, the antibodies may be labeled, and the presence of label associated with the cell detected, e.g., using FMAT or FACs analysis. In particular embodiments, the biological sample is blood, serum, plasma, bronchial lavage, or saliva. Methods of the present invention may be practiced using high throughput techniques.
Identified human antibodies may then be characterized further. For example the particular conformational epitopes with in the M2e or HA protein that are necessary or sufficient for binding of the antibody may be determined, e.g., using site-directed mutagenesis of expressed M2e or HA polypeptides. These methods may be readily adapted to identify human antibodies that bind any protein expressed on a cell surface. Furthermore, these methods may be adapted to determine binding of the antibody to the virus itself, as opposed to a cell expressing recombinant M2e or HA, or infected with the virus.
Polynucleotide sequences encoding the antibodies, variable regions thereof, or antigen-binding fragments thereof may be subcloned into expression vectors for the recombinant production of human monoclonal anti-M2e or anti-HA antibodies. In one embodiment, this is accomplished by obtaining mononuclear cells from the patient from the serum containing the identified human monoclonal anti-M2e or anti-HA antibody was obtained; producing B cell clones from the mononuclear cells; inducing the B cells to become antibody-producing plasma cells; and screening the supernatants produced by the plasma cells to determine if it contains the human monoclonal anti-M2e or anti-HA antibody. Once a B cell clone that produces a human monoclonal anti-M2e or anti-HA antibody is identified, reverse-transcription polymerase chain reaction (RT-PCR) is performed to clone the DNAs encoding the variable regions or portions thereof of the human monoclonal anti-M2e or anti-HA antibody. These sequences are then subcloned into expression vectors suitable for the recombinant production of human monoclonal anti-M2e or anti-HA antibodies. The binding specificity may be confirmed by determining the recombinant antibody's ability to bind cells expressing M2e or HA polypeptide or protein.
In particular embodiments of the methods described herein, B cells isolated from peripheral blood or lymph nodes are sorted, e.g., based on their being CD19 positive, and plated, e.g., as low as a single cell specificity per well, e.g., in 96, 384, or 1536 well configurations. The cells are induced to differentiate into antibody-producing cells, e.g., plasma cells, and the culture supernatants are harvested and tested for binding to cells expressing the infectious agent polypeptide on their surface using, e.g., FMAT or FACS analysis. Positive wells are then subjected to whole well RT-PCR to amplify heavy and light chain variable regions of the IgG molecule expressed by the clonal daughter plasma cells. The resulting PCR products encoding the heavy and light chain variable regions, or portions thereof, are subcloned into human antibody expression vectors for recombinant expression. The resulting recombinant antibodies are then tested to confirm their original binding specificity and may be further tested for pan-specificity across various strains of isolates of the infectious agent.
Thus, in one embodiment, a method of identifying human monoclonal anti-M2e or anti-HA antibodies is practiced as follows. First, full length or approximately full length M2 or HA cDNAs are transfected into a cell line for expression of M2 or HA protein. Secondly, individual human plasma or sera samples are tested for antibodies that bind the cell-expressed M2 or HA. And lastly, MAbs derived from plasma- or serum-positive individuals are characterized for binding to the same cell-expressed M2 or HA. Further definition of the fine specificities of the MAbs can be performed at this point.
These methods may be practiced to identify a variety of different HuM2e antibodies, including antibodies specific for (a) epitopes in a linear M2e peptide, (b) common epitopes in multiple variants of M2e, (c) conformational determinants of an M2 homotetramer, and (d) common conformational determinants of multiple variants of the M2 homotetramer. The last category is particularly desirable, as this specificity is perhaps specific for all A strains of influenza.
These methods may be practiced to identify a variety of different human monoclonal anti-HA antibodies, including antibodies specific for (a) epitopes in a linear HA peptide, (b) common epitopes in multiple variants of HA, (c) conformational determinants of an HA protein or homotrimer, and (d) common conformational determinants of multiple variants of the HA protein or homotrimer. The last category is particularly desirable, as this specificity is perhaps specific for all A strains of influenza.
Polynucleotides that encode the human monoclonal anti-M2e or anti-HA antibodies or portions thereof of the present invention may be isolated from cells expressing human monoclonal anti-M2e or anti-HA antibodies, according to methods available in the art and described herein, including amplification by polymerase chain reaction using primers specific for conserved regions of human antibody polypeptides. For example, light chain and heavy chain variable regions may be cloned from the B cell according to molecular biology techniques described in WO 92/02551; U.S. Pat. No. 5,627,052; or Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996). In certain embodiments, polynucleotides encoding all or a region of both the heavy and light chain variable regions of the IgG molecule expressed by the clonal daughter plasma cells expressing the human monoclonal anti-M2e or anti-HA antibody are subcloned and sequenced. The sequence of the encoded polypeptide may be readily determined from the polynucleotide sequence.
Isolated polynucleotides encoding a polypeptide of the present invention may be subcloned into an expression vector to recombinantly produce antibodies and polypeptides of the present invention, using procedures known in the art and described herein.
Binding properties of an antibody (or fragment thereof) to M2e or infected cells or tissues may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS). Immunoassay methods may include controls and procedures to determine whether antibodies bind specifically to M2e from one or more specific strains of Influenza A, and do not recognize or cross-react with normal control cells.
Following pre-screening of serum to identify patients that produce antibodies to an infectious agent or polypeptide thereof, e.g., M2 or HA, the methods of the present invention typically include the isolation or purification of B cells from a biological sample previously obtained from a patient or subject. The patient or subject may be currently or previously diagnosed with or suspect or having a particular disease or infection, or the patient or subject may be considered free or a particular disease or infection. Typically, the patient or subject is a mammal and, in particular embodiments, a human. The biological sample may be any sample that contains B cells, including but not limited to, lymph node or lymph node tissue, pleural effusions, peripheral blood, ascites, tumor tissue, or cerebrospinal fluid (CSF). In various embodiments, B cells are isolated from different types of biological samples, such as a biological sample affected by a particular disease or infection. However, it is understood that any biological sample comprising B cells may be used for any of the embodiments of the present invention.
Once isolated, the B cells are induced to produce antibodies, e.g., by culturing the B cells under conditions that support B cell proliferation or development into a plasmacyte, plasmablast, or plasma cell. The antibodies are then screened, typically using high throughput techniques, to identify an antibody that specifically binds to a target antigen, e.g., a particular tissue, cell, infectious agent, or polypeptide. In certain embodiments, the specific antigen, e.g., cell surface polypeptide bound by the antibody is not known, while in other embodiments, the antigen specifically bound by the antibody is known.
According to the present invention, B cells may be isolated from a biological sample, e.g., a tumor, tissue, peripheral blood or lymph node sample, by any means known and available in the art. B cells are typically sorted by FACS based on the presence on their surface of a B cell-specific marker, e.g., CD19, CD138, and/or surface IgG. However, other methods known in the art may be employed, such as, e.g., column purification using CD19 magnetic beads or IgG-specific magnetic beads, followed by elution from the column. However, magnetic isolation of B cells utilizing any marker may result in loss of certain B cells. Therefore, in certain embodiments, the isolated cells are not sorted but, instead, phicol-purified mononuclear cells isolated from tumor are directly plated to the appropriate or desired number of specificities per well.
In order to identify B cells that produce an infectious agent-specific antibody, the B cells are typically plated at low density (e.g., a single cell specificity per well, 1-10 cells per well, 10-100 cells per well, 1-100 cells per well, less than 10 cells per well, or less than 100 cells per well) in multi-well or microtitre plates, e.g., in 96, 384, or 1536 well configurations. When the B cells are initially plated at a density greater than one cell per well, then the methods of the present invention may include the step of subsequently diluting cells in a well identified as producing an antigen-specific antibody, until a single cell specificity per well is achieved, thereby facilitating the identification of the B cell that produces the antigen-specific antibody. Cell supernatants or a portion thereof and/or cells may be frozen and stored for future testing and later recovery of antibody polynucleotides.
In certain embodiments, the B cells are cultured under conditions that favor the production of antibodies by the B cells. For example, the B cells may be cultured under conditions favorable for B cell proliferation and differentiation to yield antibody-producing plasmablast, plasmacytes, or plasma cells. In particular embodiments, the B cells are cultured in the presence of a B cell mitogen, such as lipopolysaccharide (LPS) or CD40 ligand. In one specific embodiment, B cells are differentiated to antibody-producing cells by culturing them with feed cells and/or other B cell activators, such as CD40 ligand.
Cell culture supernatants or antibodies obtained therefrom may be tested for their ability to bind to a target antigen, using routine methods available in the art, including those described herein. In particular embodiments, culture supernatants are tested for the presence of antibodies that bind to a target antigen using high-throughput methods. For example, B cells may be cultured in multi-well microtitre dishes, such that robotic plate handlers may be used to simultaneously sample multiple cell supernatants and test for the presence of antibodies that bind to a target antigen. In particular embodiments, antigens are bound to beads, e.g., paramagnetic or latex beads) to facilitate the capture of antibody/antigen complexes. In other embodiments, antigens and antibodies are fluorescently labeled (with different labels) and FACS analysis is performed to identify the presence of antibodies that bind to target antigen. In one embodiment, antibody binding is determined using FMATTM analysis and instrumentation (Applied Biosystems, Foster City, Calif.). FMATTM is a fluorescence macro-confocal platform for high-throughput screening, which mix-and-read, non-radioactive assays using live cells or beads.
In the context of comparing the binding of an antibody to a particular target antigen (e.g., a biological sample such as infected tissue or cells, or infectious agents) as compared to a control sample (e.g., a biological sample such as uninfected cells, or a different infectious agent), in various embodiments, the antibody is considered to preferentially bind a particular target antigen if at least two-fold, at least three-fold, at least five-fold, or at least ten-fold more antibody binds to the particular target antigen as compared to the amount that binds a control sample.
Polynucleotides encoding antibody chains, variable regions thereof, or fragments thereof, may be isolated from cells utilizing any means available in the art. In one embodiment, polynucleotides are isolated using polymerase chain reaction (PCR), e.g., reverse transcription-PCR (RT-PCR) using oligonucleotide primers that specifically bind to heavy or light chain encoding polynucleotide sequences or complements thereof using routine procedures available in the art. In one embodiment, positive wells are subjected to whole well RT-PCR to amplify the heavy and light chain variable regions of the IgG molecule expressed by the clonal daughter plasma cells. These PCR products may be sequenced.
The resulting PCR products encoding the heavy and light chain variable regions or portions thereof are then subcloned into human antibody expression vectors and recombinantly expressed according to routine procedures in the art (see, e.g., U.S. Pat. No. 7,112,439). The nucleic acid molecules encoding a tumor-specific antibody or fragment thereof, as described herein, may be propagated and expressed according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection. Thus, in certain embodiments expression of an antibody fragment may be preferred in a prokaryotic host cell, such as Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol. 178:497-515 (1989)). In certain other embodiments, expression of the antibody or an antigen-binding fragment thereof may be preferred in a eukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris); animal cells (including mammalian cells); or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma, COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells. By methods known to those having ordinary skill in the art and based on the present disclosure, a nucleic acid vector may be designed for expressing foreign sequences in a particular host system, and then polynucleotide sequences encoding the tumor-specific antibody (or fragment thereof) may be inserted. The regulatory elements will vary according to the particular host.
One or more replicable expression vectors containing a polynucleotide encoding a variable and/or constant region may be prepared and used to transform an appropriate cell line, for example, a non-producing myeloma cell line, such as a mouse NSO line or a bacterium, such as E. coli, in which production of the antibody will occur. In order to obtain efficient transcription and translation, the polynucleotide sequence in each vector should include appropriate regulatory sequences, particularly a promoter and leader sequence operatively linked to the variable domain sequence. Particular methods for producing antibodies in this way are generally well known and routinely used. For example, molecular biology procedures are described by Sambrook et al. (Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; see also Sambrook et al., 3rd ed., Cold Spring Harbor Laboratory, New York, (2001)). While not required, in certain embodiments, regions of polynucleotides encoding the recombinant antibodies may be sequenced. DNA sequencing can be performed as described in Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463 (1977)) and the Amersham International plc sequencing handbook and including improvements thereto.
In particular embodiments, the resulting recombinant antibodies or fragments thereof are then tested to confirm their original specificity and may be further tested for pan-specificity, e.g., with related infectious agents. In particular embodiments, an antibody identified or produced according to methods described herein is tested for cell killing via antibody dependent cellular cytotoxicity (ADCC) or apoptosis, and/or well as its ability to internalize.
Polynucleotides
The present invention, in other aspects, provides polynucleotide compositions. In preferred embodiments, these polynucleotides encode a polypeptide of the invention, e.g., a region of a variable chain of an antibody that binds to Influenza A, M2, M2e, or HA (soluble or recombinant). Polynucleotides of the invention are single-stranded (coding or antisense) or double-stranded DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include, but are not limited to, HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Alternatively, or in addition, coding or non-coding sequences are present within a polynucleotide of the present invention. Also alternatively, or in addition, a polynucleotide is linked to other molecules and/or support materials of the invention. Polynucleotides of the invention are used, e.g., in hybridization assays to detect the presence of an Influenza A antibody in a biological sample, and in the recombinant production of polypeptides of the invention.
Therefore, according to another aspect of the present invention, polynucleotide compositions are provided that include some or all of a polynucleotide sequences set forth herein, complements of these polynucleotide sequences, and degenerate variants of these polynucleotide sequences. In certain preferred embodiments, the polynucleotide sequences set forth herein encode polypeptides capable of preferentially binding a Influenza A-infected cell as compared to a normal control uninfected cell, including a polypeptide having a sequence set forth herein. Furthermore, the invention includes all polynucleotides that encode any polypeptide of the present invention.
In other related embodiments, the invention provides polynucleotide variants having substantial identity to the sequences set forth herein, for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this invention, as determined using the methods described herein, (e.g., BLAST analysis using standard parameters). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
Typically, polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenic binding properties of the polypeptide encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein. In additional embodiments, the present invention provides polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. As used herein, the term “intermediate lengths” is meant to describe any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.
In another embodiment of the invention, polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS. One skilled in the art will understand that the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65° C. or 65-70° C.
In preferred embodiments, the polypeptide encoded by the polynucleotide variant or fragment has the same binding specificity (i.e., specifically or preferentially binds to the same epitope or Influenza A strain) as the polypeptide encoded by the native polynucleotide. In certain preferred embodiments, the polynucleotides described above, e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that have a level of binding activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.
The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. A nucleic acid fragment of almost any length is employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are included in many implementations of this invention.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are multiple nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that encode a polypeptide of the present invention but which vary due to differences in codon usage are specifically contemplated by the invention. Further, alleles of the genes including the polynucleotide sequences provided herein are within the scope of the invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
In certain embodiments of the present invention, mutagenesis of the disclosed polynucleotide sequences is performed in order to alter one or more properties of the encoded polypeptide, such as its binding specificity or binding strength. Techniques for mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. A mutagenesis approach, such as site-specific mutagenesis, is employed for the preparation of variants and/or derivatives of the polypeptides described herein. By this approach, specific modifications in a polypeptide sequence are made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.
Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences include the nucleotide sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations are employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity; composition, stability, or primary sequence of the encoded polypeptide.
In other embodiments of the present invention, the polynucleotide sequences provided herein are used as probes or primers for nucleic acid hybridization, e.g., as PCR primers. The ability of such nucleic acid probes to specifically hybridize to a sequence of interest enable them to detect the presence of complementary sequences in a given sample. However, other uses are also encompassed by the invention, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions. As such, nucleic acid segments of the invention that include a sequence region of at least about 15 nucleotides long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein is particularly useful. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) including full length sequences, and all lengths in between, are also used in certain embodiments.
Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting, and/or primers for use in, e.g., polymerase chain reaction (PCR). The total size of fragment, as well as the size of the complementary stretch(es), ultimately depends on the intended use or application of the particular nucleic acid segment. Smaller fragments are generally used in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 15 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 12 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. Nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired, are generally preferred.
Hybridization probes are selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequences set forth herein, or to any continuous portion of the sequences, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences is governed by various factors. For example, one may wish to employ primers from towards the termini of the total sequence.
Polynucleotide of the present invention, or fragments or variants thereof, are readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments are obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Pat. No. 4,683,202, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Vectors, Host Cells and Recombinant Methods
The invention provides vectors and host cells comprising a nucleic acid of the present invention, as well as recombinant techniques for the production of a polypeptide of the present invention. Vectors of the invention include those capable of replication in any type of cell or organism, including, e.g., plasmids, phage, cosmids, and mini chromosomes. In various embodiments, vectors comprising a polynucleotide of the present invention are vectors suitable for propagation or replication of the polynucleotide, or vectors suitable for expressing a polypeptide of the present invention. Such vectors are known in the art and commercially available.
Polynucleotides of the present invention are synthesized, whole or in parts that are then combined, and inserted into a vector using routine molecular and cell biology techniques, including, e.g., subcloning the polynucleotide into a linearized vector using appropriate restriction sites and restriction enzymes. Polynucleotides of the present invention are amplified by polymerase chain reaction using oligonucleotide primers complementary to each strand of the polynucleotide. These primers also include restriction enzyme cleavage sites to facilitate subcloning into a vector. The replicable vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, and one or more marker or selectable genes.
In order to express a polypeptide of the present invention, the nucleotide sequences encoding the polypeptide, or functional equivalents, are inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art are used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.
A variety of expression vector/host systems are utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Within one embodiment, the variable regions of a gene expressing a monoclonal antibody of interest are amplified from a hybridoma cell using nucleotide primers. These primers are synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources (see, e.g., Stratagene (La Jolla, Calif.), which sells primers for amplifying mouse and human variable regions. The primers are used to amplify heavy or light chain variable regions, which are then inserted into vectors such as ImmunoZAPTM H or ImmunoZAPTM L (Stratagene), respectively. These vectors are then introduced into E. coli, yeast, or mammalian-based systems for expression. Large amounts of a single-chain protein containing a fusion of the VH and VL domains are produced using these methods (see Bird et al., Science 242:423-426 (1988)).
The “control elements” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector, e.g., enhancers, promoters, 5′ and 3′ untranslated regions, that interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, is used.
Examples of promoters suitable for use with prokaryotic hosts include the phoa promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also usually contain a Shine-Dalgarno sequence operably linked to the DNA encoding the polypeptide. Inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like are used.
A variety of promoter sequences are known for eukaryotes and any are used according to the present invention. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. Polypeptide expression from vectors in mammalian host cells aer controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker. One example of a suitable expression vector is pcDNA-3.1 (Invitrogen, Carlsbad, Calif.), which includes a CMV promoter.
A number of viral-based expression systems are available for mammalian expression of polypeptides. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus that is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
In bacterial systems, any of a number of expression vectors are selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are desired, vectors that direct high level expression of fusion proteins that are readily purified are used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase, so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega, Madison, Wis.) are also 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 to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH are used. Examples of other suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogcnase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. Other yeast promoters that are inducible promoters having the additional advantage of transcription controlled by growth conditions include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.
In cases where plant expression vectors are used, the expression of sequences encoding polypeptides are driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV are used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters are used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J., et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).
An insect system is also used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide are cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence renders the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses are then used to infect, for example, S. frugiperda cells or Trichoplusia larvae, in which the polypeptide of interest is expressed (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).
Specific initiation signals are also used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon are provided. Furthermore, the initiation codon is in the correct reading frame to ensure correct translation of the inserted polynucleotide. Exogenous translational elements and initiation codons are of various origins, both natural and synthetic.
Transcription of a DNA encoding a polypeptide of the invention is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are known, including, e.g., those identified in genes encoding globin, elastase, albumin, α-fetoprotein, and insulin. Typically, however, an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer is spliced into the vector at a position 5′ or 3′ to the polypeptide-encoding sequence, but is preferably located at a site 5′ from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-PSCA antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.
Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, plant or higher eukaryote cells described above. Examples of suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K lactis, K. fragilis (ATCC 12,424), K bulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris. (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. 
In certain embodiments, a host cell strain is chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves a “prepro” form of the protein is also used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, are chosen to ensure the correct modification and processing of the foreign protein.
Methods and reagents specifically adapted for the expression of antibodies or fragments thereof are also known and available in the art, including those described, e.g., in U.S. Pat. Nos. 4,816,567 and 6,331,415. In various embodiments, antibody heavy and light chains, or fragments thereof, are expressed from the same or separate expression vectors. In one embodiment, both chains are expressed in the same cell, thereby facilitating the formation of a functional antibody or fragment thereof.
Full length antibody, antibody fragments, and antibody fusion proteins are produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in infected cell destruction. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, which describes translation initiation region (TIR) and signal sequences for optimizing expression and secretion. After expression, the antibody is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out using a process similar to that used for purifying antibody expressed e.g., in CHO cells.
Suitable host cells for the expression of glycosylated polypeptides and antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopicius (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses are used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco are also utilized as hosts.
Methods of propagation of antibody polypeptides and fragments thereof in vertebrate cells in culture (tissue culture) are encompassed by the invention. Examples of mammalian host cell lines used in the methods of the invention are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells are transformed with the above-described expression or cloning vectors for polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines that stably express a polynucleotide of interest are transformed using expression vectors that contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells are allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells are proliferated using tissue culture techniques appropriate to the cell type.
A plurality of selection systems are used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes that are employed in tk− or aprt− cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance is used as the basis for selection; for example, dhfr, which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al.(1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, and hisD allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). The use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression is confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences are identified by the absence of marker gene function. Alternatively, a marker gene is placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
Alternatively, host cells that contain and express a desired polynucleotide sequence are identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include, for example, membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Nonlimiting examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide is preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).
Various labels and conjugation techniques are known by those skilled in the art and are used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof are cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and are used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures are conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which are used include, but are not limited to, radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
The polypeptide produced by a recombinant cell is secreted or contained intracellularly depending on the sequence and/or the vector used. Expression vectors containing polynucleotides of the invention are designed to contain signal sequences that direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
In certain embodiments, a polypeptide of the invention is produced as a fusion polypeptide further including a polypeptide domain that facilitates purification of soluble proteins. Such purification-facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Amgen, Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide are used to facilitate purification. An exemplary expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors used for producing fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).
In certain embodiments, a polypeptide of the present invention is fused with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells, the signal sequence is selected, for example, from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence is selected from, e.g., the yeast invertase leader, α factor leader (including Saccharomyces and Kluyveromyces α factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.
When using recombinant techniques, the polypeptide or antibody is produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the polypeptide or antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris is removed by centrifugation. Where the polypeptide or antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Optionally, a protease inhibitor such as PMSF is included in any of the foregoing steps to inhibit proteolysis and antibiotics are included to prevent the growth of adventitious contaminants.
The polypeptide or antibody composition prepared from the cells are purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the polypeptide or antibody. Protein A is used to purify antibodies or fragments thereof that are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the polypeptide or antibody comprises a CH 3 domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the polypeptide or antibody to be recovered.
Following any preliminary purification step(s), the mixture comprising the polypeptide or antibody of interest and contaminants are subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
Pharmaceutical Compositions
The invention further includes pharmaceutical formulations including a polypeptide, antibody, or modulator of the present invention, at a desired degree of purity, and a pharmaceutically acceptable carrier, excipient, or stabilizer (Remingion's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). In certain embodiments, pharmaceutical formulations are prepared to enhance the stability of the polypeptide or antibody during storage, e.g., in the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, e.g., buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). In certain embodiments, the therapeutic formulation preferably comprises the polypeptide or antibody at a concentration of between 5-200 mg/ml, preferably between 10-100 mg/ml.
The formulations herein also contain one or more additional therapeutic agents suitable for the treatment of the particular indication, e.g., infection being treated, or to prevent undesired side-effects. Preferably, the additional therapeutic agent has an activity complementary to the polypeptide or antibody of the resent invention, and the two do not adversely affect each other. For example, in addition to the polypeptide or antibody of the invention, an additional or second antibody, anti-viral agent, anti-infective agent and/or cardioprotectant is added to the formulation. Such molecules are suitably present in the pharmaceutical formulation in amounts that are effective for the purpose intended.
The active ingredients, e.g., polypeptides and antibodies of the invention and other therapeutic agents, are also entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remingion's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations are prepared. Suitable examples of sustained-release preparations include, but are not limited to, semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Nonlimiting examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxyburyric acid.
Formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile filtration membranes.
Diagnostic Uses
Antibodies and fragments thereof, and therapeutic compositions, of the invention specifically bind or preferentially bind to infected cells or tissue, as compared to normal control cells and tissue. Thus, these influenza A antibodies are used to detect infected cells or tissues in a patient, biological sample, or cell population, using any of a variety of diagnostic and prognostic methods, including those described herein. The ability of an anti-M2e or anti-HA specific antibody to detect infected cells depends upon its binding specificity, which is readily determined by testing its ability to bind to infected cells or tissues obtained from different patients, and/or from patients infected with different strains of Influenza A.
Diagnostic methods generally involve contacting a biological sample obtained from a patient, such as, e.g., blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy, with an Influenza A, e.g., human monoclonal anti-M2e or anti-HA antibody, and determining whether the antibody preferentially binds to the sample as compared to a control sample or predetermined cut-off value, thereby indicating the presence of infected cells. In particular embodiments, at least two-fold, three-fold, or five-fold more human monoclonal anti-M2e or anti-HA antibody binds to an infected cell as compared to an appropriate control normal cell or tissue sample. A pre-determined cut-off value is determined, e.g., by averaging the amount of human monoclonal anti-M2e or anti-HA antibody that binds to several different appropriate control samples under the same conditions used to perform the diagnostic assay of the biological sample being tested. Alternatively, or in addition, a hemagglutinin (HA) protein is substituted for an Influenza virus in the above method. The HA protein is presented on the surface of a virus, host cell (e.g. any mammalian cell), or in a recombinant and soluble form. In the HA version of this diagnostic method, the control protein is a denatured HA protein, a linear HA peptide, an unrelated protein of similar size and shape, but dissimilar sequence, or a pre-determined cut-off value.
Bound antibody is detected using procedures described herein and known in the art. In certain embodiments, diagnostic methods of the invention are practiced using human monoclonal anti-M2e or anti-HA antibodies that are conjugated to a detectable label, e.g., a fluorophore, to facilitate detection of bound antibody. However, they are also practiced using methods of secondary detection of the human monoclonal anti-M2e or anti-HA antibody. These include, for example, RIA, ELISA, precipitation, agglutination, complement fixation and immuno-fluorescence.
In certain procedures, the human monoclonal anti-M2e or anti-HA antibodies are labeled. The label is detected directly. Exemplary labels that are detected directly include, but are not limited to, radiolabels and fluorochromes. Alaternatively, or in addition, labels are moieties, such as enzymes, that must be reacted or derivatized to be detected. Nonlimiting examples of isotope labels are 99Tc, 14C, 131I, 125I, 3H, 32P and 35S. Fluorescent materials that are used include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, auramine, dansyl, umbelliferone, luciferia, 2,3-dihydrophthalazinediones, horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate dehydrogenase.
An enzyme label is detected by any of the currently utilized colorimetric, spectrophotometric, fluorospectro-photometric or gasometric techniques. Many enzymes which are used in these procedures are known and utilized by the methods of the invention. Nonlimiting examples are peroxidase, alkaline phosphatase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase, galactose oxidase plus peroxidase and acid phosphatase.
The antibodies are tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bid-diazotized benzadine and the like are used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. An enzyme is typically combined with an antibody using bridging molecules such as carbodiimides, periodate, diisocyanates, glutaraldehyde and the like. Various labeling techniques are described in Morrison, Methods in Enzymology 32b, 103 (1974), Syvanen et al., J. Biol. Chem. 284, 3762 (1973) and Bolton and Hunter, Biochem J. 133, 529(1973).
Human monoclonal anti-M2e or anti-HA antibodies of the present invention are capable of differentiating between patients with and patients without an Influenza A infection, and determining whether or not a patient has an infection, using the representative assays provided herein. According to one method, a biological sample is obtained from a patient suspected of having or known to have an influenza A infection. In preferred embodiments, the biological sample includes cells from the patient. The sample is contacted with a human monoclonal anti-M2e or anti-HA antibody, e.g., for a time and under conditions sufficient to allow the human monoclonal anti-M2e or anti-HA antibody to bind to infected cells present in the sample. For instance, the sample is contacted with a human monoclonal anti-M2e or anti-HA antibody for 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 3 days or any point in between. The amount of bound human monoclonal anti-M2e or anti-HA antibody is determined and compared to a control value, which may be, e.g., a pre-determined value or a value determined from normal tissue sample. An increased amount of antibody bound to the patient sample as compared to the control sample is indicative of the presence of infected cells in the patient sample.
In a related method, a biological sample obtained from a patient is contacted with a human monoclonal anti-M2e or anti-HA antibody for a time and under conditions sufficient to allow the antibody to bind to infected cells. Bound antibody is then detected, and the presence of bound antibody indicates that the sample contains infected cells. This embodiment is particularly useful when the human monoclonal anti-M2e or anti-HA antibody does not bind normal cells at a detectable level.
Different human monoclonal anti-M2e or anti-HA antibodies possess different binding and specificity characteristics. Depending upon these characteristics, particular human monoclonal anti-M2e or anti-HA antibodies are used to detect the presence of one or more strains of Influenza A. For example, certain antibodies bind specifically to only one or several strains of Influenza virus, whereas others bind to all or a majority of different strains of Influenza virus. Antibodies specific for only one strain of Influenza A are used to identify the strain of an infection.
In certain embodiments, antibodies that bind to an infected cell preferably generate a signal indicating the presence of an infection in at least about 20% of patients with the infection being detected, more preferably at least about 30% of patients. Alternatively, or in addition, the antibody generates a negative signal indicating the absence of the infection in at least about 90% of individuals without the infection being detected. Each antibody satisfies the above criteria; however, antibodies of the present invention are used in combination to improve sensitivity.
The present invention also includes kits useful in performing diagnostic and prognostic assays using the antibodies of the present invention. Kits of the invention include a suitable container comprising a human monoclonal anti-M2e or anti-HA antibody of the invention in either labeled or unlabeled form. In addition, when the antibody is supplied in a labeled form suitable for an indirect binding assay, the kit further includes reagents for performing the appropriate indirect assay. For example, the kit includes one or more suitable containers including enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and/or instructions are also included.
Therapeutic/Prophylactic Uses
Passive immunization has proven to be an effective and safe strategy for the prevention and treatment of viral diseases. (See Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of which are incorporated herein by reference)). Passive immunization using human monoclonal antibodies provide an immediate treatment strategy for emergency prophylaxis and treatment of influenza
Human monoclonal anti-M2e or anti-HA antibodies and fragments thereof, and therapeutic compositions, of the invention specifically bind or preferentially bind to infected cells, as compared to normal control uninfected cells and tissue. Thus, these human monoclonal anti-M2e or anti-HA antibodies are used to selectively target infected cells or tissues in a patient, biological sample, or cell population. In light of the infection-specific binding properties of these antibodies, the present invention provides methods of regulating (e.g., inhibiting) the growth of infected cells, methods of killing infected cells, and methods of inducing apoptosis of infected cells. These methods include contacting an infected cell with a human monoclonal anti-M2e or anti-HA antibody of the invention. These methods are practiced in vitro, ex vivo, and in vivo.
In various embodiments, antibodies of the invention are intrinsically therapeutically active. Alternatively, or in addition, antibodies of the invention are conjugated to a cytotoxic agent or growth inhibitory agent, e.g., a radioisotope or toxin, which is used in treating infected cells bound or contacted by the antibody.
In one embodiment, the invention provides methods of treating or preventing infection in a patient, including the steps of providing a human monoclonal anti-M2e or anti-HA antibody of the invention to a patient diagnosed with, at risk of developing, or suspected of having an Influenza A infection. The methods of the invention are used in the first-line treatment of the infection, follow-on treatment, or in the treatment of a relapsed or refractory infection. Treatment with an antibody of the invention is a stand alone treatment. Alternatively, treatment with an antibody of the invention is one component or phase of a combination therapy regime, in which one or more additional therapeutic agents are also used to treat the patient.
Subjects at risk for an influenza virus -related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to the influenza virus in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the influenza virus-related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
In various aspects, the human monoclonal anti-M2e or anti-HA is administered substantially contemporaneously with or following infection of the subject, i.e., therapeutic treatment. In another aspect, the antibody provides a therapeutic benefit. In various aspects, a therapeutic benefit includes reducing or decreasing progression, severity, frequency, duration or probability of one or more symptoms or complications of influenza infection, virus titer, virus replication or an amount of a viral protein of one or more influenza strains. still another aspect, a therapeutic benefit includes hastening or accelerating a subject's recovery from influenza infection.
Methods for preventing an increase in influenza virus titer, virus replication, virus proliferation or an amount of an influenza viral protein in a subject are further provided. In one embodiment, a method includes administering to the subject an amount of a human monoclonal anti-M2e or anti-HA antibody effective to prevent an increase in influenza virus titer, virus replication or an amount of an influenza viral protein of one or more influenza strains or isolates in the subject.
Methods for protecting a subject from infection or decreasing susceptibility of a subject to infection by one or more influenza strains/isolates or subtypes, i.e., prophylactic methods, are additionally provided. In one embodiment, a method includes administering to the subject an amount of human monoclonal anti-M2e or anti-HA antibody that specifically binds influenza M2 or HA, respectively, effective to protect the subject from infection, or effective to decrease susceptibility of the subject to infection, by one or more influenza strains/isolates or subtypes.
Optionally, the subject is further administered with a second agent such as, but not limited to, an influenza virus antibody, an anti-viral drug such as a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or a viral attachment inhibitor. The M2 ion channel inhibitor is for example amantadine or rimantadine. The neuraminidase inhibitor for example zanamivir, or oseltamivir phosphate.
Symptoms or complications of influenza infection that can be reduced or decreased include, for example, chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache or death.
For in vivo treatment of human and non-human patients, the patient is usually administered or provided a pharmaceutical formulation including a human monoclonal anti-M2e or anti-HA antibody of the invention. When used for in vivo therapy, the antibodies of the invention are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the patient's viral burden). The antibodies are administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibodies may be administered parenterally, when possible, at the target cell site, or intravenously. Intravenous or subcutaneous administration of the antibody is preferred in certain embodiments. Therapeutic compositions of the invention are administered to a patient or subject systemically, parenterally, or locally.
For parenteral administration, the antibodies are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate are also used. Liposomes are used as carriers. The vehicle contains minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies are typically formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
The dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection and the characteristics of the particular cytotoxic agent or growth inhibitory agent conjugated to the antibody (when used), e.g., its therapeutic index, the patient, and the patient's history. Generally, a therapeutically effective amount of an antibody is administered to a patient. In particular embodiments, the amount of antibody administered is in the range of about 0.1 mg/kg to about 50 mg/kg of patient body weight. Depending on the type and severity of the infection, about 0.1 mg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.
In one particular embodiment, an immunoconjugate including the antibody conjugated with a cytotoxic agent is administered to the patient. Preferably, the immunoconjugate is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cell to which it binds. In one embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the infected cell. Examples of such cytotoxic agents are described above and include, but are not limited to, maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.
Other therapeutic regimens are combined with the administration of the HuM2e antibody of the present invention. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect.
In certain embodiments, it is desirable to combine administration of an antibody of the invention with another antibody directed against another antigen associated with the infectious agent.
Aside from administration of the antibody protein to the patient, the invention provides methods of administration of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression “administering a therapeutically effective amount of an antibody”. See, for example, PCT Patent Application Publication WO96/07321 concerning the use of gene therapy to generate intracellular antibodies.
In another embodiment, human monoclonal anti-M2e or anti-HA antibodies of the invention are used to determine the structure of bound antigen, e.g., conformational epitopes, the structure of which is then used to develop a vaccine having or mimicking this structure, e.g., through chemical modeling and SAR methods. Such a vaccine could then be used to prevent Influenza A infection.
All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheetare incorporated herein by reference, in their entirety.
EXAMPLES
Example 1
Screening and Characterization of M2e-specific Antibodies Present in Human Plasma Using Cells Expressing Recombinant M2e Protein
Fully human monoclonal antibodies specific for M2 and capable of binding to influenza A infected cells and the influenza virus itself were identified in patient serum, as described below.
Expression of M2 in Cell Lines
An expression construct containing the M2 full length cDNA, corresponding to the derived M2 sequence found in Influenza subtype H3N2, was transfected into 293 cells.
The M2 cDNA is encoded by the following polynucleotide sequence and SEQ ID NO: 53:







ATGAGTCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGG


 


TGCAGATGCAACGATTCAAGTGATCCTCTTGTTGTTGCCGCAAGTATC


 


ATTGGGATCCTGCACTTGATATTGTGGATTCTTGATCGTCTTTTTTTC


 


AAATGCATTTATCGTCTCTTTAAACACGGTCTGAAAAGAGGGCCTTCT


 


ACGGAAGGAGTACCAGAGTCTATGAGGGAAGAATATCGAAAGGAACAG


 


CAGAGTGCTGTGGATGCTGACGATAGTCATTTTGTCAACATAGAGCTGG


 


AG






The cell surface expression of M2 was confirmed using the anti-M2e peptide specific MAb 14C2. Two other variants of M2, from A/Hong Kong/483/1997 (HK483) and A/Vietnam/1203/2004 (VN1203), were used for subsequent analyses, and their expression was determined using M2e-specific monoclonal antibodies of the present invention, since 14C2 binding may be abrogated by the various amino acid substitutions in M2e.
Screening of Antibodies in Peripheral Blood
Over 120 individual plasma samples were tested for antibodies that bound M2. None of them exhibited specific binding to the M2e peptide. However, 10% of the plasma samples contained antibodies that bound specifically to the 293-M2 H3N2 cell line. This indicates that the antibodies could be categorized as binding to conformational determinants of an M2 homotetramer, and binding to conformational determinants of multiple variants of the M2 homotetramer; they could not be specific for the linear M2e peptide.
Characterization of Anti-M2 MAbs
The human MAbs identified through this process proved to bind to conformational epitopes on the M2 homotetramer. They bound to the original 293-M2 transfectant, as well as to the two other cell-expressed M2 variants. The 14C2 MAb, in addition to binding the M2e peptide, proved to be more sensitive to the M2 variant sequences. Moreover, 14C2 does not readily bind influenza virions, while the conformation specific anti-M2 MAbs did.
These results demonstrate that the methods of the invention provide for the identification of M2 MAbs from normal human immune responses to influenza without a need for specific immunization of M2. If used for immunotherapy, these fully human MAbs have the potential to be better tolerated by patients that humanized mouse antibodies. Additionally, and in contrast to 14C2 and the Gemini Biosciences MAbs, which bind to linear M2e peptide, the MAbs of the invention bind to conformational epitopes of M2, and are specific not only for cells infected with A strain influenza, but also for the virus itself. Another advantage of the MAbs of the invention is that they each bind all of the M2 variants yet tested, indicating that they are not restricted to a specific linear amino acid sequence.
Example 2
Identification of M2-Specific Antibodies
Mononuclear or B cells expressing three of the MAbs identified in human serum as described in Example 1 were diluted into clonal populations and induced to produce antibodies. Antibody containing supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2. Supernatants which showed positive staining/binding were re-screened again on 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2 and on vector alone transfected cells as a control.
The variable regions of the antibodies were then rescue cloned from the B cell wells whose supernatants showed positive binding. Transient transfections were performed in 293 FT cells to reconstitute and produce these antibodies. Reconstituted antibody supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein as detailed above to identify the rescued anti-M2E antibodies. Three different antibodies were identified: 8i10, 21B15 and 23K12. A fourth additional antibody clone was isolated by the rescue screens, 4C2. However, it was not unique and had the exact same sequence as clone 8i10 even though it came from a different donor than clone 8i10.
The sequences of the kappa and gamma variable regions of these antibodies are provided below.
Clone 8i10 (Corresponds to TCN-032):
The Kappa LC variable region of the anti M2 clone 8i10 was cloned as Hind III to BsiW1 fragment (see below), and is encoded by the following polynucleotide sequences, and SEQ ID NO: 54 (top) and SEQ ID NO: 55 (bottom):








HindIII



AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTG


TTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCAC


 


CCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCA


GGTCTACACTGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGT


 


TCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCC


AGTGAACGGCCCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGGG


 


CTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGAT


GATTCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGTCACCTA


 


CTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC


GACCCTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAATGATGACAGTTG


 


                                                     BsiWI


AGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAACGTACG


TCTCAATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTCTAGTTTGCATGC






The translation of the 8i10 Kappa LC variable region is as follows, polynucleotide sequence (above, SEQ ID NO: 54, top) and amino acid sequence (below, corresponding to residues 1-131 of SEQ ID NO: 56):








HindIII



AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTG


            M  D  M  R  V  L  A  Q  L  L  G  L  L  L  L  W  L  R  G


 


CCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCA


A  R  C  D  I  Q  M  T  Q  S  P  S  S  L  S  A  S  V  G  D  R  V  T


 


TCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCC


I  T  C  R  A  S  Q  N  I  Y  K  Y  L  N  W  Y  Q  Q  R  P  G  K  A


 


CTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGAT


P  K  G  L  I  S  A  A  S  G  L  Q  S  G  V  P  S  R  F  S  G  S  G


 


CTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC


S  G  T  D  F  T  L  T  I  T  S  L  Q  P  E  D  F  A  T  Y  Y  C  Q


 


                                                     BsiWI


AGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAACGTACG


Q  S  Y  S  P  P  L  T  F  G  G  G  T  R  V  E  I  K  R  T






The amino acid sequence of the 8i10 Kappa LC variable region is as follows, with specific domains identified below (CDR sequences defined according to Kabat methods):









M D M R V L A Q L L G L L L L W L R G A R C
VK leader (SEQ ID NO: 57)



 


D I Q M T Q S P S S L S A S V G D R V T I T C
FR1 (SEQ ID NO: 58)


 


R A S Q N I Y K Y L N
CDR1(SEQ ID NO: 59)


 


W Y Q Q R P G K A P K G L I S
FR2 (SEQ ID NO: 60)


 


A A S G L Q S
CDR2 (SEQ ID NO: 61)


 


G V P S R F S G S G S G T D F T L T I T S L Q P E D F A T Y Y C
FR3(SEQ ID NO: 62)


 


Q Q S Y S P P L T
CDR3 (SEQ ID NO: 63)


 


F G G G T R V E I K
FR4 (SEQ ID NO: 64)


 


R T
Start of Kappa constant region






The following is an example of the Kappa LC variable region of 8i10 cloned into the expression vector pcDNA3.1 which already contained the Kappa LC constant region (upper polynucleotide sequence corresponds to SEQ ID NO: 65, lower polynucleotide sequence corresponds to SEQ ID NO: 66, amino acid sequence corresponds to SEQ ID NO: 56). Bases in black represents pcDNA3.1 vector sequences, underlined bases represent the cloned antibody sequences. The antibodies described herein have also been cloned into the expression vector pCEP4.








                                     NheI(894) PmeI (909) HindIII(910)



TCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTCCACCATG


AGCTTTAATTATGCTGAGTGATATCCCTCTGGGTTCGACCGATCGCAAATTTGAATTCGAAGGTGGTAC


                                                                   M


 


GACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGTG


CTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCACGGTCTACAC


 D  M  R  V  L  A  Q  L  L  G  L  L  L  L  W  L  R  G  A  R  C


 


ACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAC


TGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGTAGTG


D  I  Q  M  T  Q  S  P  S  S  L  S  A  S  V  G  D  R  V  T  I  T 


 


TTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCC


AACGGCCCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGG


  C  R  A  S  Q  N  I  Y  K  Y  L  N  W  Y  Q  Q  R  P  G  K  A 


 


CCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCA


GGATTCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGT


 P  K  G  L  I  S  A  A  S  G  L  Q  S  G  V  P  S  R  F  S  G


 


GTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTA


CACCTAGACCCTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAAT


S  G  S  G  T  D  F  T  L  T  I  T  S  L  Q  P  E  D  F  A  T  Y


 


CTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAA


GATGACAGTTGTCTCAATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTCTAGTTT


  Y  C  Q  Q  S  Y  S  P  P  L  T  F  G  G  G  T  R  V  E  I  K


 


BsiWI


CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCT


GCATGCCACCGACGTGGTAGACAGAAGTAGAAGGGCGGTAGACTACTCGTCAACTTTAGACCTTGACGGA


 R  T  V  A  A  P  S  V  F  I  F  P  P  S  D  E  Q  L  K  S  G  T  A


 


CTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT


GACAACACACGGACGACTTATTGAAGATAGGGTCTCTCCGGTTTCATGTCACCTTCCACCTATTGCGGGA


S  V  V  C  L  L  N  N  F  Y  P  R  E  A  K  V  Q  W  K  V  D  N  A  L


 


        hu Kappa constant


CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC


GGTTAGCCCATTGAGGGTCCTCTCACAGTGTCTCGTCCTGTCGTTCCTGTCGTGGATGTCGGAGTCGTCG


  Q  S  G  N  S  Q  E  S  V  T  E  Q  D  S  K  D  S  T  Y  S  L  S  S


 


ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC


TGGGACTGCGACTCGTTTCGTCTGATGCTCTTTGTGTTTCAGATGCGGACGCTTCAGTGGGTAGTCCCGG


 T  L  T  L  S  K  A  D  Y  E  K  H  K  V  Y  A  C  E  V  T  H  Q  G


 


                                                       DraI(1641)


                                               XbaI(1636) ApaI(1642)


TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAGGGTCTAGAGGGCCCGTTTAAA


ACTCGAGCGGGCAGTGTTTCTCGAAGTTGTCCCCTCTCACAATCTCCCAGATCTCCCGGGCAAATTT


L  S  S  P  V  T  K  S  F  N  R  G  E  C






The 8i10 Gamma HC variable region was cloned as a Hind III to Xho 1 fragment, and is encoded the following polynucleotide sequences, and SEQ ID NO: 67 (top) and SEQ ID NO: 68 (bottom).








HindIII



AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGT


TTCGAAGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCA


 


CCTGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTG


GGACAGGGTCCACGTTAACGTCCTCAGCCCGGGTCCTGACCACTTCGGAAGCCTCTGGGAC


 


TCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGC


AGGGAGTGGACGTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCG


 


AGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTA


TCAGGGGTCCCTTCCCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCAT


 


CAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCC


GTTAGGGAGGGAGTTCTCGGCGCAGTGGTATAGTGTTCTGTGAAGGTTCTCAGTCCAGAGG


 


CTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTT


GACTGCTACTCGAGACACTGGCGACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAA


 


                                                       Xhol


GTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAG


CATCACCACCAATGACATAGGAACTGATGACCCCGGTCCCTTGGGACCAGTGGCAGAGCTC






The translation of the 8i10 Gamma HC is as follows, polynucleotide sequence (above, SEQ ID NO: 67, top) and amino acid sequence (below, corresponding to residues 1-138 of SEQ ID NO: 69):








HindIII



AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTC


            M  K  H  L  W  F  F  L  L  L  V  A  A  P  S  W  V


 


CTGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTG


 L  S  Q  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  E  T  L


 


TCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGG


 S  L  T  C  T  V  S  G  S  S  I  S  N  Y  Y  W  S  W  I  R


 


CAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAG


 Q  S  P  G  K  G  L  E  W  I  G  F  I  Y  Y  G  G  N  T  K


 


TACAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTC


 Y  N  P  S  L  K  S  R  V  T  I  S  Q  D  T  S  K  S  Q  V


 


TCCCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCG


 S  L  T  M  S  S  V  T  A  A  E  S  A  V  Y  F  C  A  R  A


 


                                                           XhoI


TCTTGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC


 S  C  S  G  G  Y  C  I  L  D  Y  W  G  Q  G  T  L  V  T  V


 


TCGAG


 S






The amino acid sequence of the 8i10 Gamma HC is as follows with specific domains identified below (CDR sequences defined according to Kabat methods):









M K H L W F F L L L V A A P S W V L S
VH leader (SEQ ID NO: 70)



 


Q V Q L Q E S G P G L V K P S E T L S L T C T V S G S S I S
FR1 (SEQ ID NO: 71)


 


N Y Y W S
CDR1(SEQ ID NO: 72)


 


W I R Q S P G K G L E W I G
FR2 (SEQ ID NO: 73)


 


F I Y Y G G N T K Y N P S L K S
CDR2 (SEQ ID NO: 74)


 


R V T I S Q D T S K S Q V S L T M S S V T A A E S A V Y F C A R
FR3(SEQ ID NO: 75)


 


A S C S G G Y C I L D
CDR3 (SEQ ID NO: 76)


 


Y W G Q G T L V T V S
FR4 (SEQ ID NO: 77)


 


YWGQGTLVTVSS
Long FR4 (SEQ ID NO: 266)






The following is an example of the Gamma HC variable region of 8i10 cloned into the expression vector pcDNA3.1 which already contained the Gamma HC constant region (upper polynucleotide sequence corresponds to SEQ ID NO: 78, lower polynucleotide sequence corresponds to SEQ ID NO: 79, amino acid sequence corresponds to SEQ ID NO: 69). Bases in black represents pcDNA3.1 vector sequences, underlined bases represent the cloned antibody sequences.








                                                   PmeI (900)



                                             NheI (894)   Hind(910)


TGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCT


ACCGAATAGCTTTAATTATGCTGAGTGATATCCCTCTGGGTTCGACCGATCGCAAATTTGAATTCGA


 


TCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTCCTGTCCC


AGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCAGGACAGGG


       M  K  H  L  W  F  F  L  L  L  V  A  A  P  S  W  V  L  S


 


AGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTG


TCCACGTTAACGTCCTCAGCCCGGGTCCTGACCACTTCGGAAGCCTCTGGGACAGGGAGTGGAC


Q  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  E  T  L  S  L  T  C


 


CACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAG


GTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCGTCAGGGGTCCCTTC


  T  V  S  G  S  S  I  S  N  Y  Y  W  S  W  I  R  Q  S  P  G  K


 


GGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGA


CCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCATGTTAGGGAGGGAGTTCT


 G  L  E  W  I  G  F  I  Y  Y  G  G  N  T  K  Y  N  P  S  L  K


 


GCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTGAC


CGGCGCAGTGGTATAGTGTTCTGTGAAGGTTCTCAGTCCAGAGGGACTGCTACTCGAGACACTG


S  R  V  T  I  S  Q  D  T  S  K  S  Q  V  S  S  L  M  S  S  V  T


 


CGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTTACTGTATCCTT


GCGACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAACATCACCACCAATGACATAGGAA


  A  A  E  S  A  V  Y  F  C  A  R  A  S  C  S  G  G  Y  C  I  L


 


                                XhoI (1331)


GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGAGCCTCCACCAAGGGCCCATCGGTCTTC


CTGATGACCCCGGTCCCTTGGGACCAGTGGCAGAGCTCTCGGAGGTGGTTCCCGGGTAGCCAGAAG


 D  Y  W  G  Q  G  T  L  V  T  V  S  R  A  S  T  K  G  P  S  V  F


 


CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT


GGGGACCGTGGGAGGAGGTTCTCGTGGAGACCCCCGTGTCGCCGGGACCCGACGGACCAGTTCCTGA


 P  L  A  P  S  S  K  S  T  S  G  G  T  A  A  L  G  C  L  V  K  D


 


ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC


TGAAGGGGCTTGGCCACTGCCACAGCACCTTGAGTCCGCGGGACTGGTCGCCGCACGTGTGGAAGGG


Y  F  P  E  P  V  T  V  S  W  N  S  G  A  L  T  S  G  V  H  T  F  P


 


GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG


CCGACAGGATGTCAGGAGTCCTGAGATGAGGGAGTCGTCGCACCACTGGCACGGGAGGTCGTCGAAC


  A  V  L  Q  S  S  G  L  Y  S  L  S  S  V  V  T  V  P  S  S  S  L


 


GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTG


CCGTGGGTCTGGATGTAGACGTTGCACTTAGTGTTCGGGTCGTTGTGGTTCCACCTGTTCTCTCAAC


 G  T  Q  T  Y  I  C  N  V  N  H  K  P  S  N  T  K  V  D  K  R  V


 


AGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC


TCGGGTTTAGAACACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGTGGACTTGAGGACCCCCCTGG


E  P  K  S  C  D  K  T  H  T  C  P  P  C  P  A  P  E  L  L  G  G  P 


 


GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA


CAGTCAGAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAGTACTAGAGGGCCTGGGGACTCCAGTGT


  S  V  F  L  F  P  P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T


 


TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG


ACGCACCACCACCTGCACTCGGTGCTTCTGGGACTCCAGTTCAAGTTGACCATGCACCTGCCGCACC


 C  V  V  V  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V


 


AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT


TCCACGTATTACGGTTCTGTTTCGGCGCCCTCCTCGTCATGTTGTCGTGCATGGCACACCAGTCGCA


E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V


 


CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC


GGAGTGGCAGGACGTGGTCCTGACCGACTTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCGG


  L  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K  V  S  N  K  A


 


CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA


GAGGGTCGGGGGTAGCTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGTCCACATGT


 L  P  A  P  I  E  K  T  I  S  K  A  K  G  Q  P  R  E  P  Q  V  Y 


 


CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT


GGGACGGGGGTAGGGCCCTCCTCTACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTTCCGAA


T  L  P  P  S  R  E  E  M  T  K  N  Q  V  S  L  T  C  L  V  K  G  F


 


CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG


GATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATGTTCTGGTGC


  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T


 


CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGT


GGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATATCGTTCGAGTGGCACCTGTTCTCGTCCA


 P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R


 


GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA


CCGTCGTCCCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGAGACGTGTTGGTGATGTGCGTCTT


W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K


 


                                   ApaI (2339)


                                  DraII(2338)


                            XhoI (2333)   PmeI (2345)


GAGCCTCTCCCTGTCTCCGGGTAAATGAGTTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGA


CTCGGAGAGGGACAGAGGCCCATTTACTCAAGATCTCCCGGGCAAATTTGGGCGACTAGTCGGAGCT


  S  L  S  L  S  P  G  K






The framework 4 (FR4) region of the Gamma HC normally ends with two serines (SS), so that the full framework 4 region should be WGQGTLVTVSS (SEQ ID NO: 80). The accepting Xho 1 site and one additional base downstream of the Xho1 site in the vector; in which the Gamma HC constant region that the Gamma HC variable regions are cloned, supplies the last bases, which encode this final amino acid of framework 4. However, the original vector did not adjust for the silent mutation made when the Xho1 site (CTCGAG, SEQ ID NO: 81) was created and contained an “A” nucleotide downstream of the Xho1 site, which caused an amino acid change at the end of framework 4: a serine to arginine (S to R) substitution present in all the working Gamma HC clones. Thus, the full framework 4 region reads WGQGTLVTVSR (SEQ ID NO: 82). Future constructs are being created wherein the base downstream of the Xho 1 site is a “C” nucleotide. Thus, the creation of the Xho 1 site used for cloning of the Gamma HC variable region sequences in alternative embodiments is a silent mutation and restores the framework 4 amino acid sequence to its proper WGQGTLV TVSS (SEQ ID NO: 80). This is true for all M2 Gamma HC clones described herein.
Clone 21B15:
The Kappa LC variable region of the anti M2 clone 21B15 was cloned as Hind III to BsiW1 fragment, and is encoded by the following polynucleotide sequences and SEQ ID NO: 83 and SEQ ID NO: 84:








HindIII



AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGC


TTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCACG


 


CAGATGTGACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC


GTCTACACTGTAGGTCCACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGTAG


 


ACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCCTA


TGAACGGCGCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGGGGAT


 


AGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGG


TCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGTCACCTAGACC


 


GACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGT


CTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAATGATGACAGTTGTCTCA


 


                                                BsiWI


TACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGATATCAAACGTACG


ATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTATAGTTTGCATGC






The translation of the 21B15 Kappa LC variable region is as follows, polynucleotide sequence (above, SEQ ID NO: 83, top) and amino acid sequence (below, corresponding to SEQ ID NO: 298):








HindIII



AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGT


            M  D  M  R  V  L  A  Q  L  L  G  L  L  L  L  W  L  R  G


 


GCCAGATGTGACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC


 A  R  C  D  I  Q  V  T  Q  S  P  S  S  L  S  A  S  V  G  D  R  V  T


 


ATCACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCC


 I  T  C  R  A  S  Q  N  I  Y  K  Y  L  N  W  Y  Q  Q  R  P  G  K  A


 


CCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGA


 P  K  G  L  I  S  A  A  S  G  L  Q  S  G  V  P  S  R  F  S  G  S  G


 


TCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAA


 S  G  T  D  F  T  L  T  I  T  S  L  Q  P  E  D  F  A  T  Y  Y  C  Q


 


                                                      BsiWI


CAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGATATCAAACGTACG


 Q  S  Y  S  P  P  L  T  F  G  G  G  T  R  V  D  I  K  R  T






The amino acid sequence of the 21B15 Kappa LC variable region is as follows, with specific domains identified below (CDR sequences defined according to Kabat methods):









M D M R V L A Q L L G L L L L W L R G A R C
VK leader (SEQ ID NO: 57)



 


D I Q V T Q S P S S L S A S V G D R V T I T C
FR1 (SEQ ID NO: 58)


 


R A S Q N I Y K Y L N
CDR1 (SEQ ID NO: 59)


 


W Y Q Q R P G K A P K G L I S
FR2 (SEQ ID NO: 60)


 


A A S G L Q S
CDR2 (SEQ ID NO: 61)


 


G V P S R F S G S G S G T D F T L T I T S L Q P E D F A T Y Y C
FR3 (SEQ ID NO: 62)


 


Q Q S Y S P P L T
CDR3 (SEQ ID NO: 63)


 


F G G G T R V D I K
FR4 (SEQ ID NO: 64)


 


R T
Start of Kappa constant region






The primer used to clone the Kappa LC variable region extended across a region of diversity and had wobble base position in its design. Thus, in the framework 4 region a D or E amino acid could occur. In some cases, the amino acid in this position in the rescued antibody may not be the original parental amino acid that was produced in the B cell. In most kappa LCs the position is an E. Looking at the clone above (21B15) a D in framework 4 (DIKRT) (SEQ ID NO: 544) was observed. However, looking at the surrounding amino acids, this may have occurred as the result of the primer and may be an artifact. The native antibody from the B cell may have had an E in this position.
The 21B15 Gamma HC variable region was cloned as a Hind III to Xho 1 fragment, and is encoded by the following polynucleotide sequences and SEQ ID NO: 85 (top), and SEQ ID NO: 86 (bottom):








HindIII



AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTCC


TTCGAAGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCAGG


 


TGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCC


ACAGGGTCCACGTTAACGTCCTCAGCCCGGGTCCTGACCACTTCGGAAGCCTCTGGGACAGGG


 


TCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCC


AGTGGACGTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCGTCAGGG


 


CAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCT


GTCCCTTCCCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCATGTTAGGGA


 


CCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGA


GGGAGTTCTCGGCGCAGTGGTATAGTGTTCTGTGAAGGTTCTCAGTCCAGAGGGACTGCTACT


 


GCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTT


CGAGACACTGGCGACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAACATCACCACCAA


 


                                           Xhol


ACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAG


TGACATAGGAACTGATGACCCCGGTCCCTTGGGACCAGTGGCAGAGCTC






The translation of the 21B15 Gamma HC is as follows, polynucleotide sequence (above, SEQ ID NO: 87, top) and amino acid sequence (below, corresponding to residues 1-138 of SEQ ID NO: 69):

























The amino acid sequence of the 21B15 Gamma HC is as follows, with specific domains identified below (CDR sequences defined according to Kabat methods):









M K H L W F F L L L V A A P S W V L S
VH leader (SEQ ID NO: 70)



 


Q V Q L Q E S G P G L V K P S E T L S L T C T V S G S S I S
FR1 (SEQ ID NO: 71)


 


N Y Y W S
CDR1 (SEQ ID NO: 72)


 


W I R Q S P G K G L E W I G
FR2 (SEQ ID NO: 73)


 


F I Y Y G G N T K Y N P S L K S
CDR2 (SEQ ID NO: 74)


 


R V T I S Q D T S K S Q V S L T M S S V T A A E S A V Y F C A R
FR3 (SEQ ID NO: 75)


 


A S C S G G Y C I L D
CDR3 (SEQ ID NO: 76)


 


Y W G Q G T L V T V S
FR4 (SEQ ID NO: 77)


 


YWGQGTLVTVSS
Long FR4 (SEQ ID NO: 266)






Clone 23K12 (Corresponds to TCN-031):

The Kappa LC variable region of the anti M2 clone 23K12 was cloned as Hind III to BsiW1 fragment (see below), and is encoded by the following polynucleotide sequences SEQ ID NO: 88 (top) and SEQ ID NO: 89 (below).








HindIII



AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGG


TTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCC


 


TGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTC


ACGGTCTACACTGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAG


 


ACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGA


TGGTAGTGAACGGCCTGTTCAGTCTCGTAATCGTCGATAAATTTAACCATAGTCGTCTTTGGTCCCT


 


AAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGG


TTCGGGGATTTGAGGACTAGATACGACGTAGGTCAAACGTTTCACCCCAGGGTAGTTCCAAGTCACC


 


CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACCTAC


GTCACCTAGACCCTGTCTAAAGTGAGAGTGGTAGTCGCCAGACGTTGGACTTCTAAAACGTTGGATG


 


                                                            BslWI


TACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACG


ATGACAGTTGTCTCAATGTCATACGGACGGAAACCGGTCCCCTGGTTCGACCTCTAGTTTGCATGC






The translation of the 23K12 Kappa LC variable region is as follows, polynucleotide sequence (above, SEQ ID NO: 90, top) and amino acid sequence (below, corresponding to SEQ ID NO: 91).

























The amino acid sequence of the 23K12 Kappa LC variable region is as follows, with specific domains identified below (CDR sequences defined according to Kabat methods):









M D M R V L A Q L L G L L L L W L R G A R C
VK leader (SEQ ID NO: 57)



 


D I Q M T Q S P S S L S A S V G D R V T I T C
FR1 (SEQ ID NO: 58)


 


R T S Q S I S S Y L N
CDR1 (SEQ ID NO: 92)


 


W Y Q Q K P G K A P K L L I Y
FR2 (SEQ ID NO: 93)


 


A A S S L Q S G V P S R F
CDR2 (SEQ ID NO: 94)


 


S G S G S G T D F T L T I S G L Q P E D F A T Y Y C
FR3 (SEQ ID NO: 95)


 


Q Q S Y S M P A
CDR3 (SEQ ID NO: 96)


 


F G Q G T K L E I K
FR4 (SEQ ID NO: 114)


 


R T
Start of Kappa LC constant region






The 23K12 Gamma HC variable region was cloned as a Hind III to Xho 1 fragment, and is encoded by the following polynucleotide sequences and SEQ ID NO: 97 (top) and SEQ ID NO: 98 (bottom).








HindIII



AAGCTTCCACCATGGAGTTGGGGCTGTGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGT


TTCGAAGGTGGTACCTCAACCCCGACACGACCCAAAAGGAACAACGATAAAATTTTCCACAGGTCA


 


GTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTCCT


CACTCCACGTCGACCACCTCAGACCCCCTCCGAACCAGGTCGGACCCCCCAGGGACTCTTAGAGGA


 


GTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGG


CACGTCGGAGACCTAAGTGGCAGTCATCGTTGATGTACTCAACCCAGGCGGTCCGAGGTCCCTTCC


 


GGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCA


CCGACCTCACCCAGAGTCAATAAATATCACCACCATCGTGTATGATGCGTCTGAGGCACTTCCCGT


 


GATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAGAGCCG


CTAAGAGGAAGAGGTCTCTGTTGAGGTTCTTGTGTCACAAAGAAGTTTACTTGTCGGACTCTCGGC


 


AGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGGGTTACGGTTTAGACGTCT


TCCTGTGCCGACACATAATGACACGCTCTACAGACTCGTCCTACGCCCCAATGCCAAATCTGCAGA


 


                         Xhol


GGGGCCAAGGGACCACGGTCACCGTCTCGAG


CCCCGGTTCCCTGGTGCCAGTGGCAGAGCTC






The translation of the 23K12 Gamma HC variable region is as follows, polynucleotide sequence (above, SEQ ID NO: 99, top), and amino acid sequence (below, corresponding to SEQ ID NO: 100):

























The amino acid sequence of the 23K12 Gamma HC variable region is as follows, with specific domains identified below (CDR sequences defined according to Kabat methods):









M E L G L C W V F L V A I L K G V Q C
VH leader (SEQ ID NO: 101)



 


E V Q L V E S G G G L V Q P G G S L R I S C A A S G F T V S
FR1 (SEQ ID NO: 102)


 


S N Y M S
CDR1 (SEQ ID NO: 103)


 


W V R Q A P G K G L E W V S
FR2 (SEQ ID NO: 104)


 


V I Y S G G S T Y Y A D S V K
CDR2 (SEQ ID NO: 105)


 


G R F S F S R D N S K N T V F L Q M N S L R A E D T A V Y Y C A R
FR3 (SEQ ID NO: 106)


 


C L S R M R G Y G L D V
CDR3 (SEQ ID NO: 107)


 


W G Q G T T V T V S
FR4 (SEQ ID NO: 108)


 


WGQGTTVTVSS
Long FR4 (SEQ ID NO: 111)






Example 3
Identification of Conserved Antibody Variable Regions
The amino acid sequences of the three antibody Kappa LC and Gamma HC variable regions were aligned to identify conserved regions and residues, as shown below.
Amino acid sequence alignment of the Kappa LC variable regions of the three clones (SEQ ID NOs 673-675, respectively, in order of appearance):













































10









20








Translation of mp 73
A
S
T
M
D
M
R
V
L
A
Q
L
L
G
L
L
L
L
W
L
R
G
A


21B15

























Translation of mp 147
A
S
T
M
D
M
R
V
L
A
Q
L
L
G
L
L
L
L
W
L
R
G
A


8I10

























Translation of mp 137
A
S
T
M
D
M
R
V
L
A
Q
L
L
G
L
L
L
L
W
L
R
G
A


23K12












30









40





Translation of mp 73
R
C
D
I
Q
V
T
Q
S
P
S
S
L
S
A
S
V
G
D
R
V
T
I


21B15

























Translation of mp 147
R
C
D
I
Q
M
T
Q
S
P
S
S
L
S
A
S
V
G
D
R
V
T
I


8I10

























Translation of mp 137
R
C
D
I
Q
M
T
Q
S
P
S
S
L
S
A
S
V
G
D
R
V
T
I


23K12









50









60





Translation of mp 73
T
C
R
A
S
Q
N
I
Y
K
Y
L
N
W
Y
Q
Q
R
P
G
K
A
P


21B15

























Translation of mp 147
T
C
R
A
S
Q
N
I
Y
K
Y
L
N
W
Y
Q
Q
R
P
G
K
A
P


8I10

























Translation of mp 137
T
C
R
T
S
Q
S
I
S
S
Y
L
N
W
Y
Q
Q
K
P
G
K
A
P


23K12






70









80









90





Translation of mp 73
K
G
L
I
S
A
A
S
G
L
Q
S
G
V
P
S
R
F
S
G
S
G
S


21B15

























Translation of mp 147
K
G
L
I
S
A
A
S
G
L
Q
S
G
V
P
S
R
F
S
G
S
G
S


8I10

























Translation of mp 137
K
L
L
I
Y
A
A
S
S
L
Q
S
G
V
P
S
R
F
S
G
S
G
S


23K12













100









110





Translation of mp 73
G
T
D
F
T
L
T
I
T
S
L
Q
P
E
D
F
A
T
Y
Y
C
Q
Q


21B15

























Translation of mp 147
G
T
D
F
T
L
T
I
T
S
L
Q
P
E
D
F
A
T
Y
Y
C
Q
Q


8I10

























Translation of mp 137
G
T
D
F
T
L
T
I
S
G
L
Q
P
E
D
F
A
T
Y
Y
C
Q
Q


23K12




































120









130










Translation of mp 73
S
Y
S
P
P
L
T
F
G
G
G
T
R
V
D
I
K
R
T



21B15






















Translation of mp 147
S
Y
S
P
P
L
T
F
G
G
G
T
R
V
E
I
K
R
T



8I10






















Translation of mp 137
S
Y
S
M
P
—
A
F
G
Q
G
T
K
L
E
I
K
R
T



23K12









Amino acid sequence alignment of the Gamma HC variable regions of the three clones (SEQ ID NOs 676-678, respectively, in order of appearance):










































10









20





Translation of mp S1 21B15
A
S
T
M
K
H
L
W
F
F
L
L
L
V
A
A
P
S
W
V


Translation of mp 145 23K12
A
S
T
M
E
L
G
L
C
W
V
F
L
V
A
I
L
K
G
V


Translation of mp 153 8I10
A
S
T
M
K
H
L
W
F
F
L
L
L
V
A
A
P
S
W
V















30









40





Translation of mp 81 21B15
L
S
Q
V
Q
L
Q
E
S
G
P
G
L
V
K
P
S
E
T
L


Translation of mp 145 23K12
Q
C
E
V
Q
L
V
E
S
G
G
G
L
V
Q
P
G
G
S
L


Translation of mp 153 8I10
L
S
Q
V
Q
L
Q
E
S
G
P
G
L
V
K
P
S
E
T
L















50









60





Translation of mp 81 21B15
S
L
T
C
T
V
S
G
S
S
I
S
N
Y
Y
W
S
W
I
R


Translation of mp 145 23K12
R
I
S
C
A
A
S
G
F
T
V
S
S
N
Y
M
S
W
V
R


Translation of mp 153 8I10
S
L
T
C
T
V
S
G
S
S
I
S
N
Y
Y
W
S
W
I
R















70









80





Translation of mp 81 21B15
Q
S
P
G
K
G
L
E
W
I
G
F
I
Y
Y
G
G
N
T
K


Translation of mp 145 23K12
Q
A
P
G
K
G
L
E
W
V
S
V
I
Y
S
G
G
S
T
Y


Translation of mp 153 8I10
Q
S
P
G
K
G
L
E
W
I
G
F
I
Y
Y
G
G
N
T
K















90









100





Translation of mp 81 21B15
Y
N
P
S
L
K
S
R
V
T
I
S
Q
D
T
S
K
S
Q
V


Translation of mp 145 23K12
Y
A
D
S
V
K
G
R
F
S
F
S
R
D
N
S
K
N
T
V


Translation of mp 153 8I10
Y
N
P
S
L
K
S
R
V
T
I
S
Q
D
T
S
K
S
Q
V















110









120





Translation of mp 81 21B15
S
L
T
M
S
S
V
T
A
A
E
S
A
V
Y
F
C
A
R
A


Translation of mp 145 23K12
F
L
Q
M
N
S
L
R
A
E
D
T
A
V
Y
Y
C
A
R
C


Translation of mp 153 8I10
S
L
T
M
S
S
V
T
A
A
E
S
A
V
Y
F
C
A
R
A










































130








140







Translation of mp 81
S
C
S
G
G
Y
C
I
L
D
Y
W
G
Q
T
L
V
T
V
S



21B15























Translation of mp 145
L
S
R
M
R
G
Y
G
L
D
V
W
G
Q
T
T
V
T
V
S



23K12























Translation of mp 153
S
C
S
G
G
Y
C
I
L
D
Y
W
G
Q
T
L
V
T
V
S



8I10









Clones 8I10 and 21B15 came from two different donors, yet they have the same exact Gamma HC and differ in the Kappa LC by only one amino acid at position 4 in the framework 1 region (amino acids M versus V, see above), (excluding the D versus E wobble position in framework 4 of the Kappa LC).
Sequence comparisons of the variable regions of the antibodies revealed that the heavy chain of clone 8i10 was derived from germline sequence IgHV4 and that the light chain was derived from the germline sequence IgKV1.
Sequence comparisons of the variable regions of the antibodies revealed that the heavy chain of clone 21B15 was derived from germline sequence IgHV4 and that the light chain was derived from the germline sequence IgKV1.
Sequence comparisons of the variable regions of the antibodies revealed that the heavy chain of clone 23K12 was derived from germline sequence IgHV3 and that the light chain was derived from the germline sequence IgKV1.
Example 4
Production and Characterization of M2 Antibodies
The antibodies described above were produced in milligram quantities by larger scale transient transfections in 293 PEAK cells. Crude un-purified antibody supernatants were used to examine antibody binding to influenza A/Puerto Rico/8/1932 (PR8) virus on ELISA plates, and were compared to the binding of the control antibody 14C2, which was also produced by larger scale transient transfection. The anti-M2 recombinant human monoclonal antibodies bound to influenza while the control antibody did not (FIG. 9).
Binding was also tested on MDCK cells infected with the PR8 virus (FIG. 10). The control antibody 14C2 and the three anti M2E clones: 8I10, 21B15 and 23K12, all showed specific binding to the M2 protein expressed on the surface of PR8-infected cells. No binding was observed on uninfected cells.
The antibodies were purified over protein A columns from the supernatants. FACs analysis was performed using purified antibodies at a concentration of 1 ug per ml to examine the binding of the antibodies to transiently transfected 293 PEAK cells expressing the M2 proteins on the cell surface. Binding was measured testing binding to mock transfected cells and cells transiently transfected with the Influenza subtype H3N2, A/Vietnam/1203/2004 (VN1203), or A/Hong Kong/483/1997 HK483 M2 proteins. As a positive control the antibody 14C2 was used. Unstained and secondary antibody alone controls helped determined background. Specific staining for cells transfected with the M2 protein was observed for all three clones. Furthermore, all three clones bound to the high path strains A/Vietnam/1203/2004 and A/Hong Kong/483/1997 M2 proteins very well, whereas the positive control 14C2 which bound well to H3N2 M2 protein, bound much weaker to the A/Vietnam/1203/2004 M2 protein and did not bind the A/Hong Kong/483/1997 M2 protein. See FIG. 11.
Antibodies 21B15, 23K12, and 8I10 bound to the surface of 293-HEK cells stably expressing the M2 protein, but not to vector transfected cells (see FIG. 1). In addition, binding of these antibodies was not competed by the presence of 5 mg/ml 24-mer M2 peptide, whereas the binding of the control chimeric mouse V-region/human IgG1 kappa 14C2 antibody (hu14C2) generated against the linear M2 peptide was completely inhibited by the M2 peptide (see FIG. 1). These data confirm that these antibodies bind to conformational epitopes present in M2e expressed on the cell or virus surface, as opposed to the linear M2e peptide.
Example 5
Viral Binding of Human Anti-Influenza Monoclonal Antibodies
UV-inactivated influenza A virus (A/PR/8/34) (Applied Biotechnologies) was plated in 384-well MaxiSorp plates (Nunc) at 1.2 μg/ml in PBS, with 25 and was incubated at 4° C. overnight. The plates were then washed three times with PBS, and blocked with 1% Nonfat dry milk in PBS, 50 μl/well, and then were incubated at room temp for 1 hr. After a second wash with PBS, MAbs were added at the indicated concentrations in triplicate, and the plates were incubated at room temp for 1 hour. After another wash with PBS, to each well was added 25 μl of a 1/5000 dilution of horseradish peroxidase (HRP) conjugated goat anti-human IgG Fc (Pierce) in PBS/1% Milk, and the plates were left at room temp for 1 hr. After the final PBS wash, the HRP substrate 1-StepTM Ultra-TMB-ELISA (Pierce) was added at 25 μl/well, and the reaction proceeded in the dark at room temp. The assay was stopped with 25 μl/well 1N H2SO4, and light absorbance at 450 nm (A450) was read on a SpectroMax Plus plate reader. Data are normalized to the absorbance of MAb 8I10 binding at 10 μg/ml. Results are shown in FIGS. 2A and 2B.
Example 6
Binding of Human Anti-Influenza Monoclonal Antibodies to Full-Length M2 Variants
M2 variants (including those with a high pathology phenotype in vivo) were selected for analysis. See FIG. 3A for sequences.
M2 cDNA constructs were transiently transfected in HEK293 cells and analyzed as follows: To analyze the transient transfectants by FACS, cells on 10 cm tissue culture plates were treated with 0.5 ml Cell Dissociation Buffer (Invitrogen), and harvested. Cells were washed in PBS containing 1% FBS, 0.2% NaN3 (FACS buffer), and resuspended in 0.6 ml FACS buffer supplemented with 100 μg/ml rabbit IgG. Each transfectant was mixed with the indicated MAbs at 1 μg/ml in 0.2 ml FACS buffer, with 5×105 to 106 cells per sample. Cells were washed three times with FACS buffer, and each sample was resuspended in 0.1 ml containing 1 μg/ml alexafluor (AF) 647-anti human IgG H&L (Invitrogen). Cells were again washed and flow cytometry was performed on a FACSCanto device (Becton-Dickenson). The data is expressed as a percentage of the mean fluorescence of the M2-D20 transient transfectant. Data for variant binding are representative of 2 experiments. Data for alanine mutants are average readouts from 3 separate experiments with standard error. Results are shown in FIGS. 3B and 3C.
Example 7
Alanine Scanning Mutagenesis to Evaluate M2 Binding
To evaluate the antibody binding sites, alanine was substituted at individual amino acid positions as indicated by site-directed mutagenesis.
M2 cDNA constructs were transiently transfected in HEK293 cells and analyzed as described above in Example 6. Results are shown in FIGS. 4A and 4B. FIG. 8 shows that the epitope is in a highly conserved region of the amino terminus of the M2 polypeptide. As shown in FIGS. 4A, 4B and FIG. 8, the epitope includes the serine at position 2, the threonine at position 5 and the glutamic acid at position 6 of the M2 polypeptide.
Example 8
Epitope Blocking
To determine whether the MAbs 8I10 and 23K12 bind to the same site, M2 protein representing influenza strain A/HK/483/1997 sequence was stably expressed in the CHO (Chinese Hamster Ovary) cell line DG44. Cells were treated with Cell Dissociation Buffer (Invitrogen), and harvested. Cells were washed in PBS containing 1% FBS, 0.2% NaN3 (FACS buffer), and resuspended at 107 cells/ml in FACS buffer supplemented with 100 μg/ml rabbit IgG. The cells were pre-bound by either MAb (or the 2N9 control) at 10 μg/ml for 1 hr at 4° C., and were then washed with FACS buffer. Directly conjugated AF647-8I10 or -23K12 (labeled with the AlexaFluor® 647 Protein Labeling kit (Invitrogen) was then used to stain the three pre-blocked cell samples at 1 μg/ml for 106 cells per sample. Flow cytometric analyses proceeded as before with the FACSCanto. Data are average readouts from 3 separate experiments with standard error. Results are shown in FIG. 5.
Example 9
Binding of Human Anti-Influenza Monoclonal Antibodies to M2 Variants and Truncated M2 Peptides
The cross reactivity of mAbs 8i10 and 23K12 to other M2 peptide variants was assessed by ELISA. Peptide sequences are shown in FIGS. 6A and 6B. Additionally, a similar ELISA assay was used to determine binding activity to M2 truncated peptides.
In brief, each peptide was coated at 2 μg/mL to a flat bottom 384 well plate (Nunc) in 25 μL/well of PBS buffer overnight at 4° C. Plates were washed three times and blocked with 1% Milk/PBS for one hour at room temperature. After washing three times, MAb titers were added and incubated for one hour at room temperature. Diluted HRP conjugated goat anti-human immunoglobulin FC specific (Pierce) was added to each well after washing three times. Plates were incubated for one hour at room temperature and washed three times. 1-StepTM Ultra-TMB-ELISA (Pierce) was added at 25 μl/well, and the reaction proceeded in the dark at room temp. The assay was stopped with 25 μl/well 1N H2SO4, and light absorbance at 450 nm (A450) was read on a SpectroMax Plus plate reader. Results are shown in FIGS. 6A and 6B.
Example 10
In Vivo Evaluation of the Ability of Human Anti-Influenza Monoclonal Antibodies to Protect from Lethal Viral Challenge
The ability of antibodies, 23K12 and 8I10, to protect mice from lethal viral challenge with a high path avian influenza strain was tested.
Female BALB/c mice were randomized into 5 groups of 10. One day prior (Day −1 (minus one)) and two days post infection (Day +2 (plus two), 200 ug of antibody was given via 200 ul intra-peritoneal injection. On Day 0 (zero), an approximate LD90 (lethal dose 90) of A/Vietnam/1203/04 influenza virus, in a volume of 30 μl was given intra-nasally. Survival rate was observed from Day 1 through Day 28 post-infection. Results are shown in FIG. 7.
Example 11
Characterization of M2 Antibodies TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B11, and 3242_P05
FACS
Full length M2 cDNA (A/Hong Kong/483/97) were synthesized (Blue Heron Technology) and cloned into the plasmid vector pcDNA3.1 which was then transfected into CHO cells with Lipofectamine (Invitrogen) to create a stable pool of CHO-HK M2-expressing cells. For the panel of anti-M2 Mabs, 20 μl samples of supernatant from transient transfections from each of the IgG heavy and light chain combinations was used to stain the CHO-HK M2 stable cell line. Bound anti-M2 mabs were visualized on viable cells with Alexafluor 647-conjugated goat anti-Human IgG H&L antibody (Invitrogen). Flow cytometry was performed with a FACSCanto, and analysis on the accompanying FACSDiva software (Becton Dickenson).
ELISA
Purified Influenza A (A/Puerto Rico/8/34) inactivated by β-propiolactone (Advanced Biotechnologies, Inc.) was biotinylated (EZ-Link Sulfo-NHS-LC-Biotin, Pierce) and adsorbed for 16 hours at 4° C. to 384-well plates in 25 μl PBS that were pre-coated with neutravidin (Pierce). Plates were blocked with BSA in PBS, samples of supernatant from transient transfections from each of the IgG heavy and light chain combinations were added at a final dilution of 1:5, followed by HRP-conjugated goat anti-human Fc antibody (Pierce), and developed with TMB substrate (ThermoFisher).
The results of this analysis are shown below in Table 2.











TABLE 2










FACS
Virus


Transfection.

Sequence ID
M2-HK
ELISA












no
BCC well ID
Gamma
Light
MFI
OD A450















322
3241_G23
G4_005
K1_004
1697
3.02


352
3244_I10
G4_007
K2_006
434
3.01


339
3243_J07
G4_007
K1_007
131
2.94


336
3259_J21
G4_005
K2_005
1673
2.40


348
3245_O19
G3_004
K1_001
919
3.51


345
3244_H04
G3_003
K1_006
963
3.31


346

Pos Cont
Pos Cont
754
2.69




(HC)
(LC)


347

Neg Cont
Neg Cont
11
0.15




(HC)
(LC)


374
3136_G05
G4_007
K1_007
109
ND


386
3252_C13
G4_013
K1_002
449
ND


390
3255_J06
G4_013
K2_007
442
ND


400
3420_I23
G4_004
K1_003
112
ND


432
3139_P23
G4_016
K1_007a
110
1.02


412
3248_P18
G4_009
K1_006
967
0.56


413
3253_P10
G4_007
K1_004
43
0.50


434
3260_D19
G3_004a
K2_001
846
2.46


439
3362_B11
G4_010a
K1_007
218
1.83


408
3242_P05
G3_005
K2_004
596
0.50


451

Pos Cont
Pos Cont
1083
1.87




(HC)
(LC)


452

Neg Cont
Neg Cont
6
0.05




(HC)
(LC)





Positive control: supernatant from tranisent transfection with the IgG heavy and light chain combination of mAb 8I10


Negative control: supernatant from tranisent transfection with the IgG heavy and light chain combination of mAb 2N9


MFI = mean fluorescence intensity






Example 12
Human Antibodies Reveal a Protective Epitope that is Highly Conserved among Human and Non-Human Influenza A Viruses
Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain due to the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG+ memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.
Introduction
Seasonal influenza epidemics hospitalize more than 200,000 people each year in the US and kill an estimated 500,000 worldwide (Thompson, W. W. et al. (2004) JAMA 292:1333-1340). The immune system affords only partial protection from seasonal strains in most individuals because of constantly arising point mutations in the viral genome which lead to structural variability known as antigenic drift. Pandemic strains encounter even less immune resistance due to genomic reassortment events among different viruses which result in more radical shifts in viral antigenic determinants. Consequently, pandemic influenza has the potential to cause widespread illness, death, and economic disruption. Vaccines and antiviral agents are available to counter the threat of influenza epidemics and pandemics. However, the strain composition of influenza vaccines must be determined prior to the influenza season on an annual basis, and predicting in advance which strains will become dominant is challenging. Moreover, the emergence of strains that evade vaccine-induced, protective immune responses is relatively rapid which often results in inadequate protection (Carrat F, Flahault A. (2007) Influenza vaccine: the challenge of antigenic drift. Vaccine 25:6852-6862). Antiviral drugs include oseltamivir and zanamivir which inhibit the function of the viral protein neuraminidase (NA), and adamantanes which inhibit the ion channel function of the viral M2 protein (Gubareva L V, et al. (2000) Lancet 355:827-835; Wang C, et al. (1993) J Virol 67:5585-5594). Antiviral agents are effective for sensitive virus strains but viral resistance can develop quickly and has the potential to render these drugs ineffective. In the 2008-2009 US influenza season nearly 100% of seasonal H1N1 or H3N2 influenza isolates tested were resistant to oseltamivir or adamantane antivirals, respectively (CDC Influenza Survey: www.cdc.gov/flu/weekly/weeklyarchives2008-2009/weekly23.htm).
Passive immunotherapy using anti-influenza antibodies represents an alternative paradigm for preventing or treating viral infection. Evidence for the utility of this approach dates back nearly 100 years when passive serum transfer was used during the 1918 influenza pandemic with some success (Luke T C, et al. (2006) Ann Intern Med 145:599-609). While protection provided by anti-influenza monoclonal antibodies (mAbs) is typically narrow in breadth because of the antigenic heterogeneity of influenza viruses, several groups have recently reported protective mAbs that bind to conserved epitopes within the stem region of viral hemagglutinin (HA) (Okuno Y, et al. (1993) J Virol 67:2552-2558; Throsby M, et al. (2008) PLoS One. 3: e3942; Sui J, et al. (2009) Nat Struct Mol Biol 16:265-273; Corti D, et al. (2010) J Clin Invest doi:10.1172/JCI41902). These epitopes appear to be restricted to a subset of influenza viruses; these anti-HA mAbs would not be expected to provide protection against viruses of the H3 and H7 subtypes. Of these, the former comprises an important component of circulating human strains (Russell C A, et al. (2008) Science 320:340-346) while the latter includes highly pathogenic avian strains which have caused mortality in humans (Fouchier R A, et al. (2004) Proc Natl Acad Sci USA 101:1356-1361; Belser J A, et al. (2009) Emerg Infect Dis 15:859-865).
Of the three antibody targets present on the surface of the influenza virus, the ectodomain of the viral M2 protein (M2e) is much more highly conserved than either HA or NA, which makes, it an attractive target for broadly protective mAbs. Monoclonal antibodies to M2e have been shown to be protective in vivo (Wang R, et al. (2008) Antiviral Res 80:168-177; Liu W, et al. (2004) Immunol Lett 93:131-6; Fu T M, et al. (2008) Virology 385:218-226; Treanor J J, et al. (1990) J Virol 64:1375-1357; Beerli R, et al. (2009) Virology J 6:224-234), and several groups have demonstrated protection against infection with vaccine strategies based on M2e (Fu T M, et al. (2009) Vaccine 27:1440-1447; Fan J, et al. (2004) Vaccine 22:2993-3003; Slepushkin V A, et al. (1995) Vaccine 13:1399-1402; Neirynck S, et al. (1999) Nat Med 5:1157-1163; Tompkins S M, et al. (2007) Emerg Infect Dis 13:426-435; Mozdzanowska K, et al. (2003) Vaccine 21:2616-2626). In these cases, purified M2 protein or peptides derived from M2e sequence have been used as immunogens to generate anti-M2e antibodies in animals or as vaccine candidates. In the present study, mAbs were isolated directly from human B cells that bind to the M2 protein displayed on virus particles and on virus-infected cells. Further, we demonstrate that these antibodies protect mice from a lethal influenza A virus challenge and that they can recognize M2 variants derived from a wide range of human and animal influenza A virus isolates. This combination of properties may enhance the utility of these antibodies to prevent and treat influenza A virus infections.
Results and Discussion
Isolation of a Family of Anti-M2e mAbs from Human B Cells. To explore the humoral immune response to natural influenza infection in humans, we have isolated antibodies from IgG+ memory B cells of M2e-seropositive subjects. Serum samples from 140 healthy adult, United States-sourced donors were tested for reactivity with M2e expressed on the surface of HEK293 cells that were transfected with a viral M2 gene (derived from A/Fort Worth/50 H1N1). IgG+ memory B cells from 5 of the 23 M2e-seropositive subjects were cultured under conditions where they proliferated and differentiated into IgG-secreting plasma cells. B cell culture wells were screened for IgG reactivity to cell-surface M2e and immunoglobulin heavy and light chain variable region (VH and VL) genes were rescued by RT-PCR from 17 positive wells and incorporated into a human IgG1 constant region background for recombinant expression and purification. VH and VL sequences of 15 of the 17 anti-M2e mAbs cluster into two related groups (Table 3) (IMGT®, the International ImMunoGeneTics Information system®, available at www.imgt.org). In group A, assignment of the germline VH gene segment is IGHV4-59*01 while in the group B, the germline gene segment is IGHV3-66*01. The two more distantly related mAbs 62B11 and 41G23 (group C) utilize the germline V gene segment IGHV4-31*03 which has only 5 amino acid residue differences from the germline V gene segment IGHV4-59*01 of group A. All of these mAbs utilize the same light chain V gene, IGKV1-39*01 or its allele IGKV1D-39*01 and show evidence of somatic hypermutation from the germline heavy or kappa chain sequence (FIG. 12). Competitive binding experiments showed that all of these human mAbs appear to bind similar sites on native M2e expressed on the surface of Chinese hamster ovary (CHO) cells (FIG. 13). We selected for further characterization one mAb from each of groups A and B, designated TCN-031 and TCN-032, respectively.







TABLE 3







Immunoglobulin gene segment usage of human anti-M2e antibodies.










Heavy chain germline gene segments
Light chain germline gene segments














mAb
Variable
Diversity
Joining
Variable
Joining





Group A
TCN-032
IGHV4-59*01
IGHD2-15*01
IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ4*01



43J7
IGHV4-59*07
IGHD1-26*01
IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ2*01



53P10
IGHV4-59*07
IGHD1-26*01
IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ2*01



44I10
IGHV4-59*07
IGHD1-26*01
IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ2*01



55J6
IGHV4-59*01
IGHD5-18*01
IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01



52C13
IGHV4-59*01
IGHD5-18*01
IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01



39P23
IGHV4-59*01
IGHD4-23*01
IGHJ4*01
IGKV1-39*01, or IGKV1D-39*01
IGKJ1*01



36G5
IGHV4-59*01
IGHD2-8*01
IGHJ6*04
IGKV1-39*01, or IGKV1D-39*01
IGKJ3*01



48P18
IGHV4-59*01
IGHD2-15*01
IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ4*01



59J21
IGHV4-59*01
IGHD2-15*01
IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ4*01



20I23
IGHV4-59*01
IGHD6-6*01
IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01


Group C
62B11
IGHV4-31*03
IGHD4-23*01
IGHJ6*02 (a)
IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01



41G23
IGHV4-31*03
IGHD3-16*01
IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01


Group B
TCN-031
IGHV3-66*01
IGHD3-10*01
IGHJ3*01
IGKV1-39*01, or IGKV1D-39*01
IGKJ2*01



44H4
IGHV3-66*01
Cannot assign
IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01



45O19
IGHV3-66*01
Cannot assign
IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01



60D19
IGHV3-66*01
Cannot assign
IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01
IGKJ2*01





Reference sequences for each mAb heavy and light chain were analysed using IMGT/V-QUEST to determine gene usage.






High Affinity Binding to the Surface of Influenza Virus. Both TCN-031 and TCN-032 bound directly to an H1N1 virus (A/Puerto Rico/8/34) with high avidity, with half-maximal binding at about 100 ng/mL (FIG. 14a). Fab fragments prepared from TCN-031 and TCN-032 bound virus with affinities (KD) of 14 and 3 nM, respectively, as determined by surface plasmon resonance (Table 4). The human mAbs did not bind appreciably to a 23 amino acid synthetic peptide corresponding to the M2e domain of an H1N1 virus (A/Fort Worth/1/50) (FIG. 14b). A chimeric derivative of the murine anti-M2e mAb 14C2 (ch14C2), which was originally generated by immunization with purified M2 (Zebedee S L and Lamb R A. (1988) J Virol 62:2762-2772), exhibited the opposite behavior to that observed with the human mAbs, with little binding to virus but robust binding to the isolated 23mer M2e peptide with half-maximal binding to peptide at 10 ng/mL (FIGS. 14a and 14b). Interestingly, both the human mAbs and ch14C2 bound to the surface of Madin-Darby canine kidney (MDCK) cells infected with H1N1 virus (A/Puerto Rico/8/34) with similar avidities (FIG. 14c). It thus appears that viral epitopes recognized by the human anti-M2e mAbs are present and accessible on the surface of both virus and infected cells, while the epitope bound by ch14C2 is accessible only on the surface of infected cells. Our observation that the human anti-M2e mAbs do not bind appreciably to immobilized synthetic peptides derived from M2e, and further that such peptides do not compete for binding of these antibodies to M2e expressed on the surface of mammalian cells (FIG. 14d), supports the idea that secondary structure within the M2e epitope is important for binding by the human antibodies. That ch14C2 binds peptide immobilized on plastic suggests a lesser importance of higher order structure for binding of this mAb.







TABLE 4







Affinity of anti-M2e Fab fragments for influenza virus.












Fab
ka (M−1 * s−1)
kd (s−1)
KD







TCN-031
1.0e6
1.4e−2
 14 nM



TCN-032
7.4e5
2.3e−3
3.2 nM



cH14C2
5.0e2
1.8e−3
4.0 μM










Protection from Lethal Challenges with H5N1 and H1N1 viruses. We next examined the protective efficacy of the human anti-M2e mAbs TCN-031 and TCN-032 in a lethal challenge model of influenza infection in mice. Animals were challenged intranasally with 5×LD50 units of a high-pathogenicity H5N1 virus (A/Vietnam/1203/04) and both human mAbs were protective when treatment was initiated one day after viral challenge. In contrast, mice that were subjected to similar treatment regimens with a subclass-matched, irrelevant control mAb 2N9, which targets the AD2 epitope of the gp116 portion of the human cytomegalovirus gB, or with a vehicle control were protected to a lesser extent, or not at all, resulting in 70-80% survival for mice treated with human mAbs versus 20% survival for control mAb and 0% survival for vehicle (FIG. 15a). The anti-M2e mAb ch14C2 did not confer substantial protection in this model (20% survival; FIG. 15a), though this mAb has been shown to reduce the titer of virus in the lungs of mice infected with other strains of influenza virus (Treanor J J, et al. (1990) J Virol 64:1375-1357). All of the animals, including those in the TCN-031 and TCN-032 treatment groups, exhibited weight loss from days 4 to 8 post infection followed by a gradual increase in weight in the surviving animals through the end of the study on day 14 (FIG. 15b), indicating that the human anti-M2e mAbs afforded protection by reducing the severity or extent of infection rather than by completely preventing infection. Indeed, results of immunohistological and viral load analyses of lung, brain and liver tissue from additional animals in each treatment cohort are consistent with a reduction in the spread of virus beyond the lung to the brain and also possibly liver in animals that were treated with the human anti-M2e mAbs, but not with ch14C2 or the subclass-matched control mAb 2N9. The effect of the human anti-M2e mAbs on viral load in the lung versus the control mAbs was, however, more moderate (Table 5 and FIG. 16, respectively).
To test whether protection conferred by the human anti-M2e mAbs mirrors their broad binding behavior, we performed a similar in vivo challenge study with a mouse-adapted isolate of the relatively divergent H1N1 virus A/Puerto Rico/8/34. One hundred percent of PBS-treated or subclass-matched, control antibody-treated mice were killed by this virus, while a majority of the animals treated with the human anti-M2e mAbs TCN-031 and TCN-032 survived (60%; FIG. 15c). With this virus mice treated with ch14C2 provided a similar survival benefit to that of the human anti-M2e mAbs (FIG. 15c). Weight changes in each treatment group throughout the course of infection and its subsequent resolution followed a pattern that was similar to that of mice infected with the H5N1 virus (FIG. 15d).
The human anti-M2e mAbs and ch14C2 bound to cell surface-expressed M2e from A/Vietnam/1203/04 and A/Puerto Rico/8/34 viruses (FIG. 19b, Table 6) and cells infected with A/Puerto Rico/8/34 (FIG. 14c). Mechanisms for antibody-mediated protection could include killing of infected host cells by antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity (Wang R, et al. (2008) Antiviral Res 80:168-177; Jegerlehner A, et al. (2004) J Immunol 172:5598-5605). We found in vitro evidence for both of these mechanisms with the human anti-M2e mAbs and ch14C2 (FIG. 17 and Table 6). An explanation for the enhanced in vivo protection observed with the human anti-M2e mAbs as compared to ch14C2 following challenge by the high-pathogenicity avian virus A/Vietnam/1203/04 as compared with A/Puerto Rico/8/34 could be due to the unique capability of the human mAbs to bind virus directly whereas ch14C2 does not appear to bind influenza virions (FIG. 14a). Protective properties of antibodies that bind to virus might be expected to include mechanisms such as antibody-dependent virolysis (Nakamura M, et al. (2000) Hybridoma 19:427-434) and clearance via opsonophagocytosis by host cells (Huber V C, et al. (2001) J Immunol 166:7381-7388). Some of these mechanisms require efficient interaction between antibodies and host Fc receptors. In our mouse challenge experiments all of the mAbs tested had human constant regions; however other studies have shown that human antibodies can interact productively with murine Fc receptors (Clynes R A, et al. (2000) Nat Med 6:443-446).







TABLE 5







Pathological assessment of lung, liver, and brain of mice treated


with anti-M2e mAbs TCN-031 and TCN-032 after challenge with


H5N1 A/Vietnam/1203/04.














Or-

TCN-
TCN-






gans
Mouse
031
032
2N9
CH14C2
PBS
UT/C





Lung
1
++/++
++/
++/++
++/++
++/++
++/





++



+++



2
++/++
++/
++/
++/++
++/++
++/





++
+++


++



3
++/++
++/
++/++
++/++
++/
++/





++


+++
++


Brain
1
−/−
−/−
+/+
−/−
+/+++
++/









+++



2
−/−
±/+
+/+++
+/+
−/−
+/+



3
−/−
−/−
+/+
+/++
+/+++
++/









+++


Liver
1
−/−
−/−
+/+
+/−
+/+
+/+



2
−/−
−/−
+/+
+/−
+/−
+/−



3
−/−
+/−
+/+
+/+
+/+
+/+





Pathological changes and viral antigens were detected in the lungs of all virus-challenged mice. The mice had similar lung lesions across all groups, although mice in the TCN-031 and TCN-032 groups had a tendency toward less viral antigen expression in the lung. In the brain and liver, lesions were not detected in mice in the TCN-31 group and only one of three mice in the TCN-032 group showed some evidence of viral antigens in the brain.


Pathological changes/viral antigens:


+++ severe/many,


++ moderate/moderate,


+ mild/few,


± scant/rare,


− not observed/negative.













TABLE 6
















The M2e sequence at the top is from A/Brevig Mission/1/18 (H1N1) and is used as the reference sequence for alignment of the M2 ecotodomain amino acids 1-23 of 43 wild-type variants.


Grey boxes denote amino acid identity with the reference sequence and white boxes are amino acid replacement mutations.


This list of non-identical sequences, except for HK, VN, and D20, was derived from M2 sequences used in references 11 and 27.


Sequence data are from the Influenza Virus Resource at the National Center for Biotechnology Information






Binding to the Highly Conserved N-Terminal Segment of M2e. To better understand the unique viral binding property of the human anti-M2e mAbs we mapped their binding sites within the M2e domain. The lack of appreciable binding of the human mAbs to M2e-derived linear peptides precluded a synthetic peptide approach to fine structure mapping of their epitopes. Instead, binding of the mAbs to M2e alanine substitution mutants and naturally occurring M2 variants that were expressed on the surface of cDNA-transfected mammalian cells was quantified by flow cytometry. Binding experiments with a panel of M2 mutant proteins where each position in the 23 amino acid M2 ectodomain was substituted with alanine revealed that the first (S), fourth (T), and fifth (E) positions of the mature (methionine-clipped) M2 polypeptide were critical for binding of both TCN-031 and TCN-032 (FIG. 19a). In contrast, the binding of ch14C2 was selectively diminished when alanine was substituted at position 14 of mature M2 (FIG. 19a). These observations were confirmed in studies with a panel of divergent, naturally occurring M2 variants; substitution with proline at position 4 (Table 6: A/Panama/1/1966 H2N2, A/Hong Kong/1144/1999 H3N2, A/Hong Kong/1180/1999 H3N2, and A/chicken/Hong Kong/YU427/2003 H9N2) and glycine at position 5 (Table 6: A/chicken/Hong Kong/SF1/2003 H9N2) correlated with diminished binding of the human anti-M2e mAbs but not ch14C2 (FIG. 19b, Table 6). These results suggest that both TCN-031 and TCN-032 recognize a core sequence of SLLTE at positions 1-5 of the N-terminus of mature M2e. This is supported by data which show that these mAbs compete effectively with each other for binding to M2e expressed on the surface of CHO cells (FIG. 20). In contrast, our results indicate that ch14C2 binds to a site that is spatially distinct and downstream of the SLLTE core that is recognized by the human anti-M2e mAbs. Indeed, previous studies have shown that 14C2 binds a relatively broad, linear epitope with the sequence EVERTPIRNEW at positions 5-14 of processed M2e (Wang R, et al. (2008) Antiviral Res 80:168-177).
While the epitopes recognized by TCN-031 and TCN-032 are likely very similar, there were some differences between these human mAbs in their binding to several of the M2e mutants. For instance, TCN-031 appears to have a greater dependence than TCN-032 on residues 2 (L) and 3 (L) of the mature M2e sequence (FIG. 19a). The VH regions of these two human mAbs utilize different variable, diversity, and joining gene segments which may explain the minor differences in binding observed between these mAbs. Interestingly, despite the differences in their VH make-up these human mAbs utilize the same germline kappa chain V gene segments, albeit with distinct kappa chain joining segments.
Localization of the binding region of the human anti-M2e mAbs at the N-terminal region of M2e is especially significant in light of the remarkably high sequence conservation in this part of the polypeptide among influenza A viruses. The viral M gene segment that encodes M2 also encodes the internal viral protein M1 via differential splicing. However, the splice site is located downstream of the shared N-terminus of M2 and M1 resulting in two distinct mature polypeptides with an identical 8 amino acid N-terminal sequence (Lamb R A and Choppin P W (1981) Virology 112:729-737). Options for viral escape from host anti-M2e antibodies that bind this region might be limited as escape mutations in the N-terminal region would result in changes to not just M2 but also the M1 protein. Indeed, this N-terminal 8 amino acid segment of M2e shows nearly complete identity in the 1364 unique full-length M2 variants catalogued in the NCBI Influenza Database (www.ncbi.nlm.nih.gov/genomes/FLU/Database/multiple.cgi) while much lower levels of conservation are seen in M2e sequences downstream of this region (FIG. 19c). In fact, the core human anti-M2e antibody epitope SLLTE is present in  ̃98% of the 1364 unique full-length M2e sequences catalogued in the NCBI Influenza Database, including 97%, 98% and 98% of the human, swine and avian viruses, respectively. This contrasts to the much lower conservation within the linear binding sites of anti-M2e mAbs elicited by immunization with M2e peptides or proteins. For instance, 14C2 and Z3G1 (Wang R, et al. (2008) Antiviral Res 80:168-177) bind sequences that are conserved in less than 40% of influenza A viruses, and conservation within this region is even lower in avian and swine viruses (Table 7).
The linear M2e epitopes recognized by peptide-elicited antibodies may be more sensitive to escape mutations and natural substitutions that are present in some viral isolates. For example, P10L and P10H escape mutations to mAb 14C2 have been mapped to the central portion of M2e (Zharikova D, et al. (2005). J Virol 79:6644-6654) and those same substitutions also occur in M2e variants from some highly pathogenic H5N1 strains. We have found that the human mAbs TCN-031 and TCN-032 but not ch14C2 bind to the M2 variant from the H5N1 virus A/Hong Kong/483/97 (HK) which contains the P10L substitution (FIG. 19b, Table 6). Thus, monoclonal antibodies with specificities similar to that of 14C2 are likely to have limited utility as broad spectrum therapeutic agents.
In the examination of 5 human subjects we found 17 unique anti-M2e antibodies that bind the conserved N-terminal region of M2e, but did not observe IgG-reactivity with M2e-derived peptides that contain the linear epitopes recognized by 14C2 and other peptide-elicited antibodies. In contrast to the apparently uniform antibody response to M2e in naturally infected or vaccinated humans, mice immunized with M2e-derived peptides produced antibodies with a range of specificities within M2e, including the conserved N-terminus and also downstream regions (Fu T M, et al. (2008) Virology 385:218-226). It is tempting to speculate that the human immune system has evolved a humoral response that exclusively targets the highly conserved N-terminal segment of M2e rather than the more divergent, and thus less sustainably protective, downstream sites. Despite the lack of evidence for human antibodies that recognize this internal region of M2e, analysis of the evolution of the M gene suggests that this region of M2e is under strong positive selection in human influenza viruses (Furuse Y, et al. (2009) J Virol 29:67). One explanation for this finding is that selective pressure is being directed at this internal region by immune mechanisms other than antibodies. For instance, human T cell epitopes have been mapped to these internal M2e sites (Jameson J, et al. (1998) J Virol 72:8682-8689).







TABLE 7







Conservation of the viral binding site for human anti-M2e mAbs compared


with those for mAbs derived from immunized mice, in influenza A.












Human
Swine
Avian
All


mAb
(n = 506)
(n = 193)
(n = 665)
(n = 1364)














TCN-031, TCN-032
97
98
98
98


[1-SLLTE-5]


Z3G1
79
39
7
38


[2-LLTEVETPIR-11]


(Ref. 11)


14C2
75
19
2
31


[5-EVETPIRNEW-14]


(Ref. 11)









Recognition of 2009 H1N1 S-OIV. Broadly protective anti-influenza mAbs could be used in passive immunotherapy to protect or treat humans in the event of outbreaks from highly pathogenic, pandemic viral strains. A critical test of the potential for such mAbs as immunotherapeutic agents is whether they are capable of recognizing virus strains that may evolve from future viral reassortment events. As a case in point, the human anti-M2e mAbs TCN-031 and TCN-032 were tested for their ability to recognize the current H1N1 swine-origin pandemic strain (S-OIV). These mAbs were derived from human blood samples taken in 2007 or earlier, prior to the time that this strain is thought to have emerged in humans (Neumann G, et al. (2009) Nature 459:931-939). Both human mAbs bound to MDCK cells infected with A/California/4/2009 (S-OIV H1N1, pandemic) and A/Memphis/14/1996 (H1N1, seasonal) whereas ch14C2 bound only to cells infected with the seasonal virus (FIG. 21). If this broad binding behavior proves to correlate with protection, as was the case with A/Vietnam/1203/2004 and A/Puerto Rico/8/34, then these human mAbs might be useful to prevent or treat the S-OIV pandemic strain or possibly other pandemic strains that might emerge in the future.
While it is remarkable that humans have the capability to make antibodies that may confer nearly universal protection against influenza infection, the discovery of this heretofore un-described class of antibodies raises the question of why this virus is able to mount a productive infection in immunocompetent individuals at all. This apparent paradox may be explained by the nature of the protective M2e epitope and its relative immunogenicity. It has been noted by others that M2e appears to exhibit low immunogenicity in humans (Feng J, et al. (2006) Virol J 3:102; Liu W, et al. (2003) FEMS Immunol Med Microbio 35:141-146), especially when compared to the immunodominant virus glycoproteins HA and NA. Therefore, protective anti-M2e antibodies may exist in many individuals but at suboptimal titers. In support of this notion is our observation that most individuals did not display a detectable humoral response to M2e. We observed that fewer than 20% (23/140) of the individuals that we sampled in our cohort of healthy subjects had detectable serum levels of anti-M2e antibodies. The reasons for this phenomenon are not clear but a similar situation exists in HCMV where only a minority of HCMV seropositive subjects has measurable antibodies to the broadly conserved, neutralizing AD2 epitope within the gB complex of HCMV (Meyer H, et al. (1992) J Gen Virol 73:2375-2383; Ayata M, et al. (1994) J Med Virol 43:386-392; Navarro D, et al. (1997) J Med Virol 52:451-459).
An important requirement for an immunotherapeutic solution to the influenza threat will be the identification of protective epitopes that are conserved in pre-existing and emerging viruses. Using large-scale sampling of the human immune response to native influenza M2 we have identified a naturally immunogenic and protective epitope within the highly conserved N-terminal region of M2e. Human antibodies directed to this epitope, including those described in the present study, may be useful for the prevention and treatment of pandemic and seasonal influenza.
Methods
Memory B cell culture. Whole blood samples were collected from normal donors under IRB approved informed consent and peripheral blood mononuclear cells (PBMC) were purified by standard techniques. B cell cultures were set up using PBMC, B cells enriched by selection with M2-expressing cells, or IgG+ memory B cells enriched from PBMC via negative depletion of nonIgG+ cells with antibodies to CD3, CD 14, CD 16, IgM, IgA, and IgD on magnetic beads (Miltenyi, Auburn, Calif.) as previously described (Walker L, et al. (2009) Science 326:289-293). Briefly, to promote B cell activation, proliferation, terminal differentiation and antibody secretion, cells were seeded in 384-well microtiter plates in the presence of feeder cells and conditioned media generated from mitogen-stimulated human T cells from healthy donors. The culture supernatants were collected 8 days later and screened in a high throughput format for binding reactivity to M2 protein expressed on HEK 293 cells stably transfected with influenza virus M2 (A/Fort Worth/50 H1N1) using fluorescent imaging (FMAT system, Applied Biosystems).
Reconstitution of recombinant mAbs from B cell cultures. mRNA was isolated from lysed B-cell cultures using magnetic beads (Ambion). After reverse transcription (RT) with gene-specific primers, variable domain genes were PCR amplified using VH, V , and Vλ family-specific primers with flanking restriction sites (Walker L, et al. (2009) Science 326:289-293). PCR reactions producing an amplicon of the expected size were identified using 96-well E-gels (Invitrogen) and the variable domain amplicons were cloned into the pTT5 expression vector (National Research of Canada, Ottawa, Canada) containing human IgG1, Igκ, or Igλ constant regions. Each VH pool was combined with the corresponding Vκ, or Vλ pools from individual BCC wells and was transiently transfected in 293-6E cells to generate recombinant antibody. Conditioned media was harvested 3-5 days after transfection and assayed for antibody binding to M2 protein expressed on HEK 293 cells. Individual clones were isolated from positive pools and unique VH and VL genes were identified by sequencing. From these, monoclonal antibodies were subsequently expressed and re-assayed for binding activity.
ELISA. To detect viral antigen, either 10.2 μg/mL UV-inactivated H1N1 A/Puerto Rico/8/34 (PR8) virus (Advanced Biotechnologies, Inc.) was passively adsorbed to 384-well plates in 25 μL PBS/well for 16 hr at 4° C., or PR8 inactivated by β-propiolactone (Advanced Biotechnologies, Inc.) was biotinylated (EZ-Link Sulfo-NHS-LC-Biotin, Pierce) and likewise adsorbed to plates coated with neutravidin (Pierce). Virus-coated and biotinylated virus-coated plates were blocked with PBS containing 1% milk or BSA, respectively. Binding of mAbs at the indicated concentrations was detected with HRP-conjugated goat anti-human Fc antibody (Pierce) and visualized with TMB substrate (ThermoFisher). The M2e peptide, SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 680) (Genscript) was passively adsorbed at 1 μg/mL and antibody binding to the peptide was detected by the same method.
FACS analysis of virally infected cells. To detect M2e following in vitro infection, MDCK cells were treated with PR8 at multiplicity of infection (MOI) of 60:1 for 1 hr at 37° C. after which the culture media was replaced. The infected MDCK cells were further cultured for 16 hr before harvesting for cell staining with the indicated mAbs. Bound anti-M2 mAbs were visualized on viable cells with Alexafluor 647-conjugated goat anti-Human IgG H&L antibody (Invitrogen). Flow cytometry was performed on FACSCanto equipped with the FACSDiva software (Becton Dickenson). For the panel of anti-M2 mAbs, 20 μL samples of supernatant from transient transfections from each of the IgG heavy and light chain combinations was used to stain the 293 stable cell line expressing M2 of A/Hong Kong/483/97 FACS analysis was performed as above.
M2 variant analyses. Individual full length M2 cDNA mutants were synthesized with single ala mutations at each position of the ectodomain representing A/Fort Worth/1/1950 (D20), as well as were the forty-three naturally occurring variants of M2 (Blue Heron Technology). They were cloned into the plasmid vector pcDNA3.1. After transient transfection with Lipofectamine (Invitrogen), HEK293 cells were treated with 1 μg/mL of the indicated mAbs in PBS supplemented with 1% fetal bovine serum and 0.2% NaN3 (FACS buffer). Bound anti-M2 mAbs were visualized on viable cells with Alexafluor 647-conjugated goat anti-Human IgG H&L antibody (Invitrogen). Flow cytometry was performed with FACSCanto equipped with the FACSDiva software (Becton Dickenson). The relative binding to the naturally occurring variants was expressed as the percentage of the respective mAb staining of the D20 transiently transfected cells, using the formula of Normalized MFI (%) 100×(MFIexperimental-MFImock transfected)/(MFID20-MFImock transfected).
Therapeutic efficacy studies in mice. Animal studies were conducted under Institutional Animal Care and Use Committee protocols. We inoculated 6 groups of 10 mice (female 6-8 week old BALB/C) intranasally with 5×LD50 of A/Vietnam/1203/04 (FIGS. 15a and b) or 6 groups of 5 mice intranasally with 5×LD50 A/Puerto Rico/8/34 (FIGS. 15c and d). At 24, 72, and 120 hours post-infection the mice received intraperitoneal injections of 400 μg/200 μL dose of the anti-M2e mAbs TCN-031 TCN-032, control human mAb 2N9, control chimeric mAb ch14C2, PBS, or were left untreated. Mice were weighed daily for 2 weeks and were euthanized when weight loss exceeded 20% (H5N1 study shown in FIGS. 15a and 15b and H1N1 study shown in FIGS. 15c and 15d) of the pre-infection body weight.
Antibody reactivity to A/California/4/2009 infected cells. MDCK cells were infected with media alone or media containing A/California/4/2009 (H1N1) or A/Memphis/14/1996 (H1N1) at an MOI of approximately 1 and were cultured for 24 hours at 37° C. The cells were detached from the tissue culture plates with trypsin, washed extensively, and then fixed in 2% paraformaldehyde for 15 minutes. The cells were incubated with 1 μg/ml of the indicated antibodies and the primary antibody binding was detected with Alexafluor 647-conjugated goat anti-Human IgG H&L antibody (Invitrogen). The cells were analyzed with a Becton Dickinson FACSCalibur and data were processed using FlowJo software.
Competition analysis of antibody binding. Transient transfection supernatant containing antibody was screened for binding to 293 cells stably transfected with M2 from H1N1 (A/Fort Worth/50 H1N1), or mock transfected cells, in the presence or absence of the M2e peptide SLLTEVETPIRNEWGCRCNDSSD (Genscript) at 5 μg/mL. Bound anti-M2 mAbs were detected with anti-huIgG Fc FMAT Blue at 700 ng/ml in DMEM with 10% FCS and visualized by fluorescent imaging (FMAT system, Applied Biosystems).
Example 13
Screening and Characterization of HA-Specific Antibodies Present in Human Plasma that Bind Purified Whole Inactivated Influenza A Virions, Bind Recombinant Homotrimeric HA Proteins, and Neutralize Infectious Influenza A
Fully human monoclonal antibodies specific for HA and capable of binding purified whole inactivated Influenza A Virions, binding recombinant homotrimeric HA proteins, and neutralizing live influenza A were identified in patient plasma, as described below.
Expression of Recombinant Soluble HA
An expression construct was generated containing a cDNA encoding an HA precursor (HAO) polypeptide corresponding to the derived HA sequence found in the Influenza subtypes, for example, as listed in Table 9. Recombinant HA0 precursor polypeptides of the invention lack an integral membrane or transmembrane domain, and contain additional amino acids at the C-terminus of the HA0 ectodomain, for instance, corresponding to the sequence:




wherein the thrombin cleavage site is bolded and italicized, the bacteriophage T4 fibritin “foldon” or trimerization domain is underlined, the last amino acid of the trimerization domain, “G”, is the start of the boxed “V5” epitope tag, which is followed by the hexa histadine (HIS) epitope tag in bold. The hexa-HIS tag within the preceding C-terminal region is used for purification of recombinant HA proteins of the invention. Thus, recombinant HA0 precursor proteins that contain a trimerization domain are considered recombinant HA homotrimeric proteins of the invention.

Recombinant HA homotrimeric proteins of the invention retain the native signal sequence to allow efficient secretion from art-recognized cell lines maintained in vitro, e.g. 293 HEK cells as done by Immune Technology Corp. (http://www.immune-tech.com/). Moreover, within these recombinant HA homotrimeric proteins, or the HA0 precursors thereof, the native HA1/HA2 viral protease cleavage site was maintained, for instance, in all of the sequences provided in Table 9, except SEQ ID NO: 737, in which the native cleavage site positioned at amino acids 337-347 and consisting of the sequence “PQREGGRRRKR” (SEQ ID NO: 1250) was substituted with the sequence “PQTETR” (SEQ ID NO: 1251).
Furthermore, exemplary receptor binding domains of recombinant HA homotrimeric proteins, or the HA0 precursors thereof, include the following structural elements: a 190 α-helix, a 130-loop, and a 220-loop (see, sequence of Influenza A strain A/Vietnam/1203/2004) (or equivalent HA structures in other Influenza A strains that the ordinarily skilled artisan could readily obtain by accessing public databases, including GenBank, http://www.ncbi.nlm.nih.gov, and The Influenza Sequence Database, www.flu.lanl.gov, and downloading sequences) (Stevens et al. 2006. Science 312: 404-410). In other embodiments of the invention, in which the recombinant HA homotrimeric protein, or HA0 precursor thereof, encoded by this expression construct is partially or entirely expressed and administered to a subject, these receptor binding domains may be modified. The term “modified” is meant to describe the removal of one or more structural elements. Alternatively, or in addition, “modified” is meant to describe the addition, deletion, substitution, inversion, or translocation of one or more amino acids within a structural element of a receptor-binding domain of HA. For instance, a linear or discontinuous epitope to which a HuMHA antibody of the invention binds is administered to a subject at risk of contracting an influenza infection to prevent the infection. Alternatively or in addition, a linear or discontinuous epitope to which a HuMHA antibody of the invention binds is administered to a subject prior to exposure to an influenza virus to prevent influenza infection. In other embodiments a structural mimic of the conformational or discontinuous epitope is administered to a subject. When the above proteins are used for prophylactic purposes, for instance, as a vaccine, it may be advantageous to modify one or more receptor binding domains to control the resultant immune response in the subject. Exemplary structural elements of HA that are optionally modified include, but are not limited to, the 190 α-helix, the 130-loop, and the 220-loop of HA.
Recombinant homotrimeric HA proteins of the invention are encoded by the following amino acid sequences, wherein the native sequence is bolded and the sequence of SEQ ID NO: 726 is normal (see also, Table 9):








A/California/4/09









(SEQ ID NO: 727)









DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKC



 


NIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERF


 


EIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKG


 


KEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGR


 


MNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTP


 


KGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGG


 


WTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVG


 


KEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKV


 


RSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVK


 


LESTRIYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDS


 


TGHHHHHH


 


A/Solomon Islands/3/06 - H1N1








(SEQ ID NO: 728)









DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCLLKGIAPLQLGNCS



 


VAGWILGNPECELLISRESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFER


 


FEIFPKESSWPNHTTTGVSASCSHNGESSFYKNLLWLTGKNGLYPNLSKSYANNKE


 


KEVLVLWGVHHPPNIGDQRALYHKENAYVSVVSSHYSRKFTPEIAKRPKVRDQEG


 


RINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDECDAKCQTP


 


QGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGG


 


WTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVG


 


KEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKV


 


KSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVK


 


LESMGVYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLD


 


STGHHHHHH


 


A/South Carolina/1/18








(SEQ ID NO: 729)









MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKL



 


CKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEE


 


LREQLSSVSSFEKFEIFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYP


 


KLSKSYVNNKGKEVLVLWGVHHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAA


 


RPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHD


 


CNTKCQTPHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIA


 


GFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVIEKMNTQFT


 


AVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKV


 


KSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGVKLES


 


MGVYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDSTGHH


 


HHHH


 


A/Japan/305/57 - H2N2








(SEQ ID NO: 730)









DQICIGYHANNSTEKVDTNLERNVTVTHAKDILEKTHNGKLCKLNGIPPLELGDCSI



 


AGWLLGNPECDRLLSVPEWSYIMEKENPRDGLCYPGSFNDYEELKHLLSSVKHFE


 


KVKILPKDRWTQHTTTGGSRACAVSGNPSFFRNMVWLTKEGSDYPVAKGSYNNTS


 


GEQMLIIWGVHHPIDETEQRTLYQNVGTYVSVGTSTLNKRSTPEIATRPKVNGQGG


 


RMEFSWTLLDMWDTINFESTGNLIAPEYGFKISKRGSSGIMKTEGTLENCETKCQT


 


PLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNVPQIESRGLFGAIAGFIEG


 


GWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFDGITNKVNSVIEKMNTQFEA


 


VGKEFGNLERRLENLNKRMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLY


 


DKVRMQLRDNVKELGNGCFEFYHKCDDECMNSVKNGTYDYPKYEEESKLNRNEI


 


KGVKLSSMGVYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPL


 


LGLDSTGHHHHHH


 


A/Wisconsin/67/05 - H3N2








(SEQ ID NO: 731)









QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDSPHQ



 


ILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVA


 


SSGTLEFNDESFNWTGVTQNGTSSSCKRRSNNSFFSRLNWLTQLKFKYPALNVTMP


 


NNEKFDKLYIWGVHHPVTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRIRNIP


 


SRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGS


 


IPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGW


 


EGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKE


 


FSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQ


 


LRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKS


 


GSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDSTGHHHH


 


HH


 


A/swine/Ontario/01911-2/99 - H4N6








(SEQ ID NO: 732)









QNYTGNPVICLGHHAVSNGTMVKTLTDDQIEVVTAQELVESQHLPELCPSPLRLVD



 


GQTCDIVNGALGSPGCDHLNGAEWDVFIERPTAVDTCYPFDVPDYQSLRSILANNG


 


KFEFIAEEFQWNTVKQNGKSGACKRANVNDFFNRLNWLTKSDGNAYPLQNLTKV


 


NNGDYARLYIWGVHHPSTDTEQTNLYKNNPGRVTVSTQTSQTSVVPNIGSRPWVR


 


GLSSRISFYWTIVEPGDLIVFNTIGNLIAPRGHYKLNSQKKSTILNTAVPIGSCVSKCH


 


TDKGSISTTKPFQNISRISIGDCPKYVKQGSLKLATGMRSILEKATRGLFGAIAGFIE


 


NGWQGLIDGWYGFRHQNAEGTGTAADLKSTQAAIDQINGKLNRLIGKPNEKYHQI


 


EKEFEQVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERV


 


RHQLRENAEDKGNGCFEIFHQCDNSCIESIRNGTYDHDIYRDEAINNRFQIQGVKLI


 


QGYKDSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDSTG


 


HHHHHH


 


A/Hong Kong/156/97 - H5N1








(SEQ ID NO: 733)









MERTVLLLATVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILERTHNGK



 


LCDLNGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGNFN


 


DYEELKHLLSRINHFEKIQIIPKSSWSNHDASSGVSSACPYLGRSSFFRNVVWLIKKN


 


SAYPTIKRSYNNTNQEDLLVLWGVHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLV


 


PEIATRPKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKS


 


ELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNTPQR


 


ERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSCYSADKESTQKAIDG


 


VTNKVNSIINKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLM


 


ENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGT


 


YDYPQYSEEARLNREEISGVKLESMGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKD


 


GEWVLLSTFLGKPIPNPLLGLDSTGHHHHHH


 


A/Vietnam/1203/04 - H5N1








(SEQ ID NO: 734)









DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCS



 


VAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFE


 


KIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQE


 


DLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRM


 


EFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMG


 


AINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFI


 


EGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFE


 


AVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNL


 


YDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEI


 


SGVKLESIGIYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLL


 


GLDSTGHHHHHH


 


A/Indonesia/5/05 - H5N1








(SEQ ID NO: 735)









DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCS



 


VAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFE


 


KIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQE


 


DLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRM


 


EFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPM


 


GAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGF


 


IEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQF


 


EAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNL


 


YDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEI


 


SGVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLL


 


GLDSTGHHHHHH


 


A/Egypt/3300-NAMRU3/08 - H5N1








(SEQ ID NO: 736)









DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCS



 


VAGWLLGNPMCDEFLNVSEWSYIVEKINPANDLCYPGNFNNYEELKHLLSRINRFE


 


KIQIIPKSSWPDHEASLGVSSACPYQGGPSFYRNVVWLIKKNNTYPTIKKSYHNTNQ


 


EDLLVLWGIHHPNDEAEQTRIYKNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGR


 


VEFFWTILKSNDTINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPI


 


GAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGF


 


IEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQF


 


EAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNL


 


YDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEI


 


SGVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLL


 


GLDSTGHHHHHH


 


A/Common magpie/Hong Kong/5052/07 - H5N1








(SEQ ID NO: 737)









DHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCS



 


VAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFE


 


KIQIIPKDSWSDHEASLGVSSACPYQGNSSFFRNVVWLIKKGNAYPTIKKSYNNTNQ


 


EDLLVLWGIHHPNDEAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRI


 


DFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSEVEYGNCNTRCQTPM


 


GAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRERRRKRGLFGAIAGFI


 


EGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFE


 


AVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNL


 


YDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEI


 


SGVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLL


 


GLDSTGHHHHHH


 


A/Anhui/1/05 - H5N1








(SEQ ID NO: 738)









DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCS



 


VAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFE


 


KIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQE


 


DLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRM


 


DFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIG


 


AINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLRERRRKRGLFGAIAGFIE


 


GGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEA


 


VGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLY


 


DKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEIS


 


GVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLG


 


LDSTGHHHHHH


 


A/chicken/Vietnam/NCVD-016/08 - H5N1








(SEQ ID NO: 739)









DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILKDCS



 


VAGWLLGNPMCDEFLNVSEWSYIVEKASPANGLCYPGDFNDYEELKHLLSRINHL


 


KKIKIIPKSYWSNHEASSGVSAACSYLGEPSFFRNVVWLIKKNNTYPPIKVNYTNTN


 


QEDLLVLWGIHHPNDEKEQIRIYQNPNTSISVGTSTLNQRLVPKIATRPKVNGQSGR


 


MEFFWTILKPNDSINFDSNGNFIAPEYAYKIAKKGDSVIMKSELEYGNCNTKCQTP


 


MGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNAPQTETRGLFGAIAGFIEG


 


GWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGITNKVNSIIDKMNTQFEIVG


 


REFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYEKV


 


RLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLSREEISGVK


 


MESMVTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLD


 


STGHHHHHH


 


A/northern shoveler/California/HKWF115/2007 - H6N1








(SEQ ID NO: 740)









DKICIGYHANNSTTQVDTILEKNVTVTHSVELLENQKEERFCKILNKAPLDLRGCTI



 


EGWILGNPQCDLLLGDQSWSYIVERPTAQNGICYPGALNEVEELKALIGSGERVER


 


FEMFPKSTWTGVDTSSGVTKACPYNSGSSFYRNLLWIIKTKSAAYPVIKGTYNNTGS


 


QPILYFWGVHHPPDTNEQNTLYGSGDRYVRMGTESMNFAKSPEIAARPAVNGQRG


 


RIDYYWSVLKPGETLNVESNGNLIAPWYAYKFVSTNNKGAIFKSNLPIENCDATCQ


 


TIAGVLRTNKTFQNVSPLWIGECPKYVKSESLRLATGLRNVPQIETRGLFGAIAGFI


 


EGGWTGMIDGWYGYHHENSQGSGYAADKESTQKAIDGITNKVNSIIDKMNTQFEA


 


VDHEFSNLERRIDNLNKRMEDGFLDVWTYNAELLVLLENERTLDLHDANVKNLYE


 


KVKSQLRDNANDLGNGCFEFWHKCDNECIESVKNGTYDYPKYQDESKLNRQEIES


 


VKLDNLGVYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLG


 


LDSTGHHHHHH


 


A/Netherlands/219/03 - H7N7








(SEQ ID NO: 741)









DKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNVPRICSKGKRTVDLGQCG



 


LLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKET


 


MGFTYSGIRTNGTTSACRRSGSSFYAEMKWLLSNTDNAAFPQMTKSYKNTRKDPA


 


LIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNGQSGRIDFH


 


WLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSEVQVDANCEGDCYHSGGTIIS


 


NLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEIPKRRRRGLFGAIAGFIENGW


 


EGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEF


 


TEVERQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVKR


 


QLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAIQNRIQIDPVKLSSG


 


YKDSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDSTGHH


 


HHHH


 


H8N4 - A/duck/Yangzhou/02/2005








(SEQ ID NO: 742)









DRICIGYQSNNSTDTVNTLIEQKVPVTQTMELVETEKHPAYCNTDLGAPLELRDCKI



 


EAVIYGNPKCDIHLKDQGWSYIVERPSAPEGMCYPGSVENLEELRFVFSSAASYKRI


 


RLFDYSRWNVTSSGTSKACNASTGGQSFYRSINWLTKKKPDTYDFNEGTYVNNED


 


GDIIFLWGIHHPPDTKEQTTLYKNANTLSSVTTNTINRSFQPNIGPRPLVRGQQGRM


 


DYYWGILKRGETLKIRTNGNLIAPEFGYLLKGESHGRTIQNEDIPIGNCYTKCQTYA


 


GAINSSKPFQNASRHYMGECPKYVKKASLRLAVGLRNTPSVEPRGLFGAIAGFIEG


 


GWSGMIDGWYGFHHSNSEGTGMAADQKSTQEAIDKITNKVNNIVDKMNREFEVV


 


NHEFSEVEKRINMINDKIDDQIEDLWAYNAELLVLLENQKTLDEHDSNVKNLFDEV


 


KRRLSANAIDAGNGCFDILHKCDNECMETIKNGTYDHKEYEEEAKLERSKINGVKL


 


EENTTYKSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDST


 


GHHHHHH


 


A/Hong Kong/2108/03 - H9N2








(SEQ ID NO: 743)









DKICIGYQSTNSTETVDTLTKTNVPVTQAKELLHTEHNGMLCATNLGHPLILDTCTI



 


EGLIYGNPSCDLLLGGREWSYIVERPSAVNGMCYPGNVENLEELRLLFSSASSYQR


 


VQIFPDTIWNVTYSGTSSACSNSFYRSMRWLTQKDNTYPVQDAQYTNNRGKSILFM


 


WGINHPPTDTVQTNLYTRTDTTTSVTTEDINRAFKPVIGPRPLVNGLQGRIDYYWS


 


VLKPGQTLRVRSNGNLIAPWYGHILSGESHGRILKSDLNSGNCVVQCQTERGGLNT


 


TLPFHNVSKYAFGNCPKYVGVKSLKLAVGMRNVPARSSRGLFGAIAGFIEGGWPG


 


LVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFS


 


EIETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALG


 


SNAMEDGKGCFELYHKCDDRCMETIRNGTYNRGKYKEESRLERQKIEGVKLESEG


 


TYKSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDSTGHH


 


HHHH


 


A/Hong Kong/1073/99 - H9N2








(SEQ ID NO: 744)









METISLITILLVVTASNADKICIGHQSTNSTETVDTLTETNVPVTHAKELLHTEHNG



 


MLCATSLGHPLILDTCTIEGLVYGNPSCDLLLGGREWSYIVERSSAVNGTCYPGNV


 


ENLEELRTLFSSASSYQRIQIFPDTTWNVTYTGTSRACSGSFYRSMRWLTQKSGFYP


 


VQDAQYTNNRGKSILFVWGIHHPPTYTEQTNLYIRNDTTTSVTTEDLNRTFKPVIGP


 


RPLVNGLQGRIDYYWSVLKPGQTLRVRSNGNLIAPWYGHVLSGGSHGRILKTDLK


 


GGNCVVQCQTEKGGLNSTLPFHNISKYAFGTCPKYVRVNSLKLAVGLRNVPARSSR


 


GLFGAIAGFIEGGWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNI


 


VDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDVWAYNAELLVLLENQKTLDEH


 


DANVNNLYNKVKRALGSNAMEDGKGCFELYHKCDDQCMETIRNGTYNRRKYREE


 


SRLERQKIEGVKLESEGTYKSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTF


 


LGKPIPNPLLGLDSTGHHHHHH






The recombinant and soluble HA expression constructs of SEQ ID NO: 727-744 were transfected into 293 HEK cells. Recombinant HA0 protein or HA cleaved into its respective subunits HA1 and HA2 and disulphide linked was purified from culture supernatant by standard procedures using the hexa-HIS tag at the C-terminal. The purified protein was analyzed by size exclusion chromatography and/or denaturing coomasie gel to confirm a recombinant protein of the expected size was present.
Example 14
Screening of Antibodies in Peripheral Blood
One hundred and twenty-six individual serum or plasma samples were screened for the presence of IgG antibody that bound to recombinant soluble homotrimeric HA proteins (Table 9) using a micro-array scanning system, bind to whole inactivated Influenza A virions (Table 10) using standard ELISA techniques, and inhibits or neutralizes virus infection of MDCK cells with Influenza A H1N1 A/Solomon Islands/3/06 or H3N2 A/Wisconsin/67/05. A portion of the plasma samples contained IgG antibodies that bound specifically to the recombinant soluble HA homotrimeric protein, bound to inactivated virions, and neutralized virus infectivity. This indicates that the antibodies could be binding linear or discontinuous epitopes in the HA homotrimer, as well as binding to conformational determinants of multiple variants of the HA homotrimer. Soluble targets include, but are not limited to, exemplary recombinant HA proteins derived from the influenza virus strains listed in Table 2 and the inactivated virus strains listed in Table 11.







TABLE 11







Inactivated whole virions used in ELISA binding assays.










Influenza A




Subtype
Strain designation







H1N1
A/Solomon Islands/3/06



H2N2
A/Japan/305/57



H3N2
A/Wisconsin/67/05










Example 15
Identification and Rescue of HA-Specific Antibodies
IgG+ memory B cells purified from a human blood sample were cultured for 9 days in order to activate, proliferate, and differentiate these memory B cells into IgG secreting plasma cells. The B cell culture media containing the IgG was screened for the presence of IgG antibody that bound to recombinant soluble homotrimeric HA proteins (Table 9) using a micro-array scanning system, bind to whole Influenza A virus (Table 10) using standard ELISA techniques, and inhibits or neutralizes virus infection of MDCK cells with Influenza A H1N1 A/Solomon Islands/3/06 or H3N2 A/Wisconsin/67/05. As shown in Tables 12, 13, and 14, thirty-nine BCC wells were identified that contained an IgG antibody with several virus, HA, or neutralizing profiles and the variable regions of the antibody were then cloned from the corresponding B cell cultures.
Transient transfections with monoclonal heavy and light chain pairs from each BCC well were performed in 293 6E cells to reconstitute and produce the antibody. Reconstituted antibody supernatants were then screened for IgG that binds to recombinant soluble homotrimeric HA proteins (Table 9) using a micro-array scanning system, bind to whole Influenza A virus (Table 11) using standard ELISA techniques, and inhibits or neutralizes virus infection of MDCK cells with Influenza A H1N1 A/Solomon Islands/3/06, and/or H1N1 A/California/04/09, and/or H3N2 A/Wisconsin/67/05 to identify the rescued anti-HA antibody. Binding and neutralization of the human IgG antibodies to the preceding targets is compared to the binding of the proprietary positive control antibody TCN-032 (specific for the N-terminal of the matrix 2 protein of influenza A, U.S. patent application Ser. No. 12/795,618) or to the binding of the broadly HA specific, non-neutralizing mAb TCN-504 (also known as 3251K17, and described herein), and the proprietary negative control antibody TCN-202 (specific for the AD2 site I epitope on human cytomegalovirus gB) to these same targets.
The sequences of the rescued antibodies are determined. In addition, the sequence of each of the polynucleotides encoding the antibody sequences is determined.
Example 16
Binding Profiles of IgG in B Cell Culture Supernatant or Monoclonal Transfection Supernatant Using Inactivated Whole Influenza A Virions
To determine whether the human mAbs in BCC SN or monoclonal transfection supernatantbind to purified virus, Enzyme-Linked ImmunoSorbent Assays (ELISAs) were performed according to the following methods. Briefly, various purified Influenza A virus subtype strains were coated directly onto an ELISA plate. A single dilution of BCC supernatant of the mAbs shown in Table 12 or of the monoclonal transfection supernatant shown in Table 15 and various dilutions of a positive control antibody (TCN-032) were then added to the virus coated wells. Unbound antibody was washed away and the bound antibody was detected with an anti-human antibody conjugated to HRP. The presence of anti-influenza antibodies was detected when the chromogen (TMB) is oxidized by the HRP conjugate, resulting in a bluecolor. The reaction is stopped by the addition of HCl, turning antibody positive wells yellow. The yellow color has a maximal absorbance at 450 nm.
Methods:

    Coat MicrolonTM plates with 25 ul/well of 3 ug/ml inactivated influenza A virions
    Incubate plates overnight at 4° C.
    Remove plates from 4° C. and wash four times with phosphate buffered saline with Calicum and Magnesium (PBS w/Ca2+,Mg2+), using EL405× (Wash program: ELISA—4×_wash).
    Add 20 ul/well of 1% milk/PBS to plates.
    Prepare control mAb curves by 1:3 dilutions of 6 ug/ml
    5 ul of each BCC SN or monoclonal transient transfection supernatant and control mAb curves are stamped onto the plate according to the plate map.
    Incubate 2 hour (hr) at room temperature (RT).
    Wash plates four times with PBS w/Ca2+,Mg2+, using EL405× (Wash program: ELISA—4×_wash)
    Add 25 ul/well of polyclonal antibody (pAb) goat anti-human (αHuman) IgG Fc horseradish peroxidase (HRP) at a dilution of 1:5000.
    Incubate 1 hr at RT.
    Wash plates four times with PBS w/Ca2+,Mg2+, using EL405× (Wash program: ELISA—4×_wash)
    Add 25 ul/well of Ultra-TMB (3,3′,5,5′-tetramethylbenzidine) at Neat.
    Develop 30 minutes (min) at RT.
    Stop by adding 25 ul/well of hydrochloric acid (HCl) at a concentration of 0.3M.
    Read the plate at 450 nm with the Spectromax.


One or more of the following control antibodies were used in these experiments: TCN-504 (also known as 3251_K17, and described herein) TCN-032 (also known as 8I10, specific for the influenza A M2 protein), and TCN-202 (also known as 2N9, specific for the AD2 site I epitope on human cytomegalovirus gB) protein.
The following purified viruses were used in these experiments: A/Solomon Islands/3/2006(H1N1) (SEQ ID NO: 728), A/Japan/305/1957(H2N2) (SEQ ID NO: 729), A/Wisconsin/67/2005(H3N2) (SEQ ID NO: 730). As shown in Table 15 the human monoclonal antibodies in the transient transfection supernatant bind strongly to one or more of the H1N1, H2N2, and/or H3N2 viruses reproducing the virus binding profile of the IgG antibody in the original BCC SN (Table 12).
Example 17
Binding Profiles of IgG in in B Cell Culture Supernatant or Monoclonal Transfection Supernatant Using Trimeric HA
To determine whether the human mAbs in BCC SN or monoclonal transfection supernatant bind to one or more of the recombinant homotrimeric HA proteins, a micro-array scanning system was used according to the following methods. Twenty Nexterion P (Schott) glass slides are incubated overnight in a humid chamber with 3 mg/ml goat anti-human, Fc-specific antibody. Nexterion P slides contain a hydrogel that terminates in an NHS ester reactive group that binds the capture antibody. The reaction is then quenched using 50 mM ethanolamine in 50 mM NaBorate (pH 8.0) buffer for 1 hour with agitation followed by 3 washes with ultrapure water. Transfection supernatants are transferred to 384-well array source plates. Control array source plates are made in 8-point, 3-fold serial dilutions of antibody starting at 3 ug/ml and ending 0 ug/ml in BCC or mock transfection media. Both BCC or transfection supernatant array source plates and control plates are loaded onto an Aushon 2470 microarray printer along with the 20 prepared slides.
Microarrays are printed in 48 subarray blocks with variable number of features and replicates depending on number of transfection supernatants are being printed. Printed microarrays are allowed to sit for at least 1 hour after printing (to overnight) in the printer's humidity controlled chamber (80%). They are then quickly removed from the printer, washed, and spun dry to prepare the slides for sample incubations. A LifterSlip is placed on each slide and 90 uls of sample is added to each. Each slide receives either one of 18 V5-tagged HA trimerics at predetermined concentrations, a horseradish peroxidase goat anti-human H+L specific antibody at a 1 ug/ml, or a blank for a total of 20 slides. The slides are then incubated overnight in a humid chamber at room temperature. After washing, spin drying, and new LifterSlip application, 19 slides, excluding slide incubated with anti-human IgG, are incubated with anti-V5 conjugated to biotin at a 1:1000 dilution for 1 hour. Subsequently the slides are again washed, dried and prepared for an 1-hour incubation with a 1:300 dilution of NeutrAvidin-HRP. After further washing, drying and preparation all 20 slides are incubated for 1 hour with a Tyramide-AlexaFluor reagent according to kit instructions. After final washing and drying the slides are scanned on an Axon Genepix 4300A at an excitation wavelength of 594 nm and with an emission band ranging from 619 nm to 641 nm. Data is recovered using the Axon Genepix software and is then analyzed for binding profiles.
Methods:

    1. Dilute goat anti-human, FC -specific antibody (Jacksonlmmuno, 10 mg/ml) to 3 mg/ml in PBS 0.05&Tween and apply to 20 Nexterion P Slides (Schott) using 90 uls under lifterslips (Thermo).
    2. Incubate in a humid chamber overnight at room temperature
    3. Removing the lifterslip quench all slides in 50 mM ethanolamine (Fisher)) in 50 mM Sodium tetraborate decahydrate (Fisher, S248-500) at a pH of 8.0
    4. Quench for 1 hour with incubation at room temperature
    5. Wash all slides (3×2 min MilliQ water washes with agitation).
    6. Load all slides onto Aushon 2470 MicroArray printer.
    7. Prepare control plates using by spiking control antibodies into transfection media to form an 8 point 3-fold serial dilution starting at 3 ug/ml and ending in 0 ug/ml. Transfer control dilutions to 4 array source plates (Thermo).
    8. Load all array source plates, control and sample, onto Aushon 2470 microarray printer and begin deposition after checking all required fluid levels. Number of replicates is based on number of transfection supernatants being printed. Typically 1 to 10 replicates.
    9. Allow slides to sit for at least an hour, to overnight, after deposition.
    10. Immediately wash (PBS with 2% tween 20, 5 min; MilliQ water, 2 min, 3×) and spin dry (2000 RPM for 1 minute)
    11. Using lifterslips and apply 90 uls of HA at the following concentration:













Homotrimeric HA - strain
[ug/ml]
















A/California/4/09 - H1N1
5


A/Solomon Islands/3/06 - H1N1
5


A/Japan/305/57 - H2N2
5


A/Wisconsin/67/05 - H3N2
20


A/swine/Ontario/01911-2/99 - H4N6
5


A/Vietnam/1203/04 - H5N1
20


A/Indonesia/5/05 - H5N1
0.5


A/Egypt/3300-NAMRU3/08 - H5N1
0.5


A/Common magpie/Hong Kong/5052/07 - H5N1
0.5


A/Anhui/1/05 - H5N1
0.5


A/chicken/Vietnam/NCVD-016/08 - H5N1.
0.5


A/northern shoveler/California/HKWF115/2007 - H6N1
0.5


A/Netherlands/219/03 - H7N7
0.5


A/duck/Yangzhou/02/2005 - H8N4
5


A/Hong Kong/2108/03 - H9N2
5


A/South Carolina/1/18 - H1N1
20


A/Hong Kong/1073/99
20


A/Hong Kong/156/97 - H5N1
5









Use 1× PBS, 0.05% Tween 20, 10% Blocker Casein (Thermo, #37528) to bring the homotrimeric HA to the desired concentration.

    12. Incubate overnight in a humid chamber at room temperature
    13. Immediately wash (PBS with 2% tween 20, 5 min; MilliQ water, 2 min, 3×) and spin dry (2000 RPM for 1 minute)
    14. Using lifterslips incubate all but slide previously incubated with anti-human IgG(H&L)-HRP with 90 uls of anti-V5-biotin (AbD Serotec, MCA1360B) at 1 ug/ml in 1× PBS 0.05% Tween 20 for 1 hour at room temperature in a humid chamber.
    15. Immediately wash (PBS with 2% tween 20, 5 min; MilliQ water, 2 min, 3×) and spin dry (2000 RPM for 1 minute)
    16. Using lifterslips incubate with 90 uls of horseradish peroxidase conjugated NeutrAvidin (Pierce #31030) for 1 hour at room temperature in a humid chamber.
    17. Immediately wash (PBS with 2% tween 20, 5 min; MilliQ water, 2 min, 3×) and spin dry (2000 RPM for 1 minute)
    18. Prepare Tyramide Signal Amplification reagent according to kit instructions (TSA Kit #25, Invitrogen, T20935). Briefly dilute 1 ul of hydrogen peroxide solution into 200 uls of amplification buffer. Take 20 uls of hydrogen peroxide/amplification buffer solution and add to 1940 uls of fresh amplification buffer. Then add 40 uls of tyramide-Alexa Fluor resulting in a total of 2 mls of amplification reagent
    19. Incubate all 20 slides with amplification reagent for 1 hour at room temperature in a humid chamber.
    20. Immediately wash (PBS with 2% tween 20, 5 min; MilliQ water, 2 min, 3×) and spin dry (2000 RPM for 1 minute)
    21. Scan all slides on an Axon Genepix 4300A at an excitation wavelength of 594 nm and with an emission band ranging from 619 nm to 641 nm or optical scanner with similar capabilities.
    22. Lay templates on each slide using GenePix Pro 7 or similar software to recover feature data.
    23. Analyze feature data for binding profiles to each HA trimeric.


As shown in Table 16, the human monoclonal antibodies in the transient transfection supernatant bind strongly to one or more of the recombinant homotrimeric HA proteins reproducing the virus binding profile of the IgG antibody in the original BCC SN (Table 12).
Example 18
Neutralization Profiles of IgG in in B Cell Culture Supernatant or Monoclonal Transfection Supernatant
MDCK cells were plated at 3×103 cells/well in a 384-well plate in complete DMEM media (supplemented with 10% FBS, 1× penicillin/streptomycin, 1× GlutamaxTM, and 1× Sodium Pyruvate) and incubated at 37° C. overnight.
Influenza A virus was preincubated with either BCC supernatant or monoclonal transfection supernatant or a positive control neutralizing monoclonal antibody (MAb), which was diluted in pooled BCC supernatant or in mock transfection supernatant at the desired concentrations and incubated overnight ( ̃16 hours) at 37° C. Void volumes and dilutions were made in PBS with Mg2+, Ca2+, 200 mM Mannose, and 1% BSA at 37° C. with a total volume of 30 μl/well.
Each sample well contained:
(a) 20 μl/well BCC supernatant; or
(b) 20 ul/well of monoclonal transfection
(c) 3000 IU/well A/Solomon Islands/03/2006 (H1N1) in 10 μl/well; or
(e) 3000 IU/well A/California/04/2009 (H1N1) in 10 μl/well; or
(f) 3000 IU/well A/Wisconsin/67/05 (H3N2) in 10 ul/well
Prior to infection, the MDCK cells were washed once with a solution containing PBS, Mg2+, and Ca2+ at 60 μl/well. After the wash, 25 ul of the virus/mAb mixture was transferred and the infection proceeded for 4 hours at 37° C.
After 4 hours of infection, the MDCK cells are washed twice with complete DMEM. After the last wash, 25 μl/well of complete DMEM remained, and the plates were incubated overnight at 37° C.
After the overnight incubation the culture media was removed and 20 μl of BD Cytofix/CytopermTM (cat #51-2090KZ) was added to each well and incubated at room temperature (RT) for 30 minutes. Next, the wells were washed three times using the M384 Atlas plate washer.
After the final wash, 15 μl/well of 100 μg/ml Rabbit IgG (Sigma) in PBS with Mg2+, Ca2+, and 1% BSA, was added and incubated for at least 5 minutes at RT. Twenty μl/well of anti-M2e mAb TCN-032 at 2 μg/ml in PBS with Mg2+, Ca2+, and 1% BSA, were added to each well and incubated for at least 30 mins at room temperature. The wells are washed one time using the Atlas plate washer with PBS with Mg2+, Ca2+ Twenty μl/well of 2 μg/ml Alexa Fluor® 647 anti-Human IgG H+L (InvitrogenTM) and 20 μg/ml Hoechst 33342 (InvitrogenTM) was added and incubated in the dark for 45 mins at room temperature. Wells were washed three timesusing the Atlas plate washer with PBS with Mg2+, Ca2+. Twenty microliters of PBS with Mg2+, Ca2+, and 1% BSA was added to each well, and plates were sealed with black sealing tape. Plates were analyzed by scanning using an IN Cell analyzer. Specifically, the scan was performed using the IN Cell Developer Software, Protocol “384 GG Hoechst AF647 4×.” Analysis of the scan was performed using the Developer Tool Box software, Protocol “Cellular Binding Nuclei GG Density 4.”
The assay was able to detect well supernatants that individually neutralized the Influenza A infection. If an arbitrary cutoff was established at ≦150 nucleoprotein (NP)+ cells and the wells with a disrupted cell monolayer were subtracted, a total of 122 wells scored as positive.
Exemplary influenza neutralizing antibodies that were identified using this method are TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), and TCN-504 (3251_K17). The individual neutralization activities of some of these antibodies are provided in Table 17.
Several antibodies were identified that may be non-neutralizing, including, TCN-504 (3251_K17), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-559 (5097_G08), and TCN-560 (5084_P10). These antibodies, similar to the neutralizing antibodies of the invention, bind to a broad range of HA proteins, including sequence and conformational variants. In certain embodiments of the invention, non-neutralizing antibodies, including TCN-504 (3251_K17), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-559 (5097_G08), and TCN-560 (5084_P10) may be used as antibody-drug conjugates.







TABLE 12







Summary of BCC SN screening by ELISA for virus binding.









ELISA: Virus Binding (OD450)













A/Solomon
A/Japan/
A/Wisconsin/


Theraclone
BCC well
Islands/
305/57
67/05


mAb ID
ID
3/06 H1N1
H2N2
H3N2





TCN-521
3280_D18
3.70
3.10
1.10


TCN-522
3212_I12
2.12
ND
0.07


TCN-523
5248_A17
3.47
1.62
0.08


TCN-563
5237_B21
3.62
1.23
0.07


TCN-526
5084_C17
0.06
0.07
3.65


TCN-527
5086_C06
3.03
1.48
0.07


TCN-528
5087_P17
3.62
2.82
0.24


TCN-529
5297_H01
0.07
ND
3.62


TCN-530
5248_H10
3.52
1.73
0.06


TCN-531
5091_H13
3.23
0.67
3.45


TCN-532
5262_H18
0.06
0.07
3.67


TCN-533
5256_A17
3.54
1.10
0.10


TCN-534
5249_B02
3.55
2.56
0.07


TCN-535
5246_P19
3.43
1.46
0.08


TCN-536
5095_N01
3.63
0.08
3.66


TCN-537
3194_D21
3.24
ND
0.06


TCN-538
3206_O17
3.47
ND
0.07


TCN-539
5056_A08
0.06
0.06
2.85


TCN-540
5060_F05
0.07
3.62
3.65


TCN-541
5062_M11
3.44
0.06
0.25


TCN-542
5079_A16
3.66
0.08
3.13


TCN-543
5081_G23
3.63
3.62
0.07


TCN-544
5082_A19
0.32
0.07
2.71


TCN-545
5082_I15
3.32
0.06
0.47


TCN-546
5089_L08
1.95
0.06
3.69


TCN-547
5092_F11
0.06
0.07
3.68


TCN-548
5092_P01
0.09
0.09
3.62


TCN-549
5092_P04
0.09
0.08
3.58


TCN-550
5096_F06
0.06
0.06
3.65


TCN-551
5243_D01
3.35
0.19
0.07


TCN-552
5249_I23
3.57
0.71
0.06


TCN-553
5261_C18
3.60
2.54
0.07


TCN-554
5277_M05
0.06
ND
1.09


TCN-555
5246_L16
2.89
0.60
0.06


TCN-556
5089_K12
2.70
2.41
0.06


TCN-557
5081_A04
2.32
2.70
0.07


TCN-559
5097_G08
3.68
1.25
0.70


TCN-560
5084_P10
3.63
2.07
0.07


TCN-202
2N9
ND
ND
ND


TCN-504
3251_K17
ND
ND
ND


TCN-032
8I10
3.61
3.61
3.62
















TABLE 13





Summary of BCC SN screening for virus binding to recombinant homotrimeric HA.

















Trimeric HA Binding: RLU
























A/


A/





A/
A/


swine/


Egypt/




A/
Solomon
South
A/
A/
Ontario/
A/
A/
3300-




California/
Islands/
Carolina/
Japan/
Wisconsin/
01911-2/
Vietnam/
Indonesia/
NAMRU3/


Theraclone
BCC well
4/09
3/06
1/18
305/57
67/05
99
1203/
5/05
08


mAb ID
ID
H1N1
H1N1
H1N1
H2N2
H3N2
H4N6
04 H5N1
H5N1
H5N1





TCN-521
3280_D18
25726
39644
ND
6732
2298
210
196
31473
47871


TCN-522
3212_I12
5019
38914
ND
989
302
156
145
67
ND


TCN-523
5248_A17
34721
37916
ND
5111
22568
1288
3383
45815
36471


TCN-563
5237_B21
45157
30005
ND
6612
40848
4501
4836
39533
27514


TCN-526
5084_C17
203
341
ND
246
49312
253
372
46027
ND


TCN-527
5086_C06
27991
16507
ND
19261
5715
7686
21264
23838
ND


TCN-528
5087_P17
48845
44804
ND
46393
48795
47500
45577
42801
ND


TCN-529
5297_H01
434
179
ND
435
51631
235
101
44085
53394


TCN-530
5248_H10
46600
32801
ND
47049
36846
2743
7152
39774
30430


TCN-531
5091_H13
22207
51663
ND
410
7094
408
357
37443
ND


TCN-532
5262_H18
135
327
ND
176
18046
119
440
27992
24500


TCN-533
5256_A17
29280
39186
ND
10806
13823
1582
5677
44063
34299


TCN-534
5249_B02
30109
44185
ND
50626
7978
683
3435
46352
41381


TCN-535
5246_P19
48576
39442
ND
26068
34320
5950
4740
45592
39412


TCN-536
5095_N01
151
150
ND
121
34996
79
146
3969
ND


TCN-537
3194_D21
21918
44264
ND
44685
549
19
80
14858
38168


TCN-538
3206_O17
13808
33228
ND
43002
6900
406
60
26421
23553


TCN-539
5056_A08
1803
239
ND
468
715
1579
545
49
11420


TCN-540
5060_F05
57
64
ND
2969
728
178
63
31
1968


TCN-541
5062_M11
34
51
ND
83
2923
102
43
30
1162


TCN-542
5079_A16
3108
132
ND
142
1940
350
306
ND
ND


TCN-543
5081_G23
13080
511
ND
197
1358
428
488
ND
ND


TCN-544
5082_A19
281
179
ND
187
2090
316
349
3052
ND


TCN-545
5082_I15
365
266
ND
157
575
289
235
ND
ND


TCN-546
5089_L08
350
256
ND
520
30209
587
349
18432
ND


TCN-547
5092_F11
170
155
ND
40
16932
252
308
6986
ND


TCN-548
5092_P01
234
283
ND
416
48600
240
270
17626
ND


TCN-549
5092_P04
338
284
ND
387
30912
421
312
11445
ND


TCN-550
5096_F06
454
204
ND
201
26315
195
277
9100
ND


TCN-551
5243_D01
53362
53821
ND
22633
6840
733
4152
53543
47183


TCN-552
5249_I23
35312
39314
ND
23832
14769
493
2728
43559
39971


TCN-553
5261_C18
20281
16271
ND
25509
20043
2583
6560
24406
18828


TCN-554
5277_M05
173
115
ND
328
46531
78
113
32348
36126


TCN-555
5246_L16
44903
26404
ND
2131
9800
1953
876
34539
42676


TCN-556
5089_K12
14640
2846
ND
5611
9323
5604
13823
8454
ND


TCN-557
5081_A04
17603
40699
ND
43367
10218
26967
47282
45165
ND


TCN-559
5097_G08
539
7376
ND
16332
2402
1658
21670
18295
ND


TCN-560
5084_P10
49166
38758
ND
46720
49078
41599
43990
35864
ND


TCN-202
2N9
39
46
ND
85
252
75
53
24
1275


TCN-504
3251_K17
3326
24140
ND
4091
40516
14669
196
43259
44352


TCN-032
8I10
ND
ND
ND
ND
ND
ND
ND
ND
ND












Trimeric HA Binding: RLU



















A/



A/








common

A/

northern








magpie/

chicken/
A/
shoveler/

A/
A/
A/




Hong
A/
Vietnam/
Hong
California/
A/
duck/
Hong
Hong




Kong/
Anhui/1/
NCVD-
Kong/
HKWF115/
Netherlands/
Yangzhou/
Kong/
Kong/



Theraclone
5052/07
05
016/08
156/97
07
219/03
02/05
2108/03
1073/99



mAb ID
H5N1
H5N1
H5N1
H5N1
H6N4
H7N7
H8N4
H9N2
H9N2






TCN-521
13528
487
32390
ND
47991
2496
11659
93
ND



TCN-522
ND
ND
ND
ND
ND
ND
502
639
ND



TCN-523
45837
5705
48880
ND
46908
2336
28949
189
ND



TCN-563
27500
9581
40953
ND
27328
7515
46729
225
ND



TCN-526
ND
20916
179
ND
2100
1338
596
36629
ND



TCN-527
ND
21369
27047
ND
30323
8892
28803
15956
ND



TCN-528
ND
49772
38191
ND
46781
49251
38362
41966
ND



TCN-529
302
39376
410
ND
533
1165
771
42271
ND



TCN-530
42460
11874
48341
ND
45339
2896
41661
486
ND



TCN-531
ND
3606
20840
ND
43874
1969
3117
745
ND



TCN-532
134
25219
46
ND
511
327
785
29816
ND



TCN-533
44511
8065
47704
ND
41449
2473
24745
172
ND



TCN-534
32514
6861
56642
ND
52429
4340
46537
165
ND



TCN-535
45937
6875
49961
ND
48192
4757
36650
861
ND



TCN-536
ND
543
287
ND
2210
976
436
1566
ND



TCN-537
18702
335
8617
ND
5206
1052
7826
147
ND



TCN-538
18746
689
37312
ND
17448
6531
6123
109
ND



TCN-539
3957
91
100
ND
814
1698
1488
292
ND



TCN-540
113
25
70
ND
131
381
117
22
ND



TCN-541
71
17
40
ND
106
381
131
29
ND



TCN-542
ND
ND
375
ND
900
909
332
1907




TCN-543
ND
325
347
ND
2128
661
737
131




TCN-544
ND
ND
272
ND
1690
1618
464
296




TCN-545
ND
ND
392
ND
1167
1182
656
227




TCN-546
ND
2037
421
ND
1354
1451
487
12983




TCN-547
ND
1476
198
ND
1005
401
278
3986




TCN-548
ND
5381
254
ND
314
758
184
3424




TCN-549
ND
1793
498
ND
1103
1134
844
5037




TCN-550
ND
1206
361
ND
964
939
407
2279




TCN-551
45688
4187
42301
ND
52491
786
9817
224




TCN-552
42201
4156
46249
ND
38413
1241
13115
171




TCN-553
25979
12610
24158
ND
21732
1838
27998
273




TCN-554
224
10851
250
ND
296
610
583
28594




TCN-555
16060
1497
22302
ND
49507
3399
18390
262




TCN-556
ND
3703
8800
ND
11890
10606
16910
7413




TCN-557
ND
46033
45545
ND
24375
6779
50106
35309




TCN-559
ND
11888
12935
ND
7233
1983
10385
2791




TCN-560
ND
49133
45969
ND
42303
48519
44696
49385




TCN-202
162
25
69
ND
105
348
196
50




TCN-504
17038
26175
38456
ND
7869
47190
14137
454




TCN-032
ND
ND
ND
ND
ND
ND
ND
ND

















TABLE 14







Summary of BCC SN screening for virus neutralization.










% Neutralization















A/Solomon






Islands/
A/Wisconsin/



Theraclone
BCC well
3/06
67/05



mAb ID
ID
H1N1
H3N2















TCN-522
3212_I12
33
0



TCN-521
3280_D18
99
ND



TCN-523
5248_A17
69
21



TCN-563
5237_B21
78
1



TCN-526
5084_C17
0
100



TCN-527
5086_C06
12
0



TCN-528
5087_P17
0
15



TCN-529
5297_H01
0
99



TCN-530
5248_H10
64
4



TCN-531
5091_H13
65
80



TCN-532
5262_H18
11
99



TCN-533
5256_A17
56
0



TCN-534
5249_B02
86
0



TCN-535
5246_P19
82
1



TCN-536
5095_N01
75
100



TCN-537
3194_D21
89
5



TCN-538
3206_O17
35
0



TCN-539
5056_A08
97
62



TCN-540
5060_F05
100
75



TCN-541
5062_M11
89
99



TCN-542
5079_A16
98
51



TCN-543
5081_G23
100
55



TCN-544
5082_A19
95
62



TCN-545
5082_I15
98
72



TCN-546
5089_L08
0
100



TCN-547
5092_F11
0
100



TCN-548
5092_P01
0
97



TCN-549
5092_P04
0
99



TCN-550
5096_F06
0
100



TCN-551
5243_D01
100
19



TCN-552
5249_I23
57
0



TCN-553
5261_C18
87
0



TCN-554
5277_M05
42
100



TCN-555
5246_L16
48
ND



TCN-556
5089_K12
0
4



TCN-557
5081_A04
0
2



TCN-559
5097_G08
82
ND



TCN-560
5084_P10
94
ND



TCN-202
2N9
ND
ND



TCN-504
3251_K17
ND
ND



TCN-032
8I10
ND
ND
















TABLE 15







Summary of monoclonal antibody transfection supernatant


screening by ELISA for virus binding.









ELISA: Virus Binding



(OD450)















A/







Solomon
A/
A/Wis-





Islands/
Japan/
consin/



Theraclone

3/06
305/57
67/05



mAb ID
BCC well ID
H1N1
H2N2
H3N2















Monoclonal
TCN-523
5248_A17
3.59
1.69
0.09


transfection
TCN-504
3251_K17
3.65
3.65
3.65


set 1
TCN-202
2N9
0.07
0.07
0.07


Monoclonal
TCN-522
3212_I12
3.48
0.61
0.07


transfection
TCN-526
5084_C17
0.08
0.07
0.31


set 2
TCN-527
5086_C06
3.69
3.63
0.14



TCN-528
5087_P17
3.66
3.64
0.23



TCN-563
5237_B21
3.60
0.84
0.09



TCN-504
3251_K17
3.65
3.62
3.65



TCN-523
5248_A17
3.67
1.39
0.09



TCN-202
2N9
0.08
0.07
0.07


Monoclonal
TCN-531
5091_H13
0.10
0.09
3.59


transfection
TCN-530
5248_H10
3.62
3.43
0.21


set 3
TCN-529
5297_H01
0.16
0.10
3.65



TCN-533
5256_A17
3.61
3.65
0.22



TCN-532
5262_H18
0.13
0.08
3.43



TCN-504
3251_K17
3.63
3.64
3.65



TCN-523
5248_A17
3.59
3.47
0.15



TCN-202
2N9
0.10
0.08
0.08


Monoclonal
TCN-535
5246_P19
3.52
2.45
0.10


transfection
TCN-534
5249_B02
3.50
2.45
0.08


set 4
TCN-536
5095_N01
3.52
0.06
3.61



TCN-504
3251_K17
3.52
3.51
3.59



TCN-523
5248_A17
3.43
1.73
0.09



TCN-202
2N9
0.10
0.08
0.07


Monoclonal
TCN-537
3194_D21
3.56
3.48
0.11


transfection
TCN-538
3206_O17
3.59
3.37
0.12


set 5
TCN-539
5056_A08
0.07
0.07
0.13



TCN-540
5060_F05
0.09
3.61
3.62



TCN-541
5062_M11
3.63
0.07
0.07



TCN-542
5079_A16
3.60
0.07
0.23



TCN-543
5081_G23
3.63
3.64
2.23



TCN-544
5082_A19
0.07
0.07
3.65



TCN-545
5082_I15
1.36
0.10
3.62



TCN-546
5089_L08
3.67
0.07
3.67



TCN-547
5092_F11
0.09
0.09
0.13



TCN-548
5092_P01
0.13
0.09
3.63



TCN-549
5092_P04
0.09
0.08
0.46



TCN-550
5096_F06
0.13
0.08
3.62



TCN-551
5243_D01
3.65
0.10
0.27



TCN-552
5249_I23
3.61
2.68
0.27



TCN-553
5261_C18
3.56
3.28
0.17



TCN-554
5277_M05
0.18
0.11
3.40



TCN-555
5246_L16
3.57
2.10
0.13



TCN-556
5089_K12
3.64
3.64
0.34



TCN-557
5081_A04
3.58
3.59
3.47



TCN-558
5248_H10
3.52
3.31
0.20



TCN-559
5097_G08
1.16
2.29
2.39



TCN-560
5084_P10
3.52
3.55
0.30



TCN-504
3251_K17
3.60
3.59
3.60



TCN-523
5248_A17
3.62
2.56
0.14



TCN-202
2N9
0.07
0.07
0.07
















TABLE 16





Summary of monoclonal antibody transfection supernatant screening for virus binding to recombinant


homotrimeric HA.

















Trimeric HA Binding: RLU





















A/
A/


A/







A/
Solomon
South
A/
A/
swine/
A/
A/





California/
Islands/
Carolina/
Japan/305/
Wisconsin/
Ontario/
Vietnam/
Indonesia/



Theraclone

4/09
3/06
1/18
57
67/05
01911-2/
1203/04
5/05



mAb ID
BCC well ID
H1N1
H1N1
H1N1
H2N2
H3N2
99 H4N6
H5N1
H5N1





Monoclonal
TCN-523
5248_A17
17703
6785
26364
191
1646
49
80
18508


transfection set 1
TCN-504
3251_K17
18286
29844
21541
15059
14311
26491
1300
16640



TCN-202
2N9
20
67
32
27
27
29
37
57


Monoclonal
TCN-522
3212_I12
37463
17281
39613
11936
3346
1048
48169
50472


transfection set 2
TCN-526
5084_C17
3598
1468
2312
3592
27776
3487
9181
37831



TCN-527
5086_C06
21498
19685
48062
20758
35796
30085
20600
23433



TCN-528
5087_P17
44586
26908
25226
23083
46012
49173
23336
39704



TCN-563
5237_B21
21231
26475
17808
11198
23686
2248
31549
47277



TCN-504
3251_K17
15621
31447
24695
22573
21615
32496
14953
19717



TCN-523
5248_A17
18654
3270
25504
882
2062
82
26227
24527



TCN-202
2N9
52
16
195
32
46
15
121
8


Monoclonal
TCN-531
5091_H13
2830
194
1557
489
4159
296
2411
54


transfection set 3
TCN-530
5248_H10
21176
13820
17166
5174
3048
123
25172
25546



TCN-529
5297_H01
735
43
158
45
2939
45
415
1353



TCN-533
5256_A17
6877
15526
16417
18618
16132
180
11677
28339



TCN-532
5262_H18
23601
59
311
441
9967
29
452
2786



TCN-504
3251_K17
18614
32672
27617
35957
21437
34456
13284
18559



TCN-523
5248_A17
19366
11014
23710
4554
8558
1640
22129
21973



TCN-202
2N9
205
57
731
42
51
54
967
43


Monoclonal
TCN-535
5246_P19
25157
836
22433
21491
4450
138
25244
18202


transfection set 4
TCN-534
5249_B02
27035
60
11830
14745
897
264
23675
948



TCN-536
5095_N01
49
30
39
260
20576
168
182
80



TCN-504
3251_K17
549
14625
21713
19576
20627
21792
25665
18154



TCN-523
5248_A17
28847
371
22983
3845
9742
850
30898
24534



TCN-202
2N9
94
34
27
334
125
157
67
19


Monoclonal
TCN-537
3194_D21
33793
760
46788
16197
1935
3282
50280
2420


transfection set 5
TCN-538
3206_O17
15956
1965
8434
6624
2070
3354
52375
13748



TCN-539
5056_A08
1053
871
5330
2565
58831
6511
53254
8947



TCN-540
5060_F05
984
1176
1356
47894
52986
5419
42500
2569



TCN-541
5062_M11
1557
2277
2583
5382
1238
4724
45517
2959



TCN-542
5079_A16
667
48021
1116
5184
711
14340
29492
1761



TCN-543
5081_G23
2338
43523
28290
2775
37048
2866
33145
1826



TCN-544
5082_A19
706
997
1331
1003
38514
2391
38620
4400



TCN-545
5082_I15
818
951
900
1355
32037
2936
42906
5269



TCN-546
5089_L08
1085
1140
955
1730
28453
4609
47108
40866



TCN-547
5092_F11
1474
1082
2016
1354
6968
1602
27666
12041



TCN-548
5092_P01
818
750
1154
1016
34194
1624
23678
29142



TCN-549
5092_P04
622
640
2340
1075
51626
1878
32265
8645



TCN-550
5096_F06
628
758
1014
731
37809
1704
41900
9001



TCN-551
5243_D01
46133
43523
4344
25191
936
3120
41915
1289



TCN-552
5249_I23
23459
48559
37374
51986
18451
9084
18494
43382



TCN-553
5261_C18
23323
49581
41300
39848
25248
12230
19214
31183



TCN-554
5277_M05
4034
992
1261
2621
25329
5402
36582
44935



TCN-555
5246_L16
20223
44571
48009
16743
14660
4522
19300
33927



TCN-556
5089_K12
20865
23409
32732
22805
44715
19346
17421
15229



TCN-557
5081_A04
44419
26960
44865
27282
46082
41525
21320
22246



TCN-558
5248_H10
20220
36107
40709
34434
26611
10003
23527
24632



TCN-559
5097_G08
8079
41254
2233
46993
15279
35761
37607
51869



TCN-560
5084_P10
31134
22427
30462
25855
55383
16751
19475
16971



TCN-504
3251_K17
17599
30086
31975
23254
38088
54789
21485
18485



TCN-523
5248_A17
34415
37587
52010
22019
13690
1699
27819
55048



TCN-202
2N9
733
525
5331
1883
581
2692
30002
1973












Trimeric HA Binding: RLU





















A/



A/









common

A/

northern








A/
magpie/

chicken/
A/
shoveler/

A/
A/
A/




Egypt/
Hong

Vietnam/
Hong
California/
A/
duck/
Hong
Hong




3300-
Kong/
A/
NCVD-
Kong/
HKWF115/
Netherlands/
Yangzhou/
Kong/
Kong/



Theraclone
NAMRU3/
5052/07
Anhui/1/
016/08
156/97
07
219/03
02/05
2108/03
1073/99



mAb ID
08 H5N1
H5N1
05 H5N1
H5N1
H5N1
H6N4
H7N7
H8N4
H9N2
H9N2





Monoclonal
TCN-523
25494
6235
60
10813
ND
9046
28
229
41
21719


transfection
TCN-504
16113
29696
29046
27765
ND
5791
23828
28934
78
25703


set 1
TCN-202
76
21
20
14
ND
13
55
26
24
21


Monoclonal
TCN-522
55413
21393
2031
49343
ND
54364
2170
1646
203
11800


transfection
TCN-526
39894
1758
43991
3294
ND
3789
5659
4305
23267
490


set 2
TCN-527
23595
43794
15238
20112
ND
19272
49149
17921
25527
557



TCN-528
36834
45257
21207
22573
ND
28370
47229
19792
33621
322



TCN-563
23915
52783
1568
36304
ND
21901
5423
6275
364
49230



TCN-504
21365
16850
30975
27266
ND
1277
19272
17751
23
25832



TCN-523
30672
7214
139
17590
ND
21214
407
383
18
31283



TCN-202
18
11
9
22
ND
30
26
19
7
33


Monoclonal
TCN-531
363
37
76
44
ND
96
384
3220
158
370


transfection
TCN-530
25523
5576
97
17979
ND
23513
118
227
44
10219


set 3
TCN-529
1673
33
1258
33
ND
110
33
48
1197
46



TCN-533
18587
25210
301
24551
ND
7806
209
711
33
23808



TCN-532
2821
20
2402
29
ND
78
42
57
2767
48



TCN-504
17969
17814
29988
10261
ND
8335
19600
19647
36
27781



TCN-523
29080
640
546
18903
ND
29404
3144
2017
51
24193



TCN-202
63
16
33
14
ND
73
247
66
30
57


Monoclonal
TCN-535
25318
477
24
20929
ND
18057
159
428
18
21197


transfection
TCN-534
8314
127
20
11352
ND
21287
361
79
27
731


set 4
TCN-536
97
27
18
19
ND
95
246
48
24
115



TCN-504
24796
8675
14053
19176
ND
2609
6707
19872
7
18770



TCN-523
33351
2131
48
23995
ND
25087
443
463
38
24575



TCN-202
40
21
13
42
ND
117
173
77
22
81


Monoclonal
TCN-537
33495
2068
2580
4476
10681
2376
3654
25529




transfection
TCN-538
40969
3141
2745
2948
5167
8895
4374
4065




set 5
TCN-539
24338
1436
3244
3496
1450
2862
15729
7281





TCN-540
15589
1799
2192
1514
1551
2461
4386
6078





TCN-541
23234
1936
2998
11039
1975
2684
13649
21203





TCN-542
14763
1674
7407
5458
786
2369
8049
11598





TCN-543
17583
1070
2626
5877
1433
2512
12873
13130





TCN-544
19414
1116
3770
3069
1491
2915
4647
9444





TCN-545
26538
1388
4876
3230
1560
2319
3125
13197





TCN-546
53039
1916
33344
2940
1243
2429
3743
11391





TCN-547
16784
856
1238
1315
1955
2618
2089
3335





TCN-548
37002
813
15578
1059
1555
2104
2131
5735





TCN-549
18581
1340
4113
1601
1848
996
8475
9452





TCN-550
26791
993
7680
1802
955
2305
3248
4713





TCN-551
17368
1123
2643
792
792
1764
4837
3097





TCN-552
21246
34619
9010
34451
36317
43861
7416
15937





TCN-553
19470
39224
8548
34718
36337
32461
7532
12545





TCN-554
42395
1734
42805
2708
1441
3352
4522
5808





TCN-555
20001
33642
12236
35215
36302
28717
12656
8544





TCN-556
16603
20140
13795
22676
24620
34817
43640
22400





TCN-557
26351
54874
19980
23323
27061
49611
53747
24015





TCN-558
21861
49655
9783
33423
47113
29396
5841
19531





TCN-559
47369
12900
41835
33850
28953
6632
4852
42614





TCN-560
18730
30485
18628
18174
29287
35866
32120
27927





TCN-504
20625
37328
37168
34166
26597
39179
30626
35074





TCN-523
35484
33905
6079
51296
28380
46941
6750
25599





TCN-202
13876
1366
10033
2570
945
3036
18026
13796
















TABLE 17







Summary of monoclonal antibody transfection supernatant screening


for virus neutralization.









% Neutralization















A/







Solomon
A/Cali-
A/Wis-





Islands/
fornia/
consin/



Theraclone

3/06
4/09
67/05



mAb ID
BCC well ID
H1N1
H1N1
H3N2















Monoclonal
TCN-523
5248_A17
82.71
ND
ND


transfection
TCN-504
3251_K17
0.00
ND
0.00


set 1
TCN-202
2N9
0.00
ND
0.00


Monoclonal
TCN-522
3212_I12
39.46
ND
0.84


transfection
TCN-526
5084_C17
10.58
ND
94.26


set 2
TCN-527
5086_C06
17.55
ND
0.00



TCN-528
5087_P17
23.92
ND
0.00



TCN-563
5237_B21
88.85
ND
0.00



TCN-504
3251_K17
7.19
ND
0.00



TCN-523
5248_A17
80.68
ND
0.00



TCN-202
2N9
7.00
ND
0.00


Monoclonal
TCN-531
5091_H13
0.00
ND
97.30


transfection
TCN-530
5248_H10
96.95
ND
34.54


set 3
TCN-529
5297_H01
0.00
ND
99.59



TCN-533
5256_A17
96.12
ND
32.51



TCN-532
5262_H18
0.00
ND
99.73



TCN-504
3251_K17
0.00
ND
10.74



TCN-523
5248_A17
62.19
ND
34.00



TCN-202
2N9
0.00
ND
17.77


Monoclonal
TCN-535
5246_P19
88.60
97.28
0.00


transfection
TCN-534
5249_B02
65.66
96.37
0.00


set 4
TCN-536
5095_N01
81.63
18.76
99.88



TCN-504
3251_K17
29.68
0.00
0.00



TCN-523
5248_A17
65.54
93.19
0.00



TCN-202
2N9
ND
ND
ND


Monoclonal
TCN-537
3194_D21
0
71
0


transfection
TCN-538
3206_O17
21
66
0


set 5
TCN-539
5056_A08
0
0
99



TCN-540
5060_F05
3
0
98



TCN-541
5062_M11
88
0
4



TCN-542
5079_A16
99
15
8



TCN-543
5081_G23
98
0
73



TCN-544
5082_A19
0
0
97



TCN-545
5082_I15
88
0
99



TCN-546
5089_L08
98
0
100



TCN-547
5092_F11
0
0
88



TCN-548
5092_P01
0
0
96



TCN-549
5092_P04
0
0
100



TCN-550
5096_F06
0
0
100



TCN-551
5243_D01
99
74
6



TCN-552
5249_I23
0
81
14



TCN-553
5261_C18
44
83
5



TCN-554
5277_M05
0
0
100



TCN-555
5246_L16
49
89
5



TCN-556
5089_K12
0
0
15



TCN-557
5081_A04
7
10
0



TCN-558
5248_H10
84
96
7



TCN-559
5097_G08
7
10
12



TCN-560
5084_P10
0
0
4



TCN-504
3251_K17
0
0
0



TCN-523
5248_A17
39
86
7



TCN-202
2N9
0
0
0









Other Embodiments
Although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A composition comprising:
(a) an isolated human monoclonal antibody, wherein said antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 771 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 778; and
(b) an isolated human monoclonal antibody, wherein said antibody comprises a VH CDR1 region comprising the amino acid sequence of NYYWS (SEQ ID NO: 72); a VH CDR2region comprising the amino acid sequence of FIYYGGNTKYNPSLKS (SEQ ID NO: 74); a VH CDR3 region comprising the amino acid sequence of ASCSGGYCILD (SEQ ID NO: 76); a VL CDR1 region comprising the amino acid sequence of RASQNIYKYLN (SEQ ID NO: 59); a VL CDR2 region comprising the amino acid sequence of AASGLQS (SEQ ID NO: 61); a VL CDR3region comprising the amino acid sequence of and QQSYSPPLT (SEQ ID NO: 63),
wherein at least one of the antibodies is directly conjugated to either a detectable label or therapeutic agent.

2. A passive immunization composition comprising the composition of claim 1.

3. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutical carrier.