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NZ614215B2 - Compositions and methods for the therapy and diagnosis of influenza - Google Patents
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NZ614215B2 - Compositions and methods for the therapy and diagnosis of influenza - Google Patents

Compositions and methods for the therapy and diagnosis of influenza Download PDF

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Publication number
NZ614215B2
NZ614215B2 NZ614215A NZ61421512A NZ614215B2 NZ 614215 B2 NZ614215 B2 NZ 614215B2 NZ 614215 A NZ614215 A NZ 614215A NZ 61421512 A NZ61421512 A NZ 61421512A NZ 614215 B2 NZ614215 B2 NZ 614215B2
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NZ
New Zealand
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seq
amino acid
acid sequence
antibody
heavy chain
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NZ614215A
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NZ614215A (en
Inventor
Thomas C Cox
Andres G Grandea
Phil Hammond
Gordon King
Jennifer Mitcham
Matthew Moyle
Ole Olsen
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Theraclone Sciences Inc
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Priority claimed from PCT/US2012/024971 external-priority patent/WO2012112489A2/en
Publication of NZ614215A publication Critical patent/NZ614215A/en
Publication of NZ614215B2 publication Critical patent/NZ614215B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C07K16/1018—
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

Abstract

Disclosed is a composition comprising (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; wherein the antibodies comprise the VH/VL chains or CDRs of the sequences as defined in the specification. odomain (M2e) polypeptide of an influenza virus; wherein the antibodies comprise the VH/VL chains or CDRs of the sequences as defined in the specification.

Description

COMPOSITIONS AND METHODS FOR THE THERAPY AND DIAGNOSIS OF INFLUENZA RELATED APPLICATIONS This application claims the benefit of provisional application USSN 61/442,733, filed February 14, 2011, the contents of which are herein incorporated by reference in their entirety.
ORATION OF SEQUENCE G ' The contents ofthe text file named “37418-518001WO_ST25.txt,” which was d on January 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 ofinfluenza infection. The invention is more specifically related to compositions containing a combination of human dies raised against either the influenza hemagglutinin or matrix 2 protein. Such itions are useful in pharmaceutical compositions for the prevention and ent of influenza, and for the diagnosis and monitoring ofinfluenza infection.
BACKGROUND OF THE INVENTION za virus infects 5-20% of the population and results in 30,000-50,000 deaths each year in the US. Disease caused by influenza A viral infections is typified by its cyclical . Antigenic drift and shifl allow for ent 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, prophtyflactic, 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 dy raised against an e of the Influenza hemagglutinin protein and an isolated human onal antibody raised against an epitope ofthe 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 ns common to all za strains.
Specifically, the invention provides a composition including: (a) an ed human antibody that specifically binds to an epitope of the lutinin (HA) rotein of an influenza virus; and (b) an ed human monoclonal antibody that specifically binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an nza virus. In certain embodiments of this composition, the ed human monoclonal antibody that specifically binds an e of the M2e polypeptide is TCN—032 (8110), 21315, TCN-031 (23K12), 3241_GZ3, ,3244_110, 3243_JO7, 21, 3245_Ol9, 3244_H04, 3136_GOS, 3252_C13, 3255~JO6, 3420_I23, 3139_P23, 3248__P18, 3253_P10, 3260_D19, 3362_B11, or 3242_P05. Moreover, the ed human antibody that specifically binds an epitope of the HA glycoprotein is optionally TCN-522 (3212_Il2), 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:A17I), TCN-534 (5249_BOZ), 5 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN—538 (3206_017), TCN—539 (5056_A08), TCN-540 (5060_F05), 1 (5062_M11), 2 (5079_A16), TCN— 543 (5081_GZ3), TCN-544 (5082_A19), TCN-545 115), 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_123), 3 (5261_C18), TCN-554 (5277_M05), TCN—555 (5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN 558 (5248_H10b), TCN-559 GO8), TCN-560 (5084_P10), TCN-504 (3251_K17), SCO6- 141, SCO6-255, SC06-257, SC06-260, SCO6-261, SC06-262, SCO6—268, SCO6—272, SCO6- 296, SCO6-301, SC06-307, SC06-310, SC06-314, SC06—323, SC06—325, SCO6—327, SCO6- 328, SC06-329, SCO6-l331, SC06-332, SC06-334, SC06-336, SC06-339, SC06-342, SCO6- 343, SC06-344, CR6141, CR6255, , CR6260, CR6261, CR6262, CR6268, CR6272, CR6296, CR6301, CR6307, CR6310, CR6314, ,~CR6325, CR6327, CR6328, CR6329, CR6331, , CR6334, CR6336, 036839, CR6342, CR6343, CR6344, 2A, D7, D8, F10, 017, H40, A66, D80, E88, E90, or [$98. . [07] The epitope of the HA glycoprotein is optionally GVTNKVNSIIDK (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO: 283), GVTNKENSIIDK (SEQ ID NO: 202), WO 12489 GVTNKVNRIIDK (SEQ ID NO: 201), QITNKVNSVIEK (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 HA] and HA2 subunit. Exemplary epitopes of the HA glycoprotein e the HA] subunit, HA2 subunit, or both the HA1 and HA2 subunits. Alternatively, or in addition, the epitope of the M2e ptide is a discontinuous epitOpe. For example, the epitope of the M2e polypeptide includes the amino acid at pdsitions 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 ed 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 es the following amino acid sequences: VH CDR]: SEQ ID N05: 247, 571, 586, 597, 603, 609, 615, 627, 633, 637, 643, 649, 658, 664, 670, 303, 251, 242, or 222; VH CDR2: SEQ ID N05: 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 N03: 568, 573, 588, 593, 599, 605, 611, 617, 629, 635, 639, 645, 651, 656, 660, 666, 672, 725, 246, 290, or 220; VL CDRl: SEQ ID N05: 569, 574, 577, 580, 583, 589,694,612, 618, 621, 624, 640, 646, 652, 661, 667, 285, 289, 245, 224, or 219; VL CDR2: 8EQ ID NOs: 570, 575, 578, 581, 584, 590, 595, 601, 607, 613, 619, 622, 625, 631, 653, 6625668, 305,2,23 or 231 ;VL CDR3: SEQ ID N05: 289, 576, 579, 582, 585, 591, 596, 602, 608, 6134, 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, ing a heavy chain variable (VH) domain and a light chain variable (VL) , 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 ing amino acid sequences: VH CDR]: SEQ ID NOS: 72, 103, 179, 187, 203, 211, 228, 252, 260, 268, 284, 293, or 301; VH CDR2: SEQ ID N03: 74, 105, 180, 188, 204, 212, 229, 237, 253, 261, 269, 285, or 294; VH CDR3 SEQ ID N05: 76, 107, 181, 189, 197, 205, 213, 230, 238, 254, 262, 270, 286, or 295; VL CDR]: 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 ion provides a composition including: (a) an isolated human anti—HA antibody, or an antigen-binding fragment f, including a heavy chain variable region (VI-I) domain and a light chain variable (VL) domain, wherein the VB 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 CDRl: SEQ ID N03: 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 N05: 568, 573, 588, 593, 599, 605, 611, 617, 629, 635, 639, 645, 65,1 656, 660, 666, 672, 725, 246, 290, or 220; VL CDR]: SEQ ID N05: 569, 574, 577, 580,583,589, 594, 612, 618, 621, 624, 640, 646, 652, 661, 667, 285, 289, 245, 224, or 219; V1. DDR2: 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 (M26) antibody, or antigen-binding nt 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 3), and wherein each CDR includes the following amino acid sequences: VH CDR]: SEQ ID N05: 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 CDRl: SEQ ID N05: 59, 92, 184, 192, 208, 192,233,241, 265, or 273; VL CDR2: SEQ ID N05: 61, 94, 185, 193, 209, 217, 226, 234, 258, 274, or 282; and VL CDR3} SEQ ID N05: 63, 96, 186, 194, 210, 218, 243, 259, 267, 275, 291, or 300. ' 1 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 ing amino acid sequences: SEQ ID NOS 309,313, 317,321, 9, 333, 337, 341,345,349, 353, 357, 361, 9, 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) , 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, WO 12489 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 (M26) antibody, or antigen- binding fragment thereof, including a heavy chain variable (VH) domain, Wherein the VH domain es the following amino acid sequences: SEQ ID N05 44, 277, 276, 50, 236, 235, 116, 120, 124, 128, 132, 136, 140, 144,148, 152, 156, 160,164, 168, 172, or 176, anda 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 compositionsdescribed herein containing an isolated human anti~HA dy, 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 (8110), 21315, TCN-031 (23K12), 3241_G23, 3244_110, 07, 3259_J-2l, 3245O_19, 04, 3136_G05, 3252_C13, 3255_J06, 3420_123, 3139_P2,3 3248_P18, 3253P_10, 3260_D19, 3362_B11, 3242_P05, TCN—522 (3212_Il2), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN—563 (5237_B21), 6 (5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529 H01), TCN-530 (5248_H10), TCN—531 (5091_H13), TCN-532 H18), TCN— 533 (5256_A17), TCN—534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_Ol7), TCN-539 (5056_A08), 0 (5060_F05), TCN-541 (5062_M1 1), TCN—542 A16), TCN-543 G23), TCN—544 (5082_A19), TCN-545 (5082_115), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), 9 (5092_P04), TCN—550 (5096_F06), TCN-551 (5243_D01), TCN-552 (5249_123), 3 (5261_C18), TCN—554 M05), 5 (5246_L16), TCN-556 (5089_K12), 7 (5081_A04), TCN 558 (5248_H10b), TCN-559 (5097_G08), TCN- 560 (5084_P10), TCN—504 (3251_K17), SC06-141, SC06-255, SC06-257, 60, SCO6- 261, SC06-262, SC06-268, SC06-272 SC06-296, S‘CO6-301, SC06-307, SC06-310, SCO6— 314, SC06—323, SC06-325, SCO6—327, SC06-328,2SC06-329, SC06331, SC06-332, SCO6- 334, 36, 39, SCO6-342, 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 e 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), GITNKENS¥IEK;(SEQ ID NO: 257), GITNKVNSIIDK (SEQ ID NO: 225), KITSKYNlSIIVDK.(SEQ ID NO: 216), MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID N6: 1), and MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1) provided in its native conformation.
The multivalent vaccine also es a composition including: (a) a human antibody that ically 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 ion es 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 ating 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 ion also provides a method for the treatinent of an influenza virus infection in a subject in need thereof, including stering to the subject the pharmaceutical composition bed herein. The subjection may have been exposed to an influenza virus. atively, or in addition, the subject has not been diagnosed with an influenza infection.
The ceutical 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 re of the subject to an influenza virus. In certain ments of this , 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 tor or a viral ment 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 tor. In certain aspects of these methods, the M2 ion channel inhibitor is amantadine or rimantadine. In other aspects of these methods, the inidase inhibitor is zanamivir or oseltamivir phosphate. The antiviral drug may be administered prior to or after exposure of the subject to an nza 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 as bed herein. The second antibody may be administered prior to or after exposure of the subject to an Influenza virus.
The invention provides a method for determining the ce of an Influenza virus infection in a subject, including the steps of: (a) contacting a biological sample ed from the subject with any one of the antibodies or pharmaceutical compositions as described herein; (b) ing an amount of the antibody that binds to the biological sample; and (c) ing 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 ed from the subject with any one of the antibodies or pharmaceutical compositions as described herein and detecting an amount of the antibody that binds to the control sample.
The ion also provides a diagnostic kit including any one of the antibodies, compositions, or pharmaceutical compositions as described herein.
The invention further provides a prophylactic kit including a e composition described herein. Preferably, the vaccine is a multivalent vaccine. The term “multivalent e” 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 es 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 dies raised against more than one infectious agent, e.g. the influenza HA glycoprotein and the influenza M2e polypeptide. [20a] Definitions of specific embodiments of the invention as claimed herein follow. [20b] According to a first embodiment of the invention, there is provided a composition comprising: (a) an isolated human monoclonal antibody, wherein said antibody ses 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 CDR2 region comprising the amino acid ce 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 YKYLN (SEQ ID NO: 59); a VL CDR2 region comprising the amino acid sequence of AASGLQS (SEQ ID NO: 61); and a VL CDR3 region comprising the amino acid sequence of and PLT (SEQ ID NO: 63), wherein at least one of the antibody of (a) and (b) is directly conjugated to either a detectable label or a therapeutic agent. [20c] According to a second embodiment of the ion, there is provided a passive immunization composition comprising the composition of the first ment. [20d] According to a third embodiment of the invention, there is provided a ceutical composition comprising the composition of the first embodiment and a pharmaceutical carrier.
Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims.
[Text continues on page 8] _. ‘ BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the binding of three antibodies of the present ion and control hul4C2 antibody to 293-HEK cells transfected with an M2 expression uct or control vector, in the presence or absence of free M2 peptide.
Figures 2A and B are graphs showing human monoclonal antibody g to influenza A/Puerto Rico/8/32.
Figure 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).
Figures 3B and C are bar charts showing binding of human monoclonal anti-influenza antibody binding to M2 variants shown in Figure 3A.
Figures 4A and B are bar charts showing binding of human onal anti- influenza antibody binding to M2 peptides subjected to alanine scanning mutagenesis.
Figure 5 is a series of bar charts showing binding of MAbs 8i10 and 23K12 to M2 protein representing influenza strain 83/1997z. sequence that was stably expressed in the CH0 cell line DG44.
Figure 6A is a chart showing cross reactivity binding of anti—M2 antibodies to variant M2 peptides (SEQ ID NOS 680-704, tively, in order of appearance).
Figure 6B is a chart g binding activity of M2 antibodies to truncated M2 peptides (SEQ ID NOS 680, 705-724 &l9, respectively, in order of ance).
Figure 7 is a graph showing survival of influenza infected mice treated with human anti-influenza monoclonal antibodies.
Figure 8 is an illustration showing the anti-M2 antibodies bind a highly conserved region in the N-Terminus of M26 (SEQ ID NO: 19).
Figure 9 is a graph showing anti-M2 rHMAb clones from crude supernatant bound to influenza on ELISA, whereas the l anti-M2e mAb 14C2 did not readily bind virus.
Figure 10 is a series of photographs showingianti-‘M2 rHMAbs bound to cells infected with influenza. MDCK cells were or were not infected with influencza /32 and Ab binding from crude supernatant was tested 24 houfirfls later. Data were gathered from the FMAT plate scanner.
Figure l 1 is a graph showing anti-M2 rHMAb clones from crude supernatant bound to cells transfected with the za 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 ently transfected into 293 cells.
The 14C2, 8i10, 23K12, and 21B 15 mABs were teStéd for binding to the transfectants, and were detected with an AF647-conjugated anti—human IgGi secondary antibody. Shown are the mean fluorescence ities of the specific mAB‘gbound after FACS analysis.
Figures 12A-B are amino acid sequences of the variable regions of anti-M2e mAbs.
Framework regions l—4 (FR l-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 (HVIGT®, the International ImMunoGeneTics Information system® /www.imgtorg). Grey boxes denote identity with the ne sequence which is shown in light blue boxes, s denote gaps, and white boxes are amino acid replacement mutations from the germline.
Figure 13 is a graph depicting the results of a competition binding analysis of a panel of anti-M26 mAbs with TCN—O32 Fab. The indicated anti-M26 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 ug/mL TCN-O32 Fab fragment. The anti-M2e mAb bound to the cell surface was detected with goat anti-h-ngG FcAlexafluor488 FACS and .n‘kl analyzed by flow cytometry The results are derived from one experiment.
Figure 14Ais a graph ing the abilityof anti—M26 mAbs TCN-032 and TCN— 031 to bind virus particles and virus-infected cells but not MZe—derived synthetic peptide.
Purified influenza virus rto Rico/8/34) was coated at 10 ug/ml on ELISA wells and binding of anti—M2e mAbs TCN-O31, TCN-032, chl4C2, and the HCMV mAbs 2N9 was evaluated using HRP-labeled goat anti-human Fc. s shown are representative of 3 experiments.
Figure 14B is a graph depicting the ability of anti—M2e mAbs TCN—032 and TCN- 031 to bind virus les and virus-infected cells but not M2e—derived tic e. 23mer synthetic peptide of M2 derived from A/Fort Worth/I/SO was coated at 1 ug/ml on ELISA wells and binding of mAbs TCN-O31, TCN-O32, ch14C2, and 2N9 were evaluated as in panel a. Results shown are representative of 3 experiments.
Figure 14C is.a graph depicting the ability of anti-M26 mAbs TCN-O32 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—O31, TCN-032, ch14C2 and the HCMV 12 Binding of antibodies was detected using Alexafluor 647-conjugated goat anti-Human IgG H&L antibody and fied by flow cytometry. Results shown are representative of 3 experiments.
Figure 14D is a series of photographs depicting I-[EK 293 cells stably transfected with the M2 ectodomain of A/Fort Worth /1/50 (D20) were stained with transient transfection supernatant containing mAbs TCN-O3l, TCN—032, or the control chl4C2 and ed by FMAT for g to M2 in the presence or absenc’gof 5 ug/ml M2e peptide. Mock transfected cells are 293 cells stably transfected w1th vector alone. Results shown are representative of one experiment.
Figures ISA—D are graphs depicting the Therapeutic efficacy of anti-M2 mAbs TCN-0_3l and TCN—O32 in mice. Mice (n=10) were infected by intranasal inoculation with 5 x 1.1350 A/Vietnam/1203/O4 (H5N1) (panels A—B) or (n=5) with 5 x L050 A/Puerto Rico 8/34 (HlNl) (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 s shown for the treatment study of mice infected with A/Vietnam/1203/04 (HSNl) are representative of 2 experiments.
Figure 16 is,a series of graphs ing the viral titers in lung, liver, and brain of mice treated with anti-M2e mAbs TCN—O3l and TCN—O32 after challenge with H5Nl A/Vietnam/1203/04. BALB/C mice (n=19) were treated i.p. ion of a 400 ug/ZOO 11L 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-infection1n the lungs (as an indicator of local replication) andin liver and brain (as an indicator of the systemic spread which is teristic of H5N1 infection).
Figure 17 is a graph depicting the ability of TCN-031 and TCN—O32 can potentiate cytolysis by NK cells. MDCK cells were infected with A/Solomon /3/2006 (H1N1) virus, and were treated with mAbs 1, TCN-032, or the subclass—matched negative control mAb 2N9. The cells were then challenged with purified human NK cells, and the e 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.
Figure 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 l were treated with the indicated mAbs and subsequently challenged with human complement. Lysed cells were visualized by Propidium Iodide staining ed by FACS analysis. The data are representative of two experiments.
Figures l9A-C are graphs depicting binding of anti-M26 mAbs TCN-O3l and TCN- 032 to M2 mutants indicates the epitope is located in the highly conserved inal of M26. Mutants with alanine substituted at each position of the M2 ectodomain of A/Fort Worth /l/50 (D20)(A) or forty wild-type M2 mutants including ANietnam/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 d with mAbs 1, TCN-032, or the l ch14C2 and analyzed by FACS for binding to M2 at 24 hours post-transfeetion. mAbs TCN—O31 and TCN-O32 do not bind ts with amino acid substitutions at positions 1, 4, or 5 of M26. (C) The deduced epitope for TCN-O31 and TCN-032 occurs in a highly conserved region of M26 and is distinct from that found for ch14C2. Results shown for (A) and (B) are representative of 3 experiments.
Figure 20 is a graph depicting mAbs TCN-031 and TCN-032 recognize the same region on M26. The CHO transfectant stably expressing M2 for A/Hong 83/97 as stained with 10 ug/mL l, TCN—032, or 2N9, followed by detection with Alexafluor647-labeled 1 (TCN-031AF647) or TCN-O32(TCN-032AF647) and analysis by flow cytometry. The results are representative of three ments.
Figure 2.1 is a graph depicting anti-M26 mAbs TCN-03l and TCN-O32 bind cells that have been infected with HlN l fomia/4/O9. MDCK cells were infected with Influenza A strain HlNl A/Memphis/l4/96, HlNl A/Califomia/4/O9, or mock ed. Twenty four hours post-infection cells were stained with mAbs TCN-031, TCN—O32, or the control ch14C2 and analyzed by FACS for binding to M2. Results shown are for one experiment. .trl. ‘ ED DEscizIP‘T'ION Influenza viruses consist of three types: AfiB'and CE Influenza A viruses infect a wide variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In s 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. ' 2012/024971 za A viruses can be classified into subtypesx‘based on allelic variations in antigenic regions oftwo genes that encode e'glycoproteins, , hemagglutinin (HA) and neuraminidase (NA) which are required for viral attachment and cellular release.
Other major viral proteins e the nucleoprotein, the nucleocapsid structural protein, membrane proteins (M1 and M2), polymerases (PA, PB and PB2) and non-structural proteins (N51 and N82). Currently, sixteen subtypes of HA (HI-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 (HlNl, H1N2, and H3N2).
However, in recent years, the pathogenic HSNl 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 , 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, lymphopen‘ia, diarrhea and difficulties ting blood sugar levels. In contrast to al zazthe,’group most at risk is healthy adults, which make up the bulk of the population. Due tof‘ihe high pathogenicity of certain avian influenza A es,'particularly HSNl, and their demonstrated y 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 HSNl infection are available, so e therapy 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 t in humans, there is thus a need for antibodies, preferably human antibodies, capable of neutralizing influenza infection, including H5N1.
The invention provides compositions ing human dies raised against two influenza proteins, hemagglutinin (HA) and matrix 2 ectodomain (M26), and shows that these compositions can be used in medicine, in particular for diagnosis, prevention and treatment of influenza infections, including H5N1. . . . . . . m l HuM2e Antibodies The present invention provides fully human monoclonal antibodies specifically directed against M26. Optionally, the antibody is isolated form a B-cell from a human donor.
Exemplary monoclonal antibodies include TCN—032 (8110), 21B15, TCN-031 (23K12), 3241_023, 3244_110, 3243_JO7, 3259_J21, 3245_Ol9, 3244_H04, 3136_GOS, 3252_Cl3, . 3255_J06, 3420_123, 3139_P23, 3248_P1’8, 10, 3260_D19, 11, and 3242_P05.described herein. Alternatively, the monoclonal antibody is an antibody that binds to the same epitope as 2 (8110), 21B15, TCN—03l ), 3241_023, 3244_IlO, 07, 3259_J21 3245_,019 3244_H04, 3136_605', 3252_Cl3, 3255_J06, 3420_123, 3139_P23, 3248_Pl8, 3253_P10, 3260_D19 3362_B11, and 3242_P05 The antibodies respectively referred to herein are huMZe dies. 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 (M26) polypeptide of an influenza virus; b) binds to za A infected cells; or c) binds to za 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 ancewith 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 N03: 44, 277, 276, 50, 236, 235, 116,120,124,1,28 132,136,140,144,148,152, 156,160,164,168,172,0r 176 and a light chain variable having the amino acid sequence of SEQ ID N05: 46, 52,118,122,126,130,134,138,142 146,150,154,158,162,166,170, 174, or 178. Preferably, the three heavy chainCDRsinclude an amino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or more cal to the amino acid sequence of SEQ ID NOs: 72,.74, 76, 103, 105, 107, 179, 180, 181, 187, 188, 189, 3, 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 N05: 109, 1 10, 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 N05: 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, :fx” .V1’; 13:51 273, 274, 275, 282, 291, and 300 (as determined by the Chothia ). The antibody binds M26.
The heavy chain of a M26 antibody is derived from a germ line V (variable) gene such as, for example, the IgHV4 or the IgHV3 germline gene.
The M2e dies of the invention include a variable heavy chain (VH) region encoded by a human IgHV4 or the IgHV3 germline gene ce. A IgHV4 ne gene ce 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, 227504, M99679 and AB019437. The M2e: antibodies of the invention e a vH region that is encoded by a nucleic acid sequence 3that is at least 80% homologous to the IgHV4 or the IgHV3 germline gene sequence. Prefefably, 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% gous to the IgHV4 or the IgHV3 germline gene sequence. The VH region of the M26 antibody is at least 80% homologous to the amino acid sequence of the VH region encoded by the IgHV4 or the IgHV3 VH ne gene sequence. Preferably, the amino acid sequence of V” region of the M26 antibody is at least 90%, 95%, 96%, 97% gous 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 IgKVl germline gene sequence. A human IgKVI VL germline gene sequence is shown, e.g., ion numbers X59315, X59312, X59318, J00248, and Y14865. Alternatively, the M26 antibodies includes: VL‘re'gion that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgKX/l germline gene sequence.
Preferably, the nucleic acid sequence is at least 96%, 95%: 96%, 97% homologous to the IgKVl germline gene sequence, and more preferably, at least 98%, 99% homologous to the IgKV] germline gene sequence. The VL region of the M26 antibody is at least 80% homologous to the amino acid ce of the VL region encoded the IgKVl ne gene sequence. Preferably, the amino acid sequence of VL region of the M26 antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgKVl germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by e the IgKVl germline gene sequence.
In another aspect the ion provides a composition including an huM2e antibody according to the invention. In s 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 l 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 dies according to the invention are operably— linked to a therapeutic agent or a detectable label. onally, the ion 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 dy, an anti-viral siuch as a neuraminidase inhibitor, 3 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 r 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 dy of the invention is used to treat or prevent rejection influenza infection. The huM2e antibody is administered at a dose ient 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 ting a biological sample obtained from the t with a humM2e dy; detecting an amount of the antibody that binds to the ical sample; and comparing the amount of ahafi‘body-Fthat binds to the biological sample it h k to a control value.
The invention further es a diagnostic kift 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 ic against the extracellular domain of the matrix 2 (M2) polypgjlptide. The antibodies are respectively referred to herein as huM26 antibodies. a M2 is a 96 amino acid transmembrane n. present as a tramer 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 ic strain thus M2e is an attractive target for influenza therapies. In prior studies, monoclonal antibodies specific to the M2 ectodomain (M26) were derived upon immunizations with a peptide corresponding to the linear sequence of M26. In contrast, the present invention es 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 M26. 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 huM26 antibodies did not bind the linear M26 peptide, but they do bind several l 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’bf influenza A virus strains. These antibodies may be used diagnostically to identify influenza A infection and eutically to treat influenza A infection.
The huMZe antibodies of the invention have one or more of the following characteristics: the huM2e dy 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 huMZe dies of the ion ate influenza ed cells through immune effector mechanisms, such as ADCC, and promote direct viral clearance by g to influenza virons. The huM2e antibodies of the invention bind to the amino-terminal region of the M26 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 ’—l < '0 (D lliiiiIllliiiiiiiiiiiiIliliiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii 7.94 see In no: 4 SIN.301.1976 MSLLTEVETPIRSEWGCRCNDSSD SEQ ID NO:' 5 PANAMA.1.66 H2N2 MSFLPEVETPIRNEWGCRCNDSSD SEQ ID NO: 6 NEW YORK.321.1999 ' MSLLTEVETPIRNEWGCRCNDSSN SEQ ID NO: 7 CARACAS - l . 7 1 NEW 17-02 GUANGZHOU . 333 - 99 ' SWINE.HONG H3N2 MSLLTEVETPIRSEWGCRCNDSGD SEQ ID NO: 20 KONG.126.1982 HONG KONG-485.97 24 SILKY H9N2 MSLLTEVETPTRNGWECKCSDSSD SEQ ID NO: 26 CHICKEN.SHANTOU.1826.2 CHICKEN.TAIWAN.O305.04 H6N1 MSLLTEVETHTRNGWECKCSDSSD SEQ ID NO: 27 QUAIL.ARKANSAS.16309- H7N3NSA VKTPTRNGWECKCSDSSD SEQ ID NO: 28 HONG KONG.486.97 H5Nl MSLLTEVEILTRNGWGCRCSDSSD SEQ ID NO: 29 A CHICKEN.PENNSYLVANIA.1 H7N2NSB MSLLTEVEIPTRDGWECKCSDSSD SEQ ID NO: 3o 3552—l.98 ' A CHICKEN.HEILONGJIANG.4 H9N2 MSLLTEVETPTRNGWGCRCSDSSD SEQ ID NO: 31 8.01 v" lllliiiiiiiiiiii SWINE.KOREA.55.2005 MSLLTEVEIPIRNGWECKCNDSSD SEQ ID NO 32 HONG KONG.lO73.99 MSLLTEVETLTRNGWECKCSDSSD SEQ ID NO 33 WISCONSIN.3523.88 MSLLTEVETPIRNEWGCKCNDSSD SEQ ID NO 34 X—3l VACCINE STRAIN H3N2 MSFLTEVETPIRNEWGCRCNGSSD SEQ ID NO: 3.5 CHICKEN.ROSTOCK.8.1934 H7Nl MSLLTEVETPTRNGWECRCNDSSD SEQ ID NO 36 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 VETHTRNGWGCRCSDSSD SEQ ID NO: 4 o In one embodiment, the huM2e antibodies of the invention bind to a M26 that wholly or partially es the amino acid residues from position 2 to position 7 of M26 when ' i" ed in accordance with SEQ ID NO: 1. For example, the huM26 antibodies of the invention bind wholly or partially to the amino acid sequence SLLTEVET (SEQ ID NO: 41) Most preferably, the huMZe 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 M26 protein. For example, the huMZe antibodies bind to an e 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) on 2 is a serine;b) position 5 is a threonine; and c) position 6 is a ic acid. ary huM2e monoclonal antibodies that bind to this epitope are the TON-032 (8110), 21315, TCN—03l (23K12), 3241_G23, 3244_110, 3243_JO7, 3259_121, 3245_Ol9, 3244_HO4, 3136_GOS, 3252__C13, 3255_JO6, 3420_123, 3139_P23, 3248_P18, 10, 3260_D‘19, 11, and 3242_P05 antibodies described .
The TCN-032 (8110) 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 ce shown below in SEQ ID NO: 278, a long heavy chain variable region (SEQ ID NO: 276) encoded by the nucleic acid ce 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 EA. et al.(l99l, Sequences of Proteins of Immunological Interest, 5lh edit, NIH Publication no. 91-3242 US.
Department of Heath and Human Services.) are highlighted in bold in the sequences below.
The heavy chain CDRs of the 2 (8110) antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 72), NTKYNPSLKS (SEQ ID NO: 74) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the 2 (8110) 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 (81:10) antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 109), EIYYGGNTK (SEQ ID NO: 110) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the TCN-O32 (8110) 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 (8110) VH nucleotide sequence'{:;\(SEQ ID NO: 43) CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAG GGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCC CTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAG CTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTT TCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCG TCN-032 (8110) 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 TCN-032 (8110) VH short nucleotide sequence: (SEQ ID NO: 278) CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAG GGAAGGGACTGGAGTGGATTGGGTTTATCTATTAGGGTGGAAACACCAAGTACAATCCCTCC CTCAAGAGCCGCGTCACCATATCACAAGACACTECCAAGAGTCAGGTCTCCCTGACGATGAG CTCTGTGACCGCTGCGGAATCGGCCGTCTATET@TGTGGGAGAGCGTCTTGTAGTGGTGGTT ACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT TCN-032 (8110) 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 TCN-032 (8110) VH long nucleotide sequence: (SEQ ID NO: 196) CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAG GACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCC CTCAAGAGCCGCGTCACCATATCACAAGACACTTQEAAGAGTCAGGTCTCCCTGACGATGAG GACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTT ACTGTATCCTTGACTACTGGGGCCAGGGAACCCfiGGTCACCGTCTCGAGC .v’“. ‘ WO 12489 .
TCN-032 (8110) VH long amino acid sequence: (SEQ ID NO: 276) Kabat Bold, Chothia underlined H<N7€OIO HHmmH< <10 i<3><mmfi Spit-12010 OUJHi—imm m m Hm'U F'TJI—lKZC) <Omt<l<fi -'HD>D<.Qt<< ”embezx 'Mvoémru m m mt‘NWD-l b w m H 2 H m b o M a N 210 w w m m H Z > mi< > <10 0 0 0 Kmmror‘ Omt‘Oi-l U O 0 m TCN-032 (8110) VL nucleotide sequence: (SEQ ID NO: 45) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGA AAGCCCCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGG TGGAGATCAAAC 2 (8110) VL amino acid sequence: (SEQ ID NO: 46) 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 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 E‘ T L I I T s L Q P E D F A T Y Y C Q Q s Y s P P LI T F G G G T R v E I K :aA ' The 21815 dy includes a heavy chaiii 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 c acid sequence shown below in SEQ ID NO: 196, and a light chain variable region (SEQ ID NO: 46) encoded by the nucleic acid ce shown in SEQ ID NO: 48.
The amino acids encompassing the CDRs as defined by Chothia et a1. 1989, are underlined and those defined by Kabat et ‘a1., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 21B 15 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 21315 antibody have 7‘3? WO 12489 - the ing sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and PLT (SEQ ID NO: 63).
The heavy chain CDRs of the 21B 15 antibody have the following sequences per Chothia tion: GSSISN (SEQ ID NO: 109), FIYYGGNTK (SEQ ID NO: 110) and ASCSGCiYCILD (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) CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGQTGAACCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTTCGTCCATCAGTAATTAQTACTGGAGCTGGATCCGGCAGTCCCCAG GGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCC CTCAAGAGCCGCGTCACCATATCACAAGACACTTCDAAGAGTCAGGTCTCCCTGACGATGAG CTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTT ACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCG 21B15 VH amino acid sequence: (SEQ ID NO: 44) Kabat Bold, Chothia underlined H<1X7<OIO < <10 fl E O v m H m S H m m fi m 2131—1200 I M 210 w m W < I H Z H > w S <10 m<82m nmNHm mt‘KWl—l O 9 Kmmmr‘ Omr‘OH B U K G owmomm m HKUHUJ'U L-‘HJHKZO <o<b~<~<r HIVWGJK<I <wm029= (113,10me 21815 VH short nucleotide sequence: (SEQ ID NO: 278) CAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTTCGTCCATCAGTAATTACIACTGGAGCTGGATCCGGCAGTCCCCAG GGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCC CTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAQAGTCAGGTCTCCCTGACGATGAG GACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTT ACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT 21B15 VH short amino acid sequence: (SEQ ID NO: 277) Kabat Bold, Chothia underlined 0K3 HHMOH< unamb<1o 1<Bfi<mmt4 21311—126110 E 1O>momm m x m m b m S H S 2 H m x I M 0 H N 210 w N I m < m H Z < S <10 HKUHm'U r‘h'JHKzo") <1Omo<1<L-' ng>Wmt<< 3’10me w m 0 mr‘KSUv-l 0 0 «mt/itch Omt‘OH H G m 21B15 VH long tide sequence: (SEQ ID NO: 196) CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAG GGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCC CTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAG CTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTT TCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC 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) GACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGA CTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGG TGGATATCAAAC 21B15 VL amino acid sequence: (SEQ ID NO: 292) Kabat Bold, Chothia underlined D I Q V T Q S P S S L SJ 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-O3l (23K12) antibody includes a’h‘eavy bhain variable region (SEQ ID NQ: 50) encoded by the nucleic acid ce shovvn belojiv 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 c acid ce 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-03l (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-O3l (23K12) antibody have the following sequences per Kabat definition: RTSQSISSYLN (SEQ ID NO: 92), SGVPSRF (SEQ ID NO: 94) and QQSYSMPA (SEQ ID NO: 96).
The heavy chain CDRs of the TCN-03l (23K12) antibody have the following sequences per Chothia tion: GF'TVSSN (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 ) VH nucleotide sequence: (SEQ ID NO: 49) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTC AGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAG GGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCC GTGAAGGGCAGATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAA CAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGG GTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCG TCN-031 (23K12) VH amino acid sequence: (SEQ ID NO: 50) Kabat Bold, Chothia underlined om HDUOOID< UWSUF‘ID‘IO <tr1txjmmt-i 20m20< mth<n1m m m<w<<m HKUHUIC) Hmzmwm b <¢ 310 H m H 2 K m D m m D Q m < m m X 2 Z m W O > m moi—aware I."<Ii-JSC) mmi<<o NL-‘me Sorrow wzuww Q K O < H < 1 (23K12) VH short nucleotide sequence: (SEQ ID NO: 244) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTC CTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAG GGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCC GTGAAGGGCAGATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAA CAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGG GTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCACCGT TCN-03l (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 5M 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-03l (23K12) VH long nucleotide sequence: (SEQ ID NO: 195) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTC CTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAG GGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCC GTGAAGGGCAGATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAA CAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGG GTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC TCN-031 (23K12) VH long amino acid sequence: (SEQ ID NO: 235) Kabat Bold, Chothia underlined R. '2 mrxxnm meocu< 03>”er <tl'J'TJLTJUJIT‘ zomzm< mam<mm Grumman) m<w<<m HKUHmC) HKZKUIO b '<_<’ )- W H z :10 y m m 5 U w m 3 Z O hfiz mmr<<m wr'KDUm zovror w D Km<mm < < mn'r-idi‘mru mh<azm TCN-031 (23K12) VL nucleotide sequence: (SEQ ID NO: 51) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGA AAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTT TGCAACCTACTACTGTCAACAGAGTTACAGTATGGCTGCCTTTGGCCAGGGGACCAAGCTGG AGATCAAA . t TCN-03l (23K12) VL amino acid ce: (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 1 1—1 108] The 3241_023 dy (also referred to herein as G23) es a heavy chain variable region (SEQ ID NO: 1 16) encoded by the nucleic acid sequence shown below in SEQ ID NO: 1 15, and a light chain le region (SEQ ID NO: 118) encoded by the nucleic acid sequence shown in SEQ ID NO: 117.
The amino acids assing 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 023 antibody have the following sequences per Kabat definition: RASQSIGAYVN (SEQ ID NO: 184), S (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), SPR (SEQ ID NO: 183) and VGQMDKYYAMDV (SEQ ID NO: 181). The light chain CDRs of the 023 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) CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCAC TTGCACTGTCTCTGGTGGCCCCGTCAGCGGTGGTGGTTACTCCTGGAACTGGATCCGCCAAC GCCCAGGACAGGGCCTGGAGTGGGTTGGGTTCATGTTTCACAGTGGGAGTCCCCGCTACAAT CCGACCCTCAAGAGTCGAATTACCATCTCAGTCGACACGTCTAAGAACCTGGTCTCCCTGAA GCTGAGCTCTGTGACGGCCGCGGACACGGCCGTGTATTTTTGTGCGCGAGTGGGGCAGATGG ACAAGTACTATGCCATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3241_G23 VH amino acid sequence (SEQ ID NO: 116) Kabat Bold, Chothia underlined QVQLQQSGPGLVKPSQTLSLTCTVSGGPVSGGGYSWNWIRQRPGQGLEWVGFMFHSGSPRYN PTLKSRITISVDTSKNLVSLKLSSVTAADTAVYFCARVGQMDKYYAMDVWGQGTTVTVSS 3241_G23 VL nucleotide sequence (SEQ ID NO: 1-17) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTTCCTCTGTCGGAGACAGAGTCACCAT CCGGGCAAGTCAGAGCATTGGCGCCTATGTAAATTGGTATCAACAGAAAGCAGGGA AAGCCCCCCAGGTCCTGATCTTTGGTGCTTCCAATTTACAAAGCGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGACTT TGCAACTTACTTCTGTCAACAGACTTACAGTACCCCGATCACCTTCGGCCAAGGGACACGAC, TGGAGATTAAACG 3241_G23 VL amino acid sequence (SEQ ID NO: 118) Kabat Bold, Chothia underlined DIQMTQSPSSLSSSVGDRVTITCRASQSIGAYVNWYQQKAGKAPQVLIFGASNLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYFCQQTYSTPITFGQGTRLEIK The 3244_110 antibody (also ed to herein as 110) 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 a1., 1989 are underlined and those defined by Kabat et a1., 1991 are highlighted in bold in the ces below. = .~ The heavy chain CDRs of the 110 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), STKYNPSLKS (SEQ ID NO: 188) and HDAKFSGSYYVAS (SEQ ID NO: 189). The light chain CDRs of the 110 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 110 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 110 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193)Vand QQSYNTPLI (SEQ ID NO: 194). 3244_IlO VH tide ce (SEQ ID NQ: 119) CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGdTCAAéCCTTCGGACACCCTGGCCCTCAC TTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACTGGAGCTGGATCCGGCAACCCCCAG GACTGGACTGGATCGGATTCTTCTATAACGGCGGAAGCACCAAGTACAATCCCTCC CTCAAGAGTCGAGTCACCATTTCAGCGGACACGTCCAAdAACCAGTTGTCCCTGAAATTGAC i s CTCTGTGACCGCCGCAGACACGGGCGTGTATTATTpTGQGAGACATGATGCCAAATTTAGTG GGAGCTACTACGTTGCCTCCTGGGGCCAGGGAAQCCGAGTCACCGTCTCGAGC 3244_110 VH amino acid sequence (SEQ ID NO: 120) QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGFFYNGGST EYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDAKFSGSYYVASWG QGTRVTVSS 10 VL nucleotide sequence (SEQ ID NO: 121) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGCAACCTGGGA AAGCCCCTAAGGTCCTCATTTTTGGTGCAACCAACTTGCAAAGTGGGGTCCCATCTCGCTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGAGTTACAATACCCCCCTCATTTTTGGCCAGGGGACCAAGC TGGAGATCAAACG 3244_110 VL amino acid sequence (SEQ ID NO: 122) DIQMTQSPSSLSASVGDRVTISCRASS[SISTYLNWY-iQQQPGKAPKVLIFGATNLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCS 25 [SYNTPLIFGQGTKLEIK The 07 dy (also referred to herein as J07) includes a heavy chain le 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. ‘ [125] The amino acids assing 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 ing 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 1ight~chain CDRs of the JO7 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ lD NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: {94). v 3243_J07 VH tide sequence (SEQ ID NO: 123) CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACACCCTGGCCCTCAC TTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACTGGAGCTGGATCCGGCAACCCCCAG GGAGGGGACTGGACTGGATCGGATTCTTCTATAACGGCGGGAGCACCAAGTACAATCCCTCC CTCAAGAGTCGAGTCACCATATCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGAC CTCTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGTCAAATTTAGTG ACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGTCACCGTCTCGAGC 3243_J07 VH amino acid sequence (SEQ ID NO: 124) QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGFFYNGGST EYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDVKFSGSYYVASWG QGTRVTVSS 3243_J07 VL nucleotide sequence (SEQ ID NO: 125)‘ GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CTCTTGCCGGGCAAGTCAGAGCATTAGCACCIATTTAAATTGGTATCAGCAGCAACCTGGGA AAGCCCCTAAGGTCCTGATCTCTGGTGCAACCAACTTGCAAAGTGGGGTCCCATCTCGCTTC AGTGGCAGTGGATCTGGGACAGATTTCAcTcTCAQCATGAGCAGTCTGCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGAGTTACAATACccccc:CATTTTTGGCCAGGGGACCAAGC" TGGAGATCAAACG 3243_.107 VL amino acid sequence (SEQ ID NO: 126) DIQMTQSPSSLSASVGDRVTISCRASS[SISTYLNWYQQQF’GKAPKVLISGATNLS QSG GSGSGTDFTLTISSLQPEDFATYYCS gg [SYNTPLIFGQGTKLEIK The 32S9_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 dy have the following sequences per Kabat definition: SYNWI (SEQ ID NO: 203), HIYDYGRTFYNSSLQS (SEQ ID NO: 204) and YYAMDL (SEQ ID NO: 205). The light-'rehain CDRs of the J21 antibody have the following sequences per Kabat definition: RASQS-IDKFEN (SEQ ID NO: 208), GASNLHS (SEQ ID NO: 209) and QQSFSVPA (SEQ ID NO: 210). ’ The heavy chain CDRs of the J21 dy 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).
.. A 3259_J21 VH nucleotide sequence (SEQLID NO: 127) CAGGTGCAGCTGCAGGAGTCGGGCCCACGAGTGGTGAGGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCGGGGGGCTCCATCAGTTCTTACAACTGGATTTGGATCCGGCAGCCCCCTG GACTGGAGTGGATTGGGCACATATATGACTATGGGAGGACCTTCTACAACTCCTCC CTCCAGAGTCGACCTACCATATCTGTAGACGCGTCCAAGAATCAGCTCTCCCTGCGATTGAC CTCTGTGACCGCCTCAGACACGGCCGTCTATTACTGTGCGAGACCTCTCGGTATACTCCACT ACTACGCGATGGACCTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3259_J21 VH amino acid sequence (SEQ ID NO: 128) QVQLQESGPRVVRPSETLSLTCTVSGGSISSYNWIWIRQPPGKGLEWIGHIYDYGRTF YNSSLQSRPTISVDASKNQLSLRLTSVTASDTAVYYCARPLGILHYYAMDLWGQGT TVTVSS J21 VL nucleotide sequence (SEQ ID NO: 129) GACATCCAGATGACCCAGTCTCCATTATCCGTGTCTGTATCTGTCGGGGACAGGGTCACCAT CGCTTGCCGGGCAAGTCAGAGTATTGACAAGTTTTTAAATTGGTATCAGCAGAAACCAGGGA AAGCCCCTAAACTCCTGATCTATGGTGCCTCCAATTTGCACAGTGGGGCCCCATCAAGGTTC AGTGCCAGTGGGTCTGGGACAGACTTCACTCTAACAATCACCAATATACAGACTGAAGATTT CGCAACTTACCTCTGTCAACAGAGTTTCAGTGTCCCCGCTTTCGGCGGAGGGACCAAGGTTG AGATCAAACG ' 3259_J21 VL amino acid sequence (SEQAID NO: 130) DIQMTQSPLSVSVSVGDRVTIACRASSQSIDK‘IELNWYQQKPGKAPKLLIYGASNLHSG APSRFSASGSGTDFTLTITNIQTEDFATYLCS2g[SFSVPAFGGGTKVEIK The 3245_Ol9 antibody (also referred to herein as 019) es a heavy chain le region (SEQ ID NO: 132) d by the nucleic acid ce 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 a1., 1989 are underlined and those defined by Kabat et a1., 1991 are highlighted in bold in the sequences below.
The heavy chain CDRs of the 019 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 019 antibody have the on"; 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 019 antibody have the ing sequences per Chothia definition: GLSVSS (SEQ ID NO: 214), VFYSETRTY (SEQ ID NO: 215) and GMDV (SEQ ID NO: 213). The light chain CDRS of the 019 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), S (SEQ ID NO: 217) and QQTYSIPL (SEQ ID NO: 218). 3245_Ol9 VH nucleotide sequence (SEQ ID NQ:131) GAGGTGCAACTGGTGGAGTCTGGAGGGGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTC CTGTACGGCCTCTGGGTTAAGTGTCAGTTCCACCTACATGAACTGGGTCCGCCAGGCTCCAG GGAAGGGGCTGGAATGGGTCTCAGTTTTTTATAGTGAGACCAGGACGTACTACGCAGACTCC GTGAAGGGCCGATTCACCGTCTCCAGACACAATTCCAACAACACGCTCTATCTTCAGATGAA CAGCCTGAGAGTTGAAGACACGGCCGTGTATTATTGTGCGAGAGTCCAGAGATTGTCGTACG GTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3245_019 VH amino acid sequence (SEQ ID NO: 132) SGGGLVQPGGSLRLSCTASGLSVSSTYMNWVRQAPGKGLEWVSVFYSET RTYYADSVKGRFTVSRHNSNNTLYLQMNSLRVEDTAVYYCARVSZRLSYGMDVW GQGTTVTVSS 3245_019 VL nucleotide sequence (SEQ ID NO: 133) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGAAGAGACCAGGGA AAGCCCCTAAACTCCTGGTCTATGGTGCATCCACTTTGCAGAGTGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCGCCAGTCTGCAACCTGAAGATTC TGCAACTTACTACTGTCAACAGACTTACAGTATCCCCCTCTTCGGCCAGGGGACACGGCTGG AGATTAAACG 3245_019 VL amino acid sequence (SEQ ‘ID ND: 134) DIQMTQSPSSLSASVGDRVTITCRASQSISTY'LNWYQKRPGKAPKLLVYGASTLQSG VPSRFSGSGSGTDFTLTIASLQPEDSATYYCQQTYSIPLFGQGTRLEIK 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 ce shown below in SEQ ID NO: 135, and a light chain le region (SEQ ID NO: 138) encoded by the nucleic acid ce 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 GMDV (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 11) N0: 135) GAGGTGCAGCTGGTGGAATCTGGAGGGGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTC . AGCCTCTGGGTTAAGCGTCAGTTCCACQTACATGAACTGGGTCCGCCAGGCTCCAG GGCTGGAATGGGTCTCAGTTTTTTATAGTGAAACCAGGACGTATTACGCAGACTCC GGCCGATTCACCGTCTCCAGACACAATTCéAACAACACGCTGTATCTTCAAATGAA CAGCCTGAGAGCTGAAGACACGGCCGTGTATTATTGTGCGAGAGTCCAGAGACTGTCATACG GTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3244_H04 VH amino acid sequence (SEQ ID NO: 136) EVQLVESGGGLVQPGGSLRLSCTASGLSVSSTYMNWVRQAPGKGLEWVSVFYSET RTYYADSVKGRFTVSRHNSNNTLYLQMNSLRAEDTAVYYCARVSzRLSYGMDVW GQGTTVTVSS 3244_H04 VL nucleotide sequence (SEQ ID NO: 137) GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGAAGAGACCAGGGA AAGCCCCTAAACTCCTGGTCTATGGTGCATCCAGTTTGCAGAGTGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCGCCAGTCTGCAACCTGAAGATTC TGCAGTTTATTACTGTCAACAGACTTACAGTATCCCCCTCTTCGGCCAGGGGACACGACTGG AACG ' 3244_H04 VL amino acid sequence (SEQ TD No: 138) DIQMTQSPSSLSASVGDRVTITCRASS[SISTYLNW‘YQKRPGKAPKLLVYGASSLSQSG VPSRFSGSGSGTDFTLTIASLQPEDSAVYYCQS[TYSIPLFGQGTRLEIK The 3136__G05 antibody (also referred to herein as GOS) 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‘3ar‘e ghted in bold in the sequences below. :3.“ . i The heavy chain CDRs of the GOS 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_GOS 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 3 l36_G05 antibody have the ing sequences per a definition: GGSISS (SEQ ID NO: 206), YVYNRGSTK (SEQ ID NO: 232) and NGRSSTSWGIDV (SEQ ID NO: 230). The light chain CDRs of the 3136_GOS 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 ce (SEQ ID NO: 139) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCGGAGACCCTGTCCCTCAC CTGCAGTGTCTCTGGTGGCTCCATTAGTAGTGALETCTGGAGTTGGATCCGACAGCCCCCAG GGAAGGGACTGGAGTGGATTGGGTATGTCTATAACIAGAGGGAGCACTAAGTACAGTCCCTCC AGTCGAGTCACCATATCAGCAGACATGTCCAAGAACCAGTTTTCCCTGAATATGAG TTCTGTGACCGCTGCGGACACGGCCGTGTATEACTGTGCGAAAAATGGTCGAAGTAGCACCA GTTGGGGCATCGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCGAGC 3136_G05 VH amino acid sequence (SEQ ID NO: 140) QVQLQESGPGLVKPSETLSLTCSVSGGSISSDFWSWIRQPPGKGLEWIGYVYNRGST EYSPSLKSRVTISADMSKNQFSLNMSSVTAADTAVYYCAKNGRSSTSWGIDVWGK GTFVTVSS 3136_G05 VL nucleotide sequence (SEQ ID NO: 141) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGACTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTACATTGGTATCAGCAGAAACCAGGGA AAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC AGTAGATCAGGAACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGATGACTT TGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCCCTCACTTTCGGCCCTGGGACCAAAG TGGATATGAAACG 3136_G05 VL amino acid sequence (SEQ ID NO: 142) DIQMTQSPSSLSASVGDRLTITCRASS2SISTYLIJWYQQKPGKAPKLLIYAASSLS[SGV PSRFSGSRSGTDFTLTISSLQPDDFATYYCSQQSYSPPLTFGPGTKVDMK 2012/024971 The 3252_Cl3 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 ined and those defined by Kabat et a1., 1991:?‘3’ ghted in bold in the sequences '1 I below.
The heavy chain CDRs of the C13 antibody—have the following sequences per Kabat tion: 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 'l-IVGGl—ITYGIDY (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 PIT (SEQ ID NO: 243). 3252_Cl3 VH nucleotide sequence (SEQ ID NO: 143) CAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTGCCTCCATCAGTAGTGACTACTGGAGCTGGATCCGGCTGCCCCCAG ‘ GGAAGGGACTGGAGTGGATTGGGTATATCTATAAfAGAGGGAGTACCAAGTACACCCCCTCC CTGAAGAGTCGAGTCACCATATCACTAGACACGGCCGAGAACCAGTTCTCCCTGAGGCTGAG GTCGGTGACCGCCGCAGACACGGCCATCTATTAGTQTGCGAGACATGTAGGTGGCCACACCT ATGGAATTGATTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC 13 VH amino acid sequence (SEQ ID NO: 144) QVQLQESGPGLVKPSETLSLTCTVSGASISSDYWSWIRLPPGKGLEWIGYIYNRGSTK YTPSLKSRVTISLDTAENQFSLRLRSVTAADTAIYYCARHVGGHTYGIDYWGQGTL VTVSS 3252_C13 VL nucleotide sequence (SEQ ID NO: 145) GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCCTCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAACTATTTAAATTGGTATCAACACAAACCTGGGG AAGCCCCCAAGCTCCTGAACTATGCTGCGTCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGCCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTTCAACCTGAAGATTT TGCCACTTACTACTGTCAACAGAGTTACAATACTCCGATCACCTTCGGCCAAGGGACACGAC TGGAAATTAAACG 6‘ r 3252_Cl3 VL amino acid sequence (SEQ ID NO: 146) DIQMTQSPSSLSASVGDRVTITCRASS[SISNYLNWYQHKPGEAPKLLNYLASSLQLSG VPSRFSASGSGTDFTLTISSLQPEDFATYYC! TPITFGQGTRLEIK 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 ce 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 a1., 1991 are highlighted in bold in the sequences below.’ 43‘: The heavy chain CDRs of the J06 antibody haventhe following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), YIYNRGILSTKYTIPSLKS (SEQ ID NO: 237) and HVGGHTYGIDY (SEQ ID NO: 238). The light chain CDRs of the J06 antibody have the following sequences per Kabat tion: 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 tion: 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 ces per Chothia definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ 1D NO: 234) and QQSYNTPIT (SEQ ID NO: 243). 3255_J06 VH nucleotide ce (SEQ ID NO: 147) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTGCCTCCATCAGTAGTGACTACTGGAGCTGGATCCGGCTGCCCCCAG GGAAGGGACTGGAGTGGATTGGGTATATCTATAATAGAGGGAGTACCAAGTACACCCCCTCC CTGAAGAGTCGAGTCACCATATCACTAGACACGGCCGAGAACCAGTTCTCCCTGAGGCTGAG GTCGGTGACCGCCGCAGACACGGCCGTCTATTACTGTGCGAGACATGTGGGTGGCCACACCT ATGGAATTGATTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC 3255_J06 VH amino acid sequence (SEQ ID NO: 148) QVQLQESGPGLVKPSETLSLTCTVSG___ASISSDYWSWIRLPPGKGLEWIGYIYNRGSTK YTPSLKSRVTISLDTAENQFSLRLRSVTAADTAVYYCARHVGGHTYGIDYWGQGT LVTVSS 3255_J06 VL nucleotide ce (SEQ ID NO 149) GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCCTCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAACTATTTAAATTGGTATCAACACAAACCTGGGG CCAAGCTCCTGAACTATGCTGCGTCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGCCAGTGGATCTGGGACAGATTTCACTCTCAGCATCAGCGGTCTTCAACCTGAAGATTT TGCCACTTACTACTGTCAACAGAGCTACAATACTCCGATCACCTTCGGCCCAGGGACACGAC TGGAAATTAAACG 3255_J06 VL amino acid sequence (SEQ ID NO: 150) SPSSLSASVGDRVTITCRASS[SISNYLNWYQHKPGEAPKLLNYAASSLgZSG VPSRFSASGSGTDFTLSISGLQPEDFATYYCSQQSYNTPITFGPGTRLEIK The 3410_123 antibody (also referred to herein as 123) 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 a et al., 1989 are underlined and those defined by Kabat et al., 1991.3a‘r'e highlighted in bold in the sequences below. . 1 The heavy chain CDRs of the 3410_123 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 23 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), AASSLHS (SEQ ID NO: 258) and PIT (SEQ ID NO: 259). - 3420_123 VH nucleotide sequence (SEQ ID NO: 151) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCGTCAC CTGCAAAGTCTCTGGTGACTCCATCAGTAGTTATTCCTGGAGCTGGATCCGGCAGCCCCCAG GGAAGGGACTGGAGTGGGTTGGCTATTTGTATTATAGTGGGAGCACCAAGTACAACCCCTCC CTCAAGAGTCGAACCACCATATCAGTAGACACGTCCACGAACCAGTTGTCCCTGAAGTTGAG TTTTGTGACCGCCGCGGACACGGCCGTGTATTTgTGTGQGAGAACCGGCTCGGAATCTACTA CCGGCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3420_I23 VH amino acid sequence (SEQ ID NO: 152) QVQLQESGPGLVKPSETLSVTCKVSGDSISSYSWSWIRQPPGKGLEWVGYLYYSGST EYNPSLKSRTTISVDTSTNQLSLKLSFVTAADTAVYFCARTGSESTTGYGMDVWGQ G'ITVTVSS (.4; D 3420_123 VL nucleotide sequence (SEQ ID NQi, 153) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGAAACCAGGGA AAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCACAGTGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCGCTCTCACCATCAGCAGTCTGCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCGATCACCTTCGGCCAAGGGACACGAC TGGAGATTAAACG 3420_123 VL amino acid sequence (SEQ ID NO: 154) DlQMTQSPSSLSASVGDRVTITCRASS[SISTYLNWYQQKPGKAPKLLIYAASSLHSG VPSRFSGSGSGTDFALTISSLQPEDFATYYC! zg QSYSPPITFGQGTRLEIK The 23 antibody (also referred to herein as P23) es 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 ) encoded by the nucleic acid sequence shown in SEQ ID NO:157.
The amino acids encompassing the CDRs as d by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are ghted in bold in the sequences below. i 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 PLT (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 ces per Chothia definition: RASQTISTYLN (SEQ ID NO: 265), AASGLQS (SEQ ID NO: 61) and QQSYNTPLT (SEQ ID NO: 267). 3139_P23 VH tide sequence (SEQ ID NO: 155) CAGGTGCAGCTGCAGGAGTCGGGCCCAAGACTGGTGAAGCCTTCGGAGAGCCTGTCCCTCAC CTGCACTGTCTCTGGTGGCTCCATTAGTAATTCCTTCTGGGGCTGGATCCGGCAGCCCCCAG GGGAGGGACTGGAGTGGATTGGTTATGTCTATAACAGTGGCAACACCAAGTACAATCCCTCC CTCAAGAGTCGAGTCACCATTTCGCGCGACACGTCCAAGAGTCAACTCTACATGAAGCTGAG GTCTGTGACCGCCGCTGACACGGCCGTGTACTACTGTGCGAGGCATGACGACGCAAGTCATG GCATCTCCTGGGGCCACGGAACCCTGGTCACCGTCTCGAGC 3139_P23 VH amino acid sequence (SEQ ID NO: 156) QVQLQESGPRLVKPSESLSLTCTVSGGSISNSFWGWIRQPPGEGLEWIGYVYNSGNT EYNPSLKSRVTISRDTSKSQLYMKLRSVTAADTAYYYCARHDDASHGYSISWGHG" TLVTVSS - 3139_P23 VL tide sequence (SEQID NO; 157) CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGACCATTAGTACTTATTTAAATTGGTATCAACAGAAATCAGGGA AAGCCCCTAAGCTCCTGATCTATGCTGCATCCGGTTTGCAAAGTGGAGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTTCAACCTGAAGATTT TGCAACTTACTTCTGTCAACAGAGTTACAATACTCCCCTGACGTTCGGCCAAGGGACCAAGG TCAAA 3139_P23 VL amino acid sequence (SEQ ID NO: 158) DIQMTQSPSSLSASVGDRVTITCRASSETISTYLNWYQQKSGKAPKLLIYAASGLQSG VPSRFSGSGSGTDF’TLTISSLQPEDFATYFCS QQSYNTPLTFGQGTKVEIK 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 c acid sequence shown in SEQ ID NO: 161.
The amino acids encompassing the CDRS asidefined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991‘a're highlighted in bold in the sequences below. 2...
The heavy chain CDRS of the 3248_P18 antibody have the following sequences per Kabat definition: AYI-IWS (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), N (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 WO 12489 have the following sequences per Chothia definitionziiRASQSISRYLN (SEQ ID NO: 273); GASTLQN (SEQ ID NO: 274) and PA (SEQ ID NO: 275). 3248_P18 VH nucleotide sequence (SEQ ID NO: 159) CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTCGGGTGGCTCCATCAGTGCTTACCACTGGAGCTGGATCCGCCAGCCCCCAG GACTGGAGTGGATTGGGCACATCTTTGACAGTGGGAGCACTTACTACAACCCCTCC CTTAAGAGTCGAGTCACCATATCACTAGACGCGTCCAAGAACCAGCTCTCCCTGAGATTGAC CTCTGTGACCGCCTCAGACACGGCCATATATTACTGTGCGAGACCTCTCGGGAGTCGGTACT ATTACGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3248_P18 VH amino acid sequence (SEQ ID NO: 160) QVQLQESGPGLVKPSETLSLTCTVSGGSISAYHWSWIRQPPGKGLEWIGHIFDSGST XYNPSLKSRVTISLDASKNQLSLRLTSVTASDTAIYYCARPLGSRYYYGMDVWGQG TTVTVSS 3248_P18 VL nucleotide sequence (SEQ ID NO: 161) GACATCCAGATGACCCAGTCTCCGTCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGTATTAGCAGGTATTTAAATTGGTATCAGCAGAAACCAGGGA AAGCCCCTAAGCTCCTGATCTATGGTGCCTCCACTTTGCAAAATGGGGCCCCATCAAGGTTC AGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTC CGCAACTTACCTCTGTCAACAGAGTTACAGTGTCCCTGCTTTCGGCGGAGGAACCAAGGTGG AGGTCAAA 3248_P18 VL amino acid sequence (SEQ ID NO: 162) DIQMTQSPSSLSASVGDRVTITCRASS[SISRYLNWYQQKPGKAPKLLIYGASTLQNG APSRFSGSGSGTDFTLTISSLQPEDSATYLCS2gQSYSVPAFGGGTKVEVK The 10 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 c acid ce 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. n > The heavy chain CDRs of the 3253_P10 antibody‘have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187),4,FFYN‘GGS_TKYNPSLKS (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 dy 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), S (SEQ ID NO: 282) and QQSYNTPLI (SEQ ID NO: 194). 10 VH nucleotide sequence (SEQ:ID Nd: 163) CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACACCCTGGCCCTCAC TTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACTGGAGCTGGATCCGGCAACCCCCAG GGAGGGGACTGGACTGGATCGGATTCTTCTATAACGGCGGGAGCACCAAGTACAATCCCTCC CTCAAGAGTCGAGTCACCATATCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGAC CTCTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGCCAAATTTAGTG GGAGCTACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGTCACCGTCTCGAGC 10 VH amino acid sequence (SEQ ID NO: 164) SGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGFFYNGGST EYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDAKFSGSYYVASWG QGTRVTVSS 3253_P10 VL nucleotide sequence (SEQ ID NO: 165) GACATCCAGATGACCCAGTCTCCCTCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT CTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGCAACCTGGGA AAGCCCCTAAGGTCCTGATCTCTGGTGCAACCGACTTGCAAAGTGGGGTCCCATCTCGCTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCAQCATCAGCAGTCTGCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGAGTTACAATACCCCCCICATTTTTGGCCAGGGGACCAAGC TGGAGATCAAA 3253_P10 VL amino acid sequence (SEQ ID NQ: 166) DlQMTQSPSSLSASVGDRVTISCRASS[SISTYLNWYQQQPGKAPKVLISGATDLQSG VPSRFSGSGSGTDFI‘LTISSLQPEDFATYYC! zg QSYNTPLIFGQGTKLEIK The 3260_D19 antibody (also referred to herein as D19) es 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 ing sequences per Kabat definition: DNYIN (SEQ ID NO: 284), VFYSADRTSYADSVKG (SEQ ID NO: 285) and GMDV (SEQ ID NO: 286). The light chain CDRs of the 19 antibody have the following sequences per Kabat definition: RASQSISEYLN (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 ing sequences per a definition: GFSVSD (SEQ ID NO: 287), YFYSADRTS (SEQ ID NO: 288) and VQKSYYGMDV (SEQ ID NO: 286). The light chain CDRs of the 3260_Dl9 antibody have the following sequences per Chothia definition: SRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ, ID NO: 291). 3260_D19 VH nucleotide sequence (SEQ ID NO: 167) GACATGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCCGCCGGGGGGGTCCCTGAGACTCTC CTGCGCAGCCTCTGGGTTTTCCGTCAGTGACAACTACATAAACTGGGTCCGCCAGGCTCCAG vGGAAGGGGCTGGACTGGGTCTCAGTCTTTTATAGTGCTGATAGAACATCCTACGCAGACTCC GTGAAGGGCCGATTCACCGTCTCCAGCCACGATTCCAAGAACACAGTGTACCTTCAAATGAA CAGTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTGCGAGAGTTCAGAAGTCCTATTACG GTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3260_D19 VH amino acid sequence (SEQ ID NO: 168) DMQLVESGGGLVPPGGSLRLSCAASGFSVSDNYINWVRQAPGKGLDWVSVFYSADRTSYADS VKGRFTVSSHDSKNTVYLQMNSLRAEDTAVYYCARVQKSYYGMDVWGQGTTVTVSS 3260_D19 VL nucleotide sequence (SEQ ID NO: 169) GGCATCCAGATGACCCAGTCTCCATCCTCCCTGICTGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTTAAATTGGTATCTGCAGAAACCAGGGA AAGCCCCTAAGCTCCTGATCTCTGGTGCATCCAQTTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGGCACTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGTTTGCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGACTTTCAGTATCCCTCTTTTTGGCCAGGGGACCAAGGTGG AGATCAAA 19 VL amino acid sequence (SEQ ID NO: 170) GIQMTQSPSSLSASVGDRVTITCRASS[SISRYLNWYLQKPGKAPKLLISGASSLSESGV PSRFSGTGSGTEFI‘LTISSLQPEDFATYYCS 3g zTFSIPLFGQGTKVEIK The 3362_B1 1 antibody (also referred to herein as Bl 1) includes a heavy chain variable region (SEQ ID NO: 172) encoded by the nucleic acid sequence shown below in SEQ ID NO: 17l, 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 B1 1 antibody have the following sequences per Kabat definition: RASQSISRYL-N (SEQ ID NO: 273), AASSLQS (SEQ ID NO: 234) and QQSYSTPLT (SEQ ID NO: 300).
The heavy chain CDRs of the Bl 1 antibody have the ing sequences per a definition: GDSITSGA (SEQ ID NO: 296), YIYYSGNTY (SEQ ID NO: 297) and LGYGMDV (SEQ ID NO: 295). The light chain CDRs of the BI 1 antibody have the following sequences per Chothia tion: RASQSISRYLN (SEQ ID NO: 273), AASSLQS (SEQ ID NO: 234) and PLT (SEQ ID NO: 300). 3362_B11 VH nucleotide sequence (SEQ ID NO: 171) CAGGTGCAGCTGCAGGCGTCGGGCCCAGGACTGGTGAAGCCTTCAGAGACCCTGTCCCTCAC CTGCACTGTCTCTGGTGACTCCATCACCAGTGGTGCTTACTACTGGACCTGGATCCGCCAGC ACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAACACCTACTACAAC CCGTCCCTCAAGAGTCGAGTTACCATATCACTAGACACGTCTAAGAACCAGTTCTCCCTGAA GGTGAACTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGCGAGCTGCTTCGACTT CAGTGCTAGGATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3362_B11 VH amino acid sequence (SEQ ID NO: 172) QVQLQASGPGLVKPSETLSLTCTVSGDSITSGAYYWTWIRQHPGKGLEWIGYIYYSG NTYYNPSLKSRVTISLDTSKNQFSLKVNSVTAADTAVYYCARAASTSVLGYGMDV WGQGTTVTVSS . 3362_Bll VL nucleotide sequence (SEQ ID NO: 173) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTETGCATCTGTAGGAGACAGAGTCACCAT.
CCGGGCAAGTCAGAGCATTAGCAGATATTTAAATTGGTATCAGCAGGAACCAGGGA AGGCCCCTAAGCTCCTGGTCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTCTCACQATAAGCAGTCTTCAACCTGAAGATTT TGCAACTTACTACTGTCAACAGAGTTATAGTACCCCCCTCACCTTCGGCCAAGGGACACGAC TGGAGATTAAA 3362_B11 VH amino acid sequence (SEQ ID NO: 174) DIQMTQSPSSLSASVGDRVTITCRASSQSISRYLNWYQQEPGKAPKLLVYAASSLSQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSQSYSTPLTFGQGTRLEIK The 3242_P05 dy (also referred to herein as P05) includes a heavy chain variable region (SEQ ID NO: 176) encoded by thelnucle‘ic acid sequence shown below in SEQ ID NO: 175, and a light chain variable regioii (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 ghted in bold in the sequences below.
The heavy chain CDRs of the 3242_P05 antibody have the following ces per Kabat definition: N (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 dy have the following sequences per Chothia definition: SGFSV (SEQ ID NO: 304), VFYSADRTS (SEQ ID NO: 288) and GMDV (SEQ ID NO: 286). The light chain CDRs of the 3242_P05 antibody have the following sequences per Chothia definition: Tliglight‘chain CDRs of the 3242_P05 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), S (SEQ ID NO: 226) and PLRSEQ ID NO: 291). 3242_P05 VH nucleotide sequence (SEQ ID NO: 175) GACATGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCCGCCGGGGGGGTCCCTGAGACTCTC CTGCGCAGCCTCTGGGTTTTCCGTCAGTGACAACTACATAAACTGGGTCCGCCAGGCTCCAG GGAAGGGGCTGGACTGGGTCTCAGTCTTTTATAGTGCTGATAGAACATCCTACGCAGACTCC GTGAAGGGCCGATTCACCGTCTCCAGCCACGATTCCAAGAACACAGTGTACCTTCAAATGAA CAGTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTGCGAGAGTTCAGAAGTCCTATTACG GTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC 3242_P05 VH amino acid sequence (SEQ ID NO: 176) DMQLVESGGGLVPPGGSLRLSCAASGFSVSDNYINWVRQAPGKGLDWVSVFYSAD RTSYADSVKGRF'IVSSHDSKNTVYLQMNSLRAEDTAVYYCARVS[KSYYGMDVW GQGTTVTVSS 3242_P05 VL nucleotide sequence (SEQ ID NO: 177) GGCATCCAGATGACCCAGTCTCCATCCTCCCTGTETGCATCTGTAGGAGACAGAGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTIAAATTGGTATCTGCAGAAACCAGGGA AAGCCCCTAAGCTCCTGATCTCTGGTGCATCCAGKTTGCAAAGTGGGGTCCCATCAAGGTTC AGTGGCACTGGGTCTGGGACAGAATTCACTCTCACGATCAGCAGTTTGCAACCTGAAGATTT 2012/024971 TTACTACTGTCAACAGACTTTCAGTATCCCTCTTTTTGGCCAGGGGACCAAGGTGG AGATCAAA ' 05 VL amino acid sequence (SEQ ID NO: 178) GIQMTQSPSSLSASVGDRVTITCRASS[SISRYLNWYLQKPGKAPKLLISGASSLSQSGV PSRFSGTGSGTEFTLTISSLQPEDFATYYC] zgQTFSIPLFGQGTKVEIK HuM2e dies of the invention also e 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, 1 16, 120, 124, 128, 132, 136,140, 144, 148, 152, 156, 160, 164, 168, 172, or 176. and/ora 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 , 21B15, TCN-031 (23K12), 3241_G23, 3244_IlO, 3243_J07, 3259_121, 3245_Ol9, 04, 3136_G05, 3252_C13, 3255_J06, 3420_123, 3139_P23, 3248_P18, 3253_P10, 3260_Dl9, 3362_B1 l, or 3242_P05.
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 includea varjable 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‘:Ll00§88, M29812, M95114, X56360 and M95117. An IgHV3 germline gene sequence is sliovvn, e.:g., in Accession numbers X92218, X70208, 227504, M99679 and ABOl9437. The M26 antibodies of the invention include a VH region that is encoded by'a c 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 M26 antibody is at least 80% homologous to the amino acid ce 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 M26 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 ce encoded by the IgHV4 or the IgHV3 germline gene ce.
The M2e antibodies of the invention also include a variable light chain (VL) region encoded by a human IgKVl germline gene sequence. A human IgKVl VL germline gene sequence is shown, e.g., Accession numbers X59315, X59312, X59318, J00248, and Yl4865.
Alternatively, the M2e antibodies include a VL region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgKVl germline gene sequence. ably, the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgKVl ne gene sequence, and more preferably, at least 98%, 99% homologous to the IgKVl germline gene sequence. The VL region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VL region encodedthe IgKVl germline gene sequence Preferably, the amino acid sequence of VL region of the M2e antibodyis at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgKVl germline gene sequence, and more ably, at least 98%, 99% homologous to the sequence encoded by e the IgKVl germline gene sequence.
HA Antibodies I The HA antibodies of the ion may also be capable of specifically binding to one or more fragments of influenza virus H5Nl, such as the surface roteins, hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and cellular e, or membrane proteins (M1 and M2). In a specific embodiment, the HA dies 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/0r 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 optionallyisolated before use Alternatively, HA may be expressed on the surface of cells.
For diagnostic es, the HA dies 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 (N81 and N82). The nucleotide and/or amino acid sequence of ns of various HSNl strains can be found in the GenBank-database, NCBI za 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 ses.
In another embodiment the HA antibodies of the invention are capable of specifically g to a fragment of the above-mentioned proteins and/or polypeptides, wherein the fragment at least includes an antigenic determinant ized 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 ion with iently 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 les such as polyclonal or monoclonal antibodies or the HA antibodies can be n-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single—chain antibodies (sc’fFv), bivalent single—chain dies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, and (poly)peptides that contain at least a fragment of an immunoglobulinitihat is sufficient to confer specific antigen g to influenza virus HSNl strains or a fragmenf 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 dies 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 bination, e. g., as a pharmaceutical composition sing 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 ed in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic . Optionally, the e 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 ent of an influenza virus HSNl infectiOn. ‘ if . [244] Typically, HA antibodies can bind to theirdbinding'partners, i.e. za virus H5Nl 0r fragments thereof, with an affinity constant (Kd-value) that is lower than 0.2x 10'4 M, 1.0x10'5 M, 1.0x10'6 M, 1.0x10'7 M, preferably lower than 1.0x10'8 M, more preferably lower than 1.0x10‘9 M, more preferably lower than 1.0x10 M, even more preferably lower than 1.0x10‘ll M, and in particular lower than 1.0x10’” M The affinity constants can vary for dy es. For example. affinity binding fo'rah IgM isotype refers to a binding affinity of at least about 1.0x10‘7 M. Affinity constants canfor instance be measured using e plasmon resonance, for example using‘thePIiACORE system (Pharmacia Biosensor AB, a, Sweden).
HA antibodies may bind to za 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 HSNl 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 ient shape. Furthermore, the HA antibodies may bind to influenza virus H5Nl in purified/isolated or non purified/non-isolated form.
HA antibodies exhibit neutralizing activity. Neutralizing activity can for instance be ed as described in International Patent Application (Publication No. WO 28946, the contents of which are incorporated herein in their entirety).
Alternative assays ing neutralizing activity are bed in for instance WHO Manual on Animal Influenza sis and Surveillance, ; World Health Organization, 2005, IV: I version 2002.5.
The invention relates to an ed human HA antibody that recognizes and binds to an epitope in the HA2 subunit of the influenza haemagglutinin protein (HA), terized 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 HA] 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, zed 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 r 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 nt 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 sses 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, lgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
Antigen-binding fragments include, inter alia, Fab, , 2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, -chain antibodies (scFv), bivalent single-chain antibodies, —chain phage antibodies, diabodies, triabodies, odies, (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, New York, which is incorporated herein by reference. An HA antibody or antigen-binding fragment f may have one or more g sites. If there is more than one binding site, the binding sites may be identical to one another or they may be ent. i: 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 s of HA antibodies can be specific for linear epitopes, discontinuous epitopes, or mational epitopes of proteins or protein nts, 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. es 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 dy do not significantly affect or alter the binding teristics of the HA antibody d by the nucleotide sequence or containing the amino acid sequence, i.e. the dy is still able to recognize and bind its target. The functional variant may have conservative sequence cations including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by rd techniques known in the art, such as site—directed mutagenesis and random PCR- mediated nesis, and may include'natural as well asnon-natural nucleotides and amino acids.
Conservative amino acid tutions include the. ones in which the amino acid residue is replaced with an amino acid residue having r 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., aspai’tic acid, glutarnic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., e, e, 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 t may have nservative amino acid substitutions, e.g., replacement of an amino acid with an amino acid residue having different structural or al 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 n 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 ered 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 globulin 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 ce 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 ces are also considered to be human as used herein.
Single Chain HA Antibodies The heavy chain of an HA antibody15 derived from a germ line V ble) gene such as, for e, the VH1 or VH3 germline gene (see, Tomlinson IM, Williams SC, Ignatovitch O, Corbett SJ, Winter G. V-BASE Sequence Directory. Cambridge, United Kingdom: MRC Centre for Protein Engineering ). The HA antibodies of the invention include a V“ region that is encoded by a nucleic acid ce that is at least 80% gous 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 ne gene sequence, and more preferably, at least 98%, 99% homologous to the VH1 or VH3 germline gene sequence. The V" 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 sequencefiencod‘ed by the VH1 or VH3 germline gene ce, and more preferably, at least 98%, 99% homologous to the sequence d by the VHl or VH3 germline gene sequence. 3'- In certain s of the invention the VH1 germline gene is VH1 (l;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 (V1) region encoded by a human ne gene sequence selected from the group consisting of VKI, VKII, VKIII, VKIV, VLl, VL2, and VL3 (see, Torntl’inson IM, Williams SC, Ignatovitch O, t SJ, Winter G. V-BASE Sequence Directory.- Cambridge, United m: MRC Centre for Protein ering (1997)). AlternatiVely, the HA antibodies include a VL region that is encoded by a c acid sequence that is at least 80% homologous to the germline gene ce of VKI, VKII, VKIII, VKIV, VLl, 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, VLl, VL2, or VL3, and more preferably, at least 98%, 99% homologous to the germline gene sequence of VKI, VKII, VKIII, VKIV, VLl, 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, VLl, 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, VLl, VL2, or VL3, and more ably, at least 98%, 99% homologous to the sequence encoded by the germline gene sequence of VKI, VKII, VKIII, VKIV, VLl, VL2, or VL3. _ ; In certain s of the invention the VKIA-{g'eirmline’ gene is VKI (A20), the VKII germline gene is VKII (A3), the VKIII germline gene is)VilKIII (A27), and the VKIV germline gene is VKIV (B3). In other aspects of the invention, the VLl germline gene is VLl (VI-13), VLl (VI-16), VLl (VI-17), or. VLl (VI—19). Alternatively, the VL2 germline gene is VLZ (VI-3) or VL2 (V1—4). Furthermore, the VL3 germline gene is VL3 (V2-l4).
Specific combinations of a VH- and HL—locus are provided for each HA antibody described below.
The CDR s 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 ion, HA antibodies contain two, three, four, five or all six CDR regions as sed 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 ghted in bold in the sequences below. The heavy chain CDRs of the SCO6-14l antibody have the following CDR sequences: GYYVY (HCDRI, SEQ ID NO: 247), WISAYNGNTNYAQKFQG (HCDR2, SEQ ID NO: 248) and SRSLDV (HCDR3, SEQ ID NO: 568). The light chain CDRs of the SCO6-141 antibody have the following CDR sequences: KSSQSVLYSSNNKNYLA (LCDRI, SEQ ID NO: 569), WASTRES (LCDRZ, SEQ ID NO: 570)'and QQYYSTPLT (LCDR3, SEQ ID NO: 289). '7 SC06-14l nucleotide sequence (SEQ ID NO: 311) gaggtccagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 aagg cttctgggta caccttcacc ggctactatg tgtactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 aagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240 ctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagaagtaga 300 tccctggacg tctggggcca agggaccacg gtct cgagcggtac gggcggttca 360 accg gcagcggcac tggcgggtcg acggatgttg tgatgactca gtctccagac 420 tccctggctg tgtctctggg cgagagggcc accatcaact gcaagtccag ccagagtgtt 480 agct ccaacaataa gaactactta gcttggtacc agcagaaacc aggacagcct 540 ctgc~tcatttactg tacc tccg gggtccctga ccgattcagt 600 ggcagcgggt ctgggacaga tttcactctc accatcagca gcctgcaggc tgaagatgtg 660 gcagtttatt actgtcagca atattatagt actcctctca ctttcggcgg agggaccaaa 720 gtggatatca aacgt 735 41 amino acid sequence (SEQ ID NO: 312) EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGWISAYNGNTN YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSRSLDVWGQGTTVTVSSGTGGS GGTGSGTGGSTDVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVDIK R I I ii SC06-141 VH amino acid sequence (SEQ ID NO: 309) EVQLVQSGAEVKKPGASVKVSCKASGYTFI‘GYYVYWVRQAPGQGLEWMGWISAYNGNTN YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAYYYCARSRSLDVWGQGT’I‘VTVSS SC06-141 VL amino acid ce (SEQ ID NO: 310) DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVDIKR 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: 315uand the amino acid sequence shown in SEQ ID NO: 316. The VH—locus is VH1 (1-69) and the V:L locus is VLl (VI-16).
The amino acids encompassing the CDRs are;highlighted in bold in the ces below. The heavy chain CDRs of the SCO6-255 dy have the ing CDR sequences: SYAIS , 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 antibodyhave the ing CDR sequences: SGSTFNIGSNAVD (LCDRl, SEQ ID NO: 574), SNNQRPS (LCDR2, SEQ ID NO: 575) and AAWDDILNVPV (LCDR3,‘SEQ ID NO: 576).
SC06-255 nucleotide ce (SEQ ID NO: 315) gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc agtc 60 aagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc 120 cCtggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt tccagggcag agtcacgatt accgcggacq cggg cacagtttac 240 atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg 300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gtcctatgtg 420 cagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 480 ggaagcacgt tcaacatcgg tgct gtagactggt atéggcagct cccaggaacg 540 gcccccaaac tcctcatcta tagtaataat cagcggcpct caggggtccc tgaccgattc 600 tctggctcca ggtctggcac ctcagcctcc ctggccafica-gtgggctcca gtctgaggat 660 gaggctgatt attactgtgc agcatgggat gacatcctga atgttccggt attcggcgga 720 gggaccaagc tgaccgtcct aggt 744 SC06-255 amino acid sequence (SEQ ID NO: 316) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKG'ITVTVSSG TGGSGGTGSGTGGSTSYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLI YSNNQRPSGVPDRFSGSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPVFGGGTKLTVLG _[272] SC06-255 VH amino acid sequence (SEQ ID NO: 313) SGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYA VTITADDFAGTVYMELSSLRSEDTAMYYCAKHNIGYQVRETMDVWGKG 'ITVTVSS SC06-255 VL amino acid sequence (SEQ ID NO: 314) SYVLTQPPSASGTPGQRVTlSCSGSTFNIGSNAVDWYRQLPGTAPKLLIYSNNQRPSGVPDRF SGSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPVFGGGTKLTVLG The SC06-257 PIA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 317) and a light chain variableiegiOn (SEQ ID NO: 318) encoded by the nucleic acid sequence shown in SEQ ID NO: 3'19'and the amino acid sequence shown in SEQ ID NO: 320. The VH-locus is VH1 (1-69) and the VL locus is VL2 (VI-4).
WO 12489 2012/024971 The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the 57 dy have the following CDR sequences: SYAIS (HCDRI, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG , SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SCO6— 257 antibody have the following CDR sequences: .TGTSSDVGGYNYVS (LCDRI, 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 ctgggtcctc ggtgaaagtc 60 tcttgcaagg gagg 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 gcagtctgcc 420 ctgactcagc ctgccgccgt gtctgggtct cctggacagt cgatcaccat ctcctgcact 480 ggaaccagca gtgacgttgg tggttataac tatgtctcct ggtaccaaca gcacccaggc 540 aaaqccccca aactcatgat ttatgaggtc agtaatcggc cctcaggggt ttctaatcgc 600 ttctctggct ctgg caacacggcc tccctgacca tctctgggct ccaggctgag 660 gacgaggctg attattactg cagctcatat acaagcagca gcacttatgt cttcggaact 720 . gggaccaagg tcaccgtcct aggt 744 SC06-257 amino acid ce (SEQ ID NO: 320) QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSG TGGSGGTGSGTGGSTQSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK LMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVL SC06-257 VH amino acid ce (SEQ ID NO: 317) QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYA VTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVS SC06-257 VL amino acid sequence (SEQ ID NO: 318) PAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQ RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVLG The SCO6-260 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) d 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 VLl (VI-17).
WO 12489 The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SCO6-260 antibody have the. following CDR sequences: SYAIS (HCDRl, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDRZ, SEQ ID NO: 572) and HMGYQVRETMDV (HCDRS, SEQ ID NO: 573). The light chain CDRs of the SC06- 260antibody have the following CDR ces: SGSRSNVGDNSVY , SEQ ID NO: 580), KNTQRPS (LCDR2, SEQ ID NO: 581) and VAWDDSVDGYV (LCDR3, SEQ ID NO: 582). 60 nucleotide sequence (SEQ ID NO: 323) gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc agctatgcta gggt 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 aaag ggaccacggt caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gtcCtatgtg 420 cagc caccctcagt ctctgggacc caga gggtcaccat ctcttgctct 480 ggaagccgct ccaacgtcgg agataattct gtatattggt atcaacacgt cccagaaatg 540 gcccccaaac tcctcgtcta taagaatact caacggccct caggagtccc gttt 600 tccggctcca agtctggcac ttcagcctcc ctggccatca ttggcctcca.gtccggcgat 660 gaggctgatt attattgtgt ggcatgggat gacagcgtag atggctatgt cttcggatct 720 gggaccaagg tcaccgtcct aggt 744 SC06-260 amino acid sequence (SEQ ID NO: 324) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSG TGGSGGTGSGTGGSTSYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKL ‘ LVYKNTQRPSGVPARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYVFGSGTKVTV 60 VH amino acid sequence (SEQ ID NO: 321) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYA{SWVRQAPGQGPEWMGGIIPIFGTTKYA PKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVS s ; c ‘.
SC06-260 VL amino acid sequence (SEQ ID NO: 322) SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVYKNTQRPSGVPA RFSGSKSGTSASLAHGLQSGDEADYYCVAWDDSVDGYVFGSGTKVTVLG The SC06—261 PIA-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 ce 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 VLl (VI-19).
WO 12489 The amino acids encompassing the CDRs are 3highlighted in bold in the sequences below. The heavy chain CDRs of the SCO6-26l antibody have the following CDR sequences: SYAIS (HCDRI, SEQ ID NO: 571), GTTKYAPKFQG (HCDRZ, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SCO6- 261 antibody have the following CDR sequences: SGSSSNIGNDYVS (LCDRI, SEQ ID NO: 583), DNNKRPS (LCDR2, SEQ ID NO: 584) and ATWDRRPTAYVV (LCDR3, SEQ ID NO: 535).
SC06-261 nucleotide sequence (SEQ ID NO: 327) gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc 60 tcttgcaagg gagg 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 cgtc tggggcaaag ggaccacggt caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gcagtctgtg 420 ttgacgcagc cgccctcagt gtctgcggcc ccaggacaga ccat ctcctgctct 480 agct ttgg gaatgattat gtatcctggt acCagcagct cccaggaaca 540 gcccccaaac ttta tgacaataat aagcgaccct cagggattcc tgaccgattc 600 tctggctcca agtctggcac gtcagccacc ctgggcatca‘ccggactcca gactggggac 660 gaggccaact gcgc aacatgggat cgccgcccga ctgcttatgt cggc 720 ggagggacca agctgaccgt cctaggt 747 SC06-261 amino acid sequence (SEQ ID NO: 328) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGT’I‘VTVSSG TGGSGGTGSGTGGSTQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLL IYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTV SC06-261 VH amino acid sequence (SEQ ID NO: 325) SGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYA PKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVS SC06-261 VL amino acid sequence (SEQ ID NO: 326) SVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVLG 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).
WO 12489 The amino acids encompassing the CDRs are highlightedm bold1n the sequences below. The heavy chain CDRs of the SC06—262 antibody have the following CDR sequences: GSAIS (HCDRl, SEQ ID NO: 586), GISPLFGTTNYAQKFQG (HCDR2, SEQ ID NO: 587) and EYMDV (HCDR3, SEQ ID NO: 588). .Theilight chain CDRs of the SCO6-262 antibody have the following CDR sequences: RASQGISSYLA (LCDRl, SEQ ID NO: 589), DASTLRS (LCDRZ, SEQ ID NO: 590) and QRYNSAPPI (LCDR3, SEQ ID NO: 591).
SC06-262 nucleotide sequence (SEQ ID NO: 331) caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg tttccggagt cattttcagc gcga tcagctgggt gcgacaggcc 120 cctggacaag gccttgagtg gatgggaggg atcagccctc tctttggcac aacaaattac 180 gcacaaaagt gcag gatt accgcggacc cgaa cacaacctac 240 atggaggtga acagcctgag ggac acggccgtgt atttctgtgc gcgaggtcca 300 aaatattaca gtgagtacat ggacgtctgg ggcaaaggga ccacggtcac cgtctcgagc 360 ggtacgggcg gcgg aaccggcagc ggcactggcg ggtcgacgga catccagatg 420 acccagtctc catcctccct gtctgcatct gtaggagaca gagtcaccat cacttgccgg 480 gcgagtcagg gcattagcag ttatttagcc tggtatcagc agaagccagg gaaagttcct 540 acactcctga tctatgatgc atccactttg cgatcagggg tcccatctcg tggc 600 agtggatctg cgacagattt cactctcacc atcagcagcc tgcagcctga agatgttgca 660 acttattact gtcaaaggta taacagtgcc atca ccttcggcca agggacacga 720 ctggagatta aacgt 735 SC06-262 amino acid sequence (SEQ IDN ‘NJ0332) QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGGISPLFGTTNYAQ TITADQSTNTTYMEVNSLRYEDTAVYFCARGPKYYSEYMDVWGKGTTVTVSSGT GGSGGTGSGTGGSTD]QMTQSPSSLSASVGDRVT‘ITCRASQGISSYLAWYQQKPGKVPTLLIY DASTLRSGVPSRFSGSGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFGQGTRLEIKR SC06-262 VH amino acid sequence (SEQ ID NO: 329) QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGGISPLFGTTNYA QKFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGPKYYSEYMDVWGKGTTVTVSS SC06-262 VL amino. acid sequence (SEQ ID NO: 330) DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYDASTLRSGVPSRFS GSGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFGQGTRLEIKR The SCO6—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 ce shown in SEQ ID NO: 336. The VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-l4).
The amino acids encompassing the CDRs are ghted in bold in the sequences below. The heavy chain CDRs of the SC06-268 antibody havethe following CDR sequences: SYAIS (HCDRI, SEQ ID NO: 571), GIMGMFGTTNY‘AQKFQG (HCDRZ, SEQ ID NO: 592) and SSGYYPEYFQD (HCDR3, SEQ ID NO:593). The light chain CDRs of the SC06- 268 antibody have the ing CDR sequences: SGHKLGDKYVS (LCDRl, 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 aagc cctc ggtc 6O tcctgcaagg cttctggagg caccttcagt agttatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagga atcatgggta gcac aactaactac 180 gcacagaagt tccagggcag agtcacgatt gacg aattcacgag cgcagcctac 240 atggagctga ggagcctgag atctgaggac acggccgtct actactgtgc gaggtctagt 300 ggttattacc ccgaatactt ccaggactgg ggccagggca ccctggtcac cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgca gtctgtgctg 420 actcagccac cctcagagtc cgtgtcccca ggacagacag ccagcgtcac tgga 480 cataaattgg gggataaata tgtttcgtgg tatcagcaga agccaggcca gtcccctgta 540 ttactcatct atcaagataa caggcggccc tcagggatcc ctgagcgatt cataggctcc 600 aactctggga acacagccac tctgaccatc agcgggaccc aggctctgga tgaggctgac 660 tgtc aggcgtggga cagcagcact gcggttttcg gcggagggac caagctgacc 720 gtcctaggt 729 SC06-268 amino acid sequence (SEQ ID NO: 336) SGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIMGMFGTTNY AQKFQGRVTITADEFI‘SAAYMELRSLRSEDTAVYYCARSSGYYPEYFQDWGQGTLVTVSSG TGGSGGTGSGTGGSTQSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLI YQDNRRPSGIPERFIGSNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLG SC06-268 VH amino acid sequence~ (SEQ ID NO: 333) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIMGMFGTTN YAQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCARSSGYYPEYFQDWGQGTLVTVS SC06-268 VL amino acid sequence (SEQ ID NO: 334) PPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQDNRRPSGIPERFI TATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLG The SC06-272 PIA—specific single-chain Fv antibody es 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‘VHl (1-69) and the VL locus is VL2 (VI—3). [3051* 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 (HCDRl, SEQ ID NO: 597), GIIGMFGSTNYAQNFQG (HCDRZ, SEQ ID NO: 598) and STGYYPAYLHH (HCDR3, SEQ ID NO:‘599). The light chain CDRs of the SCO6-272 antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDRl, SEQ ID NO: 577), DVSKRPS (LCDRZ, SEQ ID N6: 601) and SSYTSSSTHV (LCDR3, ' 1,.
SEQ ID NO: 602). w._.. 72 nucleotide sequence (SEQ ID NQ: 339) cagatgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 6O aagg cttctggagg ctcc 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 cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgca gtctgccctg 420 actcagcctc gctcagtgtc cgggtctcct ggacagtcag tcaccatctc ctgcactgga 480 accagcagtg atgttggtgg ctat tggt accaacagca cccaggcaaa 540 gcccccaaac tcatgattta tgatgtcagt aagcggccct caggggtccc tgatcgcttc 600 tcca agtctggcaa cacggcctcc ctgaccatct ctgggctcca ggctgaggat 660 gaggctgatt attactgcag ctcatataca agcagcagca ctcatgtctt tggg 720 gtca ccgtcctagg t 741 SC06-272 amino acid sequence (SEQ ID NO: 340) QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGGHGMFGSTYAQ NFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTGYYPAYLHHWGQGTLVTVS SGTGGSGGTGSGTGGSTQSALTQPRSVSGSPGQSYTISCTGTSSDVGGYNYVSWYQQHPG KAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISQLQAEDEADYYCSSYTSSSTHVFGTG TKVTVLG . , *3 VI . u 72 VH amino acid sequence (SEQ ID NO: 337) QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGGIIGMFGSTNY AQNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTGYYPAYLHHWGQGTLVTVSS SC06-272 VL amino acid sequence (SEQ ID NO: 338) QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVP DRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVLG The SC06-296 HA-Specific single-chain Fv antibody es a heavy chain van'able 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 96 antibody have the ing CDR sequences: SYYMH (HCDRl, SEQ ID NO: 603), WINPNSGGTNYAQKFQG (HCDRZ, SEQ ID NO: 604) and EGKWGPQAAFDI (HCDR3, SEQ ID N62 605?). The light chain CDRS of the SCO6-296 antibody have the following CDR sequencesth‘iASQSVSSSYLA (LCDRl, SEQ ID NO: 646), DASSRAT (LCDRZ, SEQ ID NO: 6079 and QQYGSSLW (LCDR3, SEQ ID NO: 608).
SC06-296 nucleotide'sequence (SEQ ID NO: 343) 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 acggccgpgt gtgc gagagagggg 300 ggac cggc ttttgatatc tggggccaag ggaeaatggt caccgtctcg 360 agcggtacgg cagg cggaaccggc agcggcagtg gcgggtcgac ggaaattgtg 420 atgacgcagt gcac cctgtctttg tctccagggg aaagagccac cctctcctgc 480 agggccagtc agagtgttag cagcagctac ttagEctdgfi acéagcagaa acctggccag 540 gctcccaggc tcctcatcta tgatgcatcc gcca ctgacatccc agacaggttc 600 agtggcagtg ggtctgggac agacttcact ctcaccatca gcagactgga gcctgaagat 660 gtgt attactgtca gcagtatggt agctcacttt ggacgttcgg ccaagggacc 720 aaggtggaga gt 738 SC06-296 amino acid sequence (SEQ ID NO: 344) EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGGTN YAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGKWGPQAAFDIWGQGTMVTV , SSGTGGSGGTGSGTGGSTEIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP RLLIYDASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFGQGTKVEIKR SC06-296VH amino acid sequence (SEQ ID NO: 341) ’ EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGGTN YAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGKWGPQAAFDIWGQGTMVT SC06-296 VL amino acid sequence (SEQ ID NO: 342) EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLA‘WYQQKPGQAPRLLIYDASSRATDIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFGQQTKVEIKR The 01 HA-specific -chain FM antibody includes a heavy chain variable . region (SEQ ID NO: 345) and a light chain variable iegion (SEQ ID NO: 346) encoded by the nucleic acid sequence shown in SEQ ID NO: 347 and the amino acid ce 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 (HCDRI, SEQ ID NO: 609), AISSSGDSTYYADSVKG (HCDRZ, 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 (LCDRl, SEQ ID NO: 612), LGSNRAS (LCDRZ, SEQ ID NO: 613) and MQALQTPL (LCDR3, SEQ ID NO: 614). 59 3:42;} .
PCT/U82012/024971 01 nucleotide sequence (SEQ ID NO: 347) gaggtgcagc tggtagagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag gatt cacctttagc atctatgcca tgagctgggt ccgccaggca 120 ccagggaagg ggctggagtg ggtctcagct attagtagta gtggtgatag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca acgccaggaa cacgctgtat 240 ctgcaaatga acagtctgag agccgaggac gtgt attactgtgc gagagcgtat 300 ggctacacgt tcgacccctg gggccaggga accctggtca ccgtctcgag cggtacgggc 360 ggttcaggcg gaaccggcag cggcactggc gggtcgacgé aaattgtgct gactcagtct 420 ccactctccc tgcchtcac ccctggagag ccggcctéda tctcctgcag gtctagtcag 480 agcctcctgc atagtaatgg atacaactat ttggattggt acthcagaa gccagggcag 540 tctccacagc tcctgatcta tttgggttct aatcgggfict tccc tgacaggttc 600 agtggcagtg gatcaggcac taca ctgaaaafca gcagagtgga ggctgaggat 660 gttggggttt attactgcat gcaagctcta caaactcccc tcactttcgg cggagggacc 720 aaggtggaga tcaaacgt 738 SC06-301 amino acid sequence (SEQ ID NO: 348) EVQLVESGGGLVQPGGSLRLSCAASGFI‘FSIYAMSWVRQAPGKGLEWVSAISSSGDSTYYAD TISRDNARNTLYLQMNSLRAEDTAVYYCARAYGYTFDPWGQGTLVTVSSGTGGSG GTGSGTGGSTEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTDPTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR SC06-301 VH amino acid sequence (SEQ ID N0: 345) EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSAISSSGDSTYYA DSVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAYGYTFDPWGQGTLVTVSS SC06-301 VL amino acid sequence (SEQ ID NO: 346) SPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR The SC06-307 cific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 349) and a light chain variableiiegipnz(SEQ ID NO: 350) encoded by the c acid sequence shown in SEQ ID NO: 37511andithe 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 (HCDRl, SEQ ID NO: 615), SISSSSSYIYYVDSVKG (HCDR2, SEQ ID NO: 616) and GGGSYGAYEGFDY , SEQ ID NO: 617). The light chain CDRs of the SC06- 307 antibody have the following CDR sequences: RASQRVSSYLA (LCDRl, SEQ ID NO: 618), GASTRAA , SEQ ID NO: 619) and PLT (LCDR3, SEQ ID NO: 620).
SC06-307 nucleotide ce (SEQ ID NO: 351) caggtccagc tggtgcagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60 gcag 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 tcgagcggta cgggcggttc aacc ggcagcggca ctggcgggtc gacggaaatt 420 actc agtctccagg caccctgtct ttgtctccag gggaaagagc caccctctcc 480 tgcagggcca gtcagcgtgt tagcagctac ttagcctggt agaa acctggccag 540 gctcccaggc tcctcatcta tggtgcatcc accagggccg ctggcatccc agacaggttc 600 agtggcagtg ggtctgggac agacttcact ctcaccatca gcagactgga gcctgaagat 660 tctgcagtgt attactgtca tggt aggacaccgc tcgg gacc 720 aaggtggaga gt 738 SC06-307 amino acid sequence (SEQ ID NO: 352) QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYVD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGGSYGAYEGFDYWGQGTLVTVSS GTGGSGGTGSGTGGSTEIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLL IYGASTRAAGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGGGTKVEIKR SC06-307 VH amino acid sequence (SEQ ID NO: 349) QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYVD SVKGRFI‘ISRDNAKNSLYLQMNSLRAEDTAVYYlgfiRGGGSYGAYEGFDYWGQGTLVTVSS SC06-307 VL amino acid sequence (SEQ, ID NO: 350) EIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYGASTRAAGIPDRFS GSGSGTDPTLTISRLEPEDSAVYYCQQYGRTPLTFGGGTKVEIKR The SCO6-310 PIA-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 c 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: SYA'IS (HCDRl, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDRZ, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SCO6- 310 antibody have the following CDR sequences: GGNNIGSKSVH (LCDRl, SEQ ID NO: 621), S (LCDR2, SEQ ID NO: 622) and QVWDSSSDHAV (LCDR3, SEQ ID NO: 623).
WO 12489 SC06-310 nucleotide sequence (SEQ. ID NO: 355) cagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaagtc 60 tcttgcaagg gagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt tccagggcag agtcacgatt accgcggacg cggg cacagtttac 240 ctga tgcg atctgaggac acggccabgt actactgtgc gaaacatatg 300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaéég ggaccacggt caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg gcgggtcgac gtcctatgtg 420 ctgactcagc caccctcggt gtcagtggcc ccaggacéga cggccaggat tacctgtggg 480 ggaaacaaca ttggaagtaa aagtgtgcac tggtacéagc agaagccagg ccaggcccct 540 gtgctggtcg tctatgatga tagcgaccgg ccctcaggga tccctgagcg attctctggc 600 tccaactctg ggaacacggc caccctgacc atcagcaggg tcgaagccgg ggatgaggcc 660 gactattact gtcaggtgtg ggatagtagt agtgatcatg ctgtgttcgg aggaggcacc 720 cagctgaccg gt 738 SC06-310 amino acid sequence (SEQ ID NO: 356) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSG TGGSGGTGSGTGGSTSYVLTQPPSVSVAPGQTARITCGGNNlGSKSVHWYQQKPGQAPVLVV YDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLG 10 VH amino acid sequence (SEQ ID NO: 353) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYA PKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVS SC06-3l0 VL amino acid sequence (SEQ ID NO: 354) q [333] SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVIIWXQQKPGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLG The SCO6—3l4 HA—specific -chain F,,v. antiboiiy includes a heavy chain le region (SEQ ID NO: 357) and a light chain vaiiab‘l‘e fiégion (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 VLl (Vi—17).
The amino acids encompassing the CDRs are highlighted in bold in the ces below. The heavy chain CDRs of the SCO6-314 antibody have the following CDR sequences: SYAIS (HCDRI, SEQ ID NO: 571), GIIPIFGTTKYAPKFQG (HCDRZ, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573). The light chain CDRs of the SCO6- 3i4 antibody have the following CDR sequences: SGSSSNIGSNYVY , SEQ ID NO: 624), RDGQRPS (LCDR2, SEQ ID NO: 625) and ATWDDNLSGPV (LCDR3, SEQ ID NO: 626).
SC06-314 nucleotide ce (SEQ ID NO: 359) gaggtgcagc tggtggagtc tggggctgag gtgaagaagc Ctgggtcctc ggtgaaagtc 60 aagg cttctggagg ccccttccgc agctatgcta gggt gcgacaggcc 120 cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240 atggagctga tgcg atctgaggac acggccatgt actactgtgc gaaacatatg 300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag cggt caccgtctcg 360 agcggtacgg cagg cggaaccggc agcggcactg cgac gtcctatgtg 420 ctgactcagc caccctcagc qtctgggacc 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 gatt attactgtgc aacatgggat gacaacctga gtggtccagt attcggcgga 720 gggaccaagc tgaccgtcct aggt '1 744 SC06-314 amino acid ce (SEQ ID NO: 360) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSG TGGSGGTGSGTGGSTSYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLI YRDGQRPSGVPDRFSGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPVFGGGTKLTVLG SC06-314 VH amino acid sequence (SEQ ID NO: 357) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYA PKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGl I VTVS SC06-314 VL amino acid sequence (SEQ ID NO: 358) SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRDGQRPSGVPDRF SGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPVFGGGTKLTVLG The SCO6-323 cific 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 ce 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 (HCDRl, SEQ ID NO: 627), DIIGMFGSTNYAQNFQG (HCDRZ, SEQ ID NO: 628) and SSGYYPAYLPH (HCDR3, SEQ ID NO: 629). The light chain CDRs of the SC06- 323 antibody have the following CDR ces: RASQSVSSSYLA (LCDRl, 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 6O aagg gagg caccttctcc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagac atcatcggta tgtttggttc aacaaactac 180 gcacagaact tccagggcag actcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac gtgt attactgtgc gagaagtagt 300 ggttattacc ctgcatacct cccccactgg ggca ccttggtcac cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgga aattgtgttg 420 acccagtctc caggcaccct gtct ccaggggdaa gagccaccct ctcctgcagg 480 gccagtcaga gtgttagcag cagctactta gcctggtacc agcagaaacc tggccaggct 540 cccaggctcc tcatctatgg tgcatccagc agggccactg gcatcccaga caggttcagt 600 ggcagtgggt ctgggacaga cttcactctc accatcagca agcc tttt 660 gcagtgtatt actgtcagca gtatggtagc tcacccagaa ctttcggcgg agggaccaag 720 gtggagatea aacgt 735 SC06-323 amino acid sequence (SEQ ID NO: 364) SGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGDIIGMFGSTNYA QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSSGTG GSGGTGSGTGGSTEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFFLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR SC06-323 VH amino acid sequence (SEQ ID NO: 361) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGDHGMFGSTNYA QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSS 23 VL amino acid sequence (SEQ ID NO: 362) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS DFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR The SCO6-325 PIA—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 (Vl-4).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SCQ6-325 antibody have the following CDR sequences: FYSMS (HCDRI, SEQ ID NO: 633), GIIPMFGTTNYAQKFQG (HCDRZ, SEQ ID NO: 634) and GDKGIYYYYMDV (HCDR3, SEQ ID NO: 635). The light chain CDRs of the SC06-325 antibody have the ing CDR sequences: TGTSSDVGGYNYVS , SEQ ID NO: 577), EVSNRPS (LCDRZ, SEQ ID NO: 578) and SSYTSSSTLV (LCDR3, SEQ ID NO: 636).
WO 12489 SC06-325 nucleotide sequence (SEQ ID NO: 367) gaggtgcagc tggtggagtc tggggctgag aagc cggggtcctc ggtgaaggtc 6O tcctgcaagg cttctggagg caccttcagc ttctattcta gggt gcgacaggcc 120 cctggacaag gacttgagtg gatgggaggg atcatcccta tgtttggtac aacaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggtcg aatccacgag ctac 240 atggaggtga gcagcctgag atctgaggac acggccgttt attactgtgc gagaggtgat 300 aagggtatct actactacta catggacgtc tggggcaaag ggaccacggt caccgtctcg, 360 agcggtacgg gcggttcagg cggaaccggc agcggdactg gcgggtcgac gcagtctgcc 420 ctgactcagc ctgcctccgt gtctgggtct cctggacagt cgatcaccat ctcctgcact 480 ggaaccagca gtgacgttgg tggttataac tcct aaca gcacccaggc 540 aaagccccca aactcatgat ttatgaggtc agtaatcggc cctcaggggt ttctaatcgc 600 ttctctggct ccaagtctgg caacacggcc tccctgacca tctctgggct ccaggctgag 660 gacgaggctg attattactg cagctcatat acaagcagca ttgt cttcggaact 720 gggaccaagg tcaccgtcct aggt ' ~ 744 SC06-325 amino acid sequence (SEQ ID-NO: 368) EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFG'I'I‘NYA QKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGDKGIYYYYMDVWGKGTTVTVSSG TGGSGGTGSGTGGSTQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPK LMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVL 25 VH amino acid sequence (SEQ ID NO: 365) EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFGTTNY AQKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGDKGIYYYYMDVWGKGTTVTVS 25 VL amino acid sequence (SEQ ID NO: 366) QSALTQPASVSGSPGQSlTISCTGTSSDVGGYNYVSWYQQ RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLG The SCO6-327 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) d by the c acid sequence shown in SEQ ID NO: 371' and the amino acid sequence shown in SEQ ID NO: 372. The VH-locusIS VHI (1 6—9) and the VI locusIS VL3 (V2- 14).
The amino acids encompassing the CDRs arehighlightedll'l bold1n the sequences below. The heavy chain CDRs of the SC06-327 antibody have the ing CDR sequences: THAIS (SEQ ID NO: 637), GIIAIFGTANYAQKFQG (SEQ ID NO: 638) and GSGYHlSTPFDN (SEQ ID NO: 639). The light chain CDRs of the SC06-327 antibody have the following CDR sequences: GGNNIGSKGVl-l (SEQ ID NO: 640), DDSDRPS (SEQ ID NO: 622) and QVWDSSSDHVV (SEQ ID NO: 642). 1'» '.
SC06-327 nucleotide sequence (SEQ ID NO: 371) gaggtgcagc tggtggagac cggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg gagg 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 caac tggggccagg gaaccctggt caccgtctcg 360 agcggtacgg gcggttcagg cggc agcggcactg gcgggtcgac gtcctatgtg 420 ctgactcagc caccctcggt gtcagtggcc ccaggacaga cggccaggat tacctgtggg 480 ggaaacaaca gtaa aggtgtgcac tggtaccagc agaagcctgg ccaggcccct 540 gtgctggtcg tctatgatga ccgg 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 VYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVL G .
SC06-327 VH amino 'acid sequence (SEQ ID NO: 369) EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGGIIAIFGTANYA QKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGSGYHISTPFDNWGQGTLVTVSS 27 VL amino acid sequence (SEQ ID NO: 370) SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG The SCO6—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 ce 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 SCO6—328 antibody have the following CDR sequences: GYAIS (HCDRI, SEQ ID NO: 643), GTTNYAQKFQG (HCDRZ, SEQ ID NO: 644) and VKDGYCTLTSCPVGWYFDL (HCDR3, SEQID NO: 645). The light chain CDRs of the SC06—328 antibody have the ing CDR sequences: RASQSVSSSYLA (LCDRI, SEQ ID NO: 646), GASSRAT , SEQ 'IDENQ: 631:) and QQYGSSLT (LCDRS, SEQ ID NO: 648). ‘ WO 12489 SC06-328 nucleotide sequence (SEQ ID NO: 375) gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 6O 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 ccgg cagcggcact 420 ggcgggtcga ttgt gatgacgcag tctccaggca ccctgtcttt gtctccaggg 480 gaaagagcca cgtg cagt cagagtgtta gcagcagcta cttagcctgg 540 taccagcaga aacctggcca ggctcccagg ctcctcatct cctc cagcagggcc 600 actggcatcc cagacaggtt cagtggcagt gggtctggga cagacttcac tctcaccatc 660 agcagactgg agcctgaaga ttttgcagtg tattactgtc agcagtatgg tagctcactc 720 actttcggcg gagggaccaa gctggagatc aaacgt g“. 756 SC06-328 amino acid sequence (SEQ ID~NO: 376) EVALVESGAEVKKPGSSVKVSCKASGHIFSGYAI‘SWVRQAPGQGLEWMGGHPIFG’ITNYAQ KFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVKDGYCTLTSCPVGWYFDLWGRGTL TGGSGGTGSGTGGSTEIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIFGASSRATGIPDRFSGSGSGTDFI‘LTISRLEPEDFAVYYCQQYGSSLT 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 29 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 us is VH1 (1—69) e VL locus is VKIII (A27).
The amino acids encompassing the CDRs highlighted in bold in the sequences below. The heavy' chain CDRs of the SC06-329 antibhdy have the following CDR sequences: SNSIS , SEQ ID NO: 649), GIFALFGTTDYAQKFQG (HCDRZ, 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 (LCDRl, SEQ ID NO: 652), GASSRAS , SEQ ID NO: 653) and QQYGSSPLT (LCDR3, SEQ ID NO: 654).
SC06-329 tide sequence (SEQ ID NO: 379) gaggtccagc tggtacagtc tggggctgag gttaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg_cttctggagg catcttcaga tcta tcagttgggt gcgacaggcc 120 cctgggcaag agtg 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 cagg 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 '[367] SC06-329 amino acid sequence (SEQ ID NO: 380) EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSlSWVRQAPGQGLEWMGGIFALFG'ITDYAQ KFQGRVTlTADESSTTVYLELSSLTSEDTAVYYCARGSGYTTRNYFDYWGQGTLVTVSSGTG SGTGGSTEIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWTQQKPGQAPRLLIY GASSRASGIPDRFSGGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR SC06-329 VH amino acid ce (SEQ ID NO: 377) EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGGIFALFGTTDYA VTITADESSTTVYLELSSLTSEDTAVYYCARGSGYTTRNYFDYWGQGTLVTVSS SC06-329 VL amino acid sequence (SEQ ID NO: 378) EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWTQQKPGQAPRLLIYGASSRASGIPDRFS GGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR The SCO6-33l HA-specific —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 VHl (1—69) and the VL locus is VL3 (V2—l4).
The amino acids assing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the 3 ody have the following CDR sequences: SYAIS (HCDRI, SEQ ID NO: 571), GIIGMFGTAifIYAQKFQG (HCDRZ, SEQ ID NO: 655) and GNYYYESSLDY (HCDR3, SEQ ID NQ3I77656).' The light chain CDRs of the SC06-331 antibody have the following CDR sequences: QCNNIGSKSVH (LCDRl, 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 ggtc 60 tcctgcaagg cttctggagg caccttcagc agctathta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcgqta tgttcggtac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatttacgag cacagcctac 240 atggagctga tgag atctgaggac acggccgtgt attactgtgc aaat 300 tatfiactatg agagtagtct cgactactgg ggccagggaa ccctggtcac cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgca gtctgtcgtg 420 ccgc tgtc agtggcccca ggacagacgg ccaggattac ctgtggggga 480 aacaacattg gaagtaaaag tgtgcactgg caga agccaggcca ggcccctgtg 540 ctggtcgtct atgatgatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc 600 aactctggga acacggccac cctgaccatc agcagggtcg aagccgggga tgaggccgac 660 tattactgtc aggtgtggga tagt gatcattatg tcttcggaac tgggaccaag 720 gtcaccgtcc taggt 735 SC06-331 amino acid sequence (SEQ ID NO: 384) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIGMFGTANYA QKFQGRVTITADEFI‘STAYMELSSLRSEDTAVYYCARGNYYYESSLDYWGQGTLVTVSSGT GGSGGTGSGTGGSTQSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVV YDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG SC06-331 VH amino acid sequence (SEQ ID NO: 381) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIGMFGTANY AQKFQGRVTITADEFTSTAYMELSSLRSEDTAV.YJhYKCARIGNYYYESSLDYWGQGTLVTVSS SC06-331 VL amino acid ce (SEQ ID NO: 382) QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG The SCO6-332 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 (HCDRl, SEQ ID NO: 658), GIIAVFGTTKYAHKFQG (HCDRZ, SEQ ID NO: 659) and GPHYYSSYMDV (HCDR3, SEQ ID NO: 660). The light chain CDRs of the 32 antibody have the following CDR sequences: RASQGISTYLA (LCDRl, SEQ ID NO: 661), S (LCDR2, SEQ ID NO: 662) and QKYNSAPS (LCDR3, SEQ ID NO: 663).
SC06-332 nucleotide ce (SEQ ID NO: 387) caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtdaaggtc 60 tcctgcaagg cttctggagg ccccttccgc gcta tcaactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg gctg tctttgggac gacaaagtac 180 gcacataagt tccagggcag agtcaccatc accgtggacé actccacaaa tacagcttac 240 atggagctgg gcagcctgaa atctgaggac acggccgtgt attactgtgc tccc 300 cactactact cctcctacat ggacgtctgg ggcgaaggga ccacggtcac cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgga catccagttg 420 acccagtctc ccct gtctgcatct gtaggagaca gagtcaccat cacttgccgg 480 cagg gcac ttatttagcc tggtatcagc agaaacccgg gaaagttcct 540 aaactcctga tctatgctgc atccactttg caatcagggg tcccatctcg gttcagtggc 600 agtggatctg ggacagattt cactctcacc atcagcagcc ctga agatgttgca 660 acttattact gtcaaaagta taacagtgcc ccttctttcg gccctgggac caaagtggat 720 atcaaacgt 729 SC06-332 amino acid sequence (SEQ ID NO: 388) SGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKYA HKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGPl-lYYSSYMDVWGEGTTVTVSSGT GGDGGTGSGTGGSTDIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIY SGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKR SC06-332 VH amino acid sequence (SEQ ID NO: 385) QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKY AHKFQGRVTITADDSTNTAYMELGSLKSEDTAVXYCABGPHYYSSYMDVWGEGTTVTVSS SC06-332 VL amino acid'sequence (SEQ ID NO: 386) DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFS GSGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKR The SC06-334 cific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 389) and a light chain le 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—l4).
The amino acids encompassing the CDRs are highlighted in bold in the sequences below. The heavy chain CDRs of the SCO6—334 antibody have the following CDR sequences: SNAVS (l-ICDR], SEQ ID NO: 664), GILGVFGSPSYAQKFQG (HCDRZ, 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 (LCDRl, 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 aagt tccagggcag agtcacgatt accgcggacg aatccaccaa cacagtccac 240 atggagctga gaggtttgag atctgaggac acggccgtgt attattgtgc gagaggtcct 300 tact actcctacat ggacgtctgg ggcaaaggga ccacggtcac cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgtc gctg 420 ccac cctcggagtc agtggcccca ggacagacgg ccaggattac ggga 480 aataacattg gaagaaatag tgtgcactgg tatcagcaga agccaggcca ggcccctgtg 540 ctggtcgtgt atgatgatag cgaccggccc tcagggatcc ctgagcgatt ttctggctcc 600 ggga 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 QKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKG'ITVTVSSG TGGSGGTGSGTGGSTSYVLTQPPSESVAPGQTARI-TCGGNNIGRNSVHWYQQKPGQAPVLVV PSGIPERFSGSKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLG SC06-334 VH amino acid sequence (SEQ ID NO: 389) EVALVETGAEVKKPGSSVKVPCKSSGSPFRSNAVS\ll/VRQAPGQGLEWVGGILGVFGSPSYA VTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKGTTVTVSS SC06-334 VL amino acid sequence (SEQ ID NO: 390) SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSi GSKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLG The SC06-336 HA—specific single-chain Fv antibody includes a heavy chain le region (SEQ ID NO: 393) and a light chain variable region (SEQ ID NO: 394) d by the c 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 (HCDRl, SEQ ID NO: 670), GIFGMFGTANYAQKFQG (HCDRZ, SEQ ID NO: 671) and SSGYYPQYFQD (HCDR3, SEQ ID NOVI' .2 The light chain CDRs of the SCO6— 336 antibody have the following CDR sequences: R'ASQS'VSSSYLA (LCDRl, SEQ ID NO: 646), GASSRAT (LCDRZ, SEQ ID NO: 631) andi'QQYGSSSLT (LCDR3, SEQ ID NO: 308). ' SC06-336 nucleotide ce (SEQ ID Nb: 395‘) cagatgcagc tggtacaatc tggagctgag gtgaagaagc ctgégtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctatgéta tcagctgggt ggcc 120 cctggacaag ggcttgagtg gatgggaggg atcttcg§ta3tgtttgggac 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 aacc tggccaggct 540 cccagactcc tcatgtatgg tgcatccagc agggccactg gcatcccaga caggttcagt 600 ggcagtgggt ctgggacaga cttcactctc accatcagca agcc tgaagatttt 660 gcagtgtatt actgtcagca gtatggtagc tcatcgctca ctttcggcgg agggaccaag 720 ctggagatca aacgt 735 SC06-336 amino acid sequence (SEQ ID NO: 396) QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIFGMFGTANY AQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSSGYYPQYFQDWGQGTLVTVSSG TGGSGGTGSGTGGSTEIVM’TQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRL LMYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKR SC06-336 VH amino acid ce (SEQ ID NO: 393) QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIFGMFGTAN YAQKFQGRVTITADEFTSAAYMELSSLGSEDTAMLYYCARSSGYYPQYFQDWGQGTLVTVS S 1“}.
SC06-336 VL amino acid sequence (SEQ; ID NO: 394) EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWzYQQKPGQAPRLLMYGASSRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKR The SC06-339 HA—specific single—chain Fv antibody includes a heavy chain le region (SEQ ID NO: 397) and a light chain le region (SEQ ID NO: 398) d 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 (HCDRI, SEQ ID NO: 303), GIIAIFHTPKYAQKFQG (HCDRZ, SEQ ID NO: 306) and GSTYDFSSGLDY , SEQ ID NO: 725). The light chain CDRs of the 39 antibody have the ing CDR sequences: GGNNIGSKSVH (LCDRl, SEQ ID NO: 621), DDSDRPS , SEQ ID NO: 622) and QVWDSSSDHVV (LCDR3, SEQ ID NO: 642). 4: ,‘C- 1‘ SC06-339 nucleotide ce (SEQ ID NO: 399) gaggtgcagc tggtggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 6O tcctgcaagg cttctggagg catcttcaac agttatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggc atcatcgcta tctttcatac accaaagtac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa ctac 240 atggaactga gaagcctgaa atctgaggac acggccctgt attactgtgc gagagggtcc 300 gatt tttcgagtgg ccttgactac tggggccagg gaaecctggt caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcaéfig gcgggtcgac aggg 420 ctgactcagc caccctcggt ggcc ccaggacaga cggccaggat tacctgtggg 480 ggaaacaaca ttggaagtaa aagtgtgcac tggtaccagc agaagccagg ccaggcccct 540 gtcctagtcg tctatgatga tagcgaccgg ccctpaggga tccctgagcg attctctggc 600 tccaactctg ggaacacggc caccctgacc atcagcaggd tcgaagccgg ggatgaggcc 660 tact gtcaggtgtg tagt agtgatcatg tcgg cggagggacc 720 aagctgaccg tcctaggt 738 SC06-339 amino acid sequence (SEQ ID NO: 400) SGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGGIIAIFHTPKYAQ 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 SCO6-342 cific single-chain Fv?‘§ntibody includes a heavy chain variable region (SEQ ID NO: 401) and a light chain variable’regiohl(SEQ ID NO: 402) encoded by the nucleic acid sequence shown in SEQ ID NO: 4’03gand the amino acid sequence shown in SEQ ID NO: 404. The VII-locus is VI-Il (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 ing CDR sequences: SYAIS (HCDRI, SEQ ID NO: 251), GVIPIFRTANYAQNFQG (HCDR2, SEQ ID NO: 249) and LNYHDSGTYYNAPRGWFDP (HCDR3, SEQ ID NO: 246). The light chain CDRs of the SCO6—342 antibody have the following CDR sequences: KSSQSILNSSNNKNYLA (LCDRl, SEQ ID NO: 245), WASTRES , SEQ ID NO: 570) and QQYYSSPPT (LCDR3, SEQ ID NO: 250)., 2012/024971 SC06-342 nucleotide sequence (SEQ ID NO: 403) cagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtc 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 ataa cgccccccgg ggctggttcg acccctgggg ccagggaacc 360 ctggtcaccg tctcgagcgg tacgggcggt tcaggcggaa ccggcagcgg cactggcggg 420 tcgacggaca tccagatgac ccagtctcca gactccctgg ctgtgtctct gaag 480 atca actgcaagtc cagccagagt attttaaaca gctccaacaa taagaactac 540 ttagcttggt accagcagaa acag 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 ce (SEQ IDNO: 404) QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGGVIPIFRTANYA QNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYHDSGTYYNAPRGWFDPWGQGTLV TVSSGTGGSGGTGSGTGGSTDIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQ QKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPPTFGQ 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 GVPDRFSGSGSGTDFFLTISSLQAEDVAVYYCQQYYSSPPTFGQGTKVEIKR The SC06-343 HA—specific single—chain Fv antibody es 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-locus13 VH1 (l -69) and the VL locus15 VL3 (V2- 14).
The amino acids encompassing the CDRs are highlightedin bold1n the sequences below. The heavy chain CDRs of the 43 antibody have the ing CDR sequences: YYAMS (HCDRl, 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: SNSVH (LCDRl, SEQ ID NO: 224), S (LCDR2, SEQ ID NO: 223) and QVWGSSSDH (LCDR3, SEQ ID NO: 227). 2012/024971 SC06-343 nucleotide sequence (SEQ ID NO: 407) caggtccagc tggtgcagtc tggagctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagt caccttcagt tactatgcta tgagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg agga atcagcccta tgtttgggac aacaacctac 180 gcacagaagt tccagggcag agtcacgatt actgcggacg actccacgag tacagcctac 240 atggaggtga ggagcctgag atctgaggac gtgt attactgtgc gagatcttcg 300 aattactatg atagtgtata tgactactgg ggccagggaa ceqtggtcac cgtctcgagc 360 ggcg gttcaggcgg aaccggcagc ggcactggcg ggtcgacgca gtctgtcgtg 420 acgcagccgc cctcggagtc agtggcccca ggacagacgg ttac ctgtggggga 480 cataacattg gaagtaatag tgtgcactgg taccagcaga agccaggcca ggcccctgtg 540 ctggtcgtgt atgataatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc 600 aactctggga ccac cctgaccatc agcagggtcg aagccgggga tgaggccgac 660 tattactgtc aggtgtgggg tagtagtagt gaccattatq gaac caag 720 gtcaccgtcc taggt 735 43 amino acid sequence (SEQ ID NO: 408) QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGGISPMFG'I'I‘TY AQKFQGRVTlTADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVSSG TGGSGGTGSGTGGSTQSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVV YDNSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLG SC06-343 VH amino acid sequence (SEQ ID NO: 405) QVQLVQSGAEVKKPGSSVKVSCKASGVVTFSYYAMSWVRQAPGQGLEWMGGISPMFGTTT YAQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVS SC06-343 VL amino acid sequence (SEQ ID NO: 406) QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDNSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLG The SCO6-344 HA-specific single—chain Fvvantibohdy includes a heavy chain le 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: 41 land the amino acid sequence shown in SEQ ID NO: 412. The VH-locus is VH1 (1—69) and the VL locus is V_L1 (V1—I3).
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 ces: NYAMS (HCDRI, 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 (LCDRl, 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 aagc ctgggtcctc ggtgagagtc 60 tcctgcaagg cttctggaag catcttcaga aactatgcta gggt ggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcgcta tttttgggac accaaagtac 180 gcacagaagt tccagggcag agtcacgatt gacg aatcgacgag cactgtctac 240 atggaactga gcggactgag atctgaggac acggccatgt attactgtgc tccc 300 cactataatt ttggttcggg gagttatttc gactactggg gccagggaac cacc 360 gtctcgagcg gtacgggcgg ttcaggcgga accggcagcg gcactggcgg gact 420 gtgttgacac agccgccctc agtgtctggg gccccagggc agagggtcac catctcctgc 480 actgggagca gctccaacat cggggcaggt tatgatgtac actggtacca gcagcttcca 540 ggaacagccc ccaaactcct catctatggt aacagcaat; ggccctcagg ggtccctgac 600 cgattctctg gctccaagtc tggcacgtca ctgg gcatcaccgg actccagact 660 ggggacgagg ccgattatta ctgcggaaca tgggatagca gcctgagtgc cttc 720 ggaactggga ccaaggtcac cgtcctaggt 750 SC06-344 amino acid sequence (SEQ ID NO: 412) QVQLVQSGAEVKKPGSSVRVSCKASGSlFRNYAMSWVRQAPGQGLEWMGGIIAIFGTPKYA QKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVTVSS GTGGSGGTGSGTGGSTTVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK LLIYGNSNRPSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVT SC06-344 VH amino acid sequence (SEQ ID NO: 409) SGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGGIIAIFGTPKY AQKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVT SC06-344 VL amino acid sequence (SEQ ID NO: 410) TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDV FSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLG IgG HA Antibodies The CR6141 HA-specific IgG antibody includes atheavy 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 PIA—specific IgG antibody also includes a light chain variable region (SEQ ID NO: 414) d 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 tide 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 ctga gcagcctgag atctgaagac acggctgtgt gtgc gagaagtaga 300 tccctggacg tctggggcca cacg gtcaccgtct cgagtgctag caccaagggc 360 cccagcgtgt tccccctggc ccccagcagc aagagcacca gcggcggcac agccgccctg 420 ctgg tgaaggacta cttccccgag accg tgagctggaa cagcggcgcc 480 ttgaccagcg gcgtgcacac cttccccgcc gtgctgdaga gcagcggcct gtacagcctg 540 agcagcgtgg tgaccgtgcc cagcagcagc ctgggcaccc agacctacat ctgcaacgtg 600 aaccacaagc ccagcaacac caaggtggac aaacgcgtgg agcccaagag ctgcgacaag 660 acct gccccccctg ccctgccccc gagctgctgg gcggaccctc cgtgttcctg 720 ttccccccca agcccaagga catg cgga aggt cgtg 780 gtggtggacg tgagccacga ggaccccgag gtgaagttca actggtacgt ggacggcgtg 840 gaggtgcaca acgccaagac ccgg 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 agcc ggtggcagca gggcaacgtg 1260 ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gagc 1320 ctgagccccg gcaag 1335 CR6141 Heavy Chain amino acid sequence (SEQ ID NO: 413) EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGWISAYNGNTN YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSRSLDVWGQG'ITVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VKGFYPSDIAVEWESNGQPENNYK'I'I‘PPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK .- CR6141 VH amino acid sequence (SEQ ID-NO: 199) EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGWISAYNGNTN YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSRSLDVWGQG'I'I’VTVSS 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 gggc atctacccgg 180 gggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtact 300 cctctcactt tcggcggagg gaccaaagtg aaac gtgcggccgc acccagcgtg 360 ttcatcttcc ccccctccga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 420 ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480 aaca gccaggagag cgtgaccgag caggacagca aggactccac ctacagcctg 540 agcagcaccc tgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600 gtgacccacc agggcctgag cagccccgtg accaagagct tcaaccgggg cgagtgt 657 CR6141 Light Chain amino acid sequence (SEQ ID NO: 416) DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQK’YSTP‘LTFGGGTKVDIKRAAAPSVFIFPPS 77 ~ '5‘" DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CR6141 VL amino acid sequence (SEQ ID NO: 414) SPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFFLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVDIKR The CR6255 PIA—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 ce 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 tide 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 agtc tgag gtgaagaagc ctgggtcctc ggtgaaagtc 6O tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg gatgggaggg ccta tttttggtac atac 180 gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240 atggagctga gcagcctgcg atctgaggac atgt actactgtgc gaaacatatg 300 cagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt-caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc 420 acag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt gaccagcggc gtgcacacpt tccccgccgt gctgcagagc 540 agcggcctgt acagcctgag cagcgtggtg accgtgccda gcagcagcct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc agcaacaééa aggtggacaa acgcgtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc 720 tccg tgttcctgtt cccccccaag cccaaggada ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg agccacdagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga gtac 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 agag caacggccag cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag cctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca ctac 1320 acccagaaga gcctgagcct gagccccggc aag 1353 CR6255 Heavy Chain amino acid sequence (SEQ ID NO: 419) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAIS‘WVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 'J‘" 9' CR6255 VH amino acid sequence (SEQ'ID NO: 417) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKY RVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVS CR6255 Light Chain nucleotide sequence (SEQ ID NO: 421) tcctatgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcacgtt caacatcgga agtaatgctg tagactggta ccggcagctc 120 ccaggaacgg aact cctcatctat agtaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccag gtctggcacc tcagcctccc tggccatcag ccag 240 tctgaggatg aggctgatta ttactgtgca gcatgggatg acatcctgaa ggta 300 ttcggcggag ggaccaagct gaccgtccta ggtgcggccg caggccagcc caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc acaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa cagc 480 agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600 tgccaggtga cccacgaggg cgtg gagaagaccg tggcccccac cagc 660 CR6255 Light Chain amino acid sequence (SEQ ID NO: 422) SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIYSNNQRPSGVPDRFS GSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPVFGGGTKLTVLGAAAGQPKAAPSVTLF PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET’I’I‘PSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6255 VL amino acid sequence (SEQ IDz-NO: 420) SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIYSNNQRPSGVPDRFS GSRSGTSASLAlSGLQSEDEADYYCAAWDDILNVPVFGGGTKLTVLG 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 PIA-specific lgG antibody also es 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 tide sequence (SEQ ID NO: 424) caggcccagc tggtgcagtc tgggggtgag gtgaagaagc ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg aggg ccta gtac aacaaaatac 180 gcaccgaagt tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240 atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg 300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaég ggaccacggt caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc ccégcagcaa cagc 420 ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt cggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gggc 720 ggaccctccg tgtt cccccccaag cccaaggaca ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac 840 gtgg tgga caac 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 gaca 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 TLMISRTPEVTCVVVDVSHEDPEVKFNWXVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT'I’PPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ’4': CR6257 VH amino acid sequence (SEQ ID ‘NO: 423) SGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKY APKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVS CR6257 Light Chain nucleotide sequence (SEQ ID NO: 427) gccc tgactcagcc tgccgccgtg tctgggtctc ctggacagtc gatcacéatc 60 tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180 tctaatcgct gctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc tata caagcagcag cacttatgtc 300 ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc aggccaacaa ggccaccctg 420 ctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa cagc 480 agccccgtga aggccggcgt cacc 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) QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNY-VSWYQQ RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVLGAAAGQPKAAPSVTL FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE'ITTPSKQSNNKYAASSYLSL TPEQWKSHRSYS CQVTHEGSTVEKTVAP’I‘ECS CR6257 VL amino acid sequence (SEQ ID NO: 426) QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQ 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 tgag gtgaagaagc otéggtcctc ggtgaaagtc 60 tcttgcaagg gagg ccgc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg gatgggaggg atcatcccta gtac aacaaaatac 180 gcaccgaagt tccagggcag agtcacgatt gaeg atttcgcggg cacagtttac 240 atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg 300 gggtaccagg tgcgcgaaac cgtc tggggcaaag ggaccacggt caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 aaca 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 cgtg agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960 taca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc 1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc ggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag aaca actacaagac caccccccct 1200 gtgctggaca gcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 cagg gcaacgtgtt cagctgcagc cacg aggccctgca ctac 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 ce (SEQ ID NO: 429) l \.
EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS CR6260 Light Chain nucleotide sequence_(SEQ ID NO: 433) tcctatgtgc tgactcagcc accctcagtc tctgggaccc ccgggcagag ggtcaccatc 60 tcttgctctg gaagccgctc cgga gataattctg ggta tcaacacgtc 120 ccagaaatgg cccccaaact cctcgtctat aagaatactc aacggccctc aggagtccct 180 gcccggtttt ccggctccaa gtctggcact tcagcctccc tggccatcat ccag 240 tccggcgatg aggctgatta ttattgtgtg gcatgggatg acagcgtaga tggctatgtc 300 ttcggatctg ggaccaaggt caccgtccta ggtgcggccg agcc caaggccgct 360 gtga ccctgttccc cccctcctcc gaggagctgc acaa ggccaccctg 420 gtgtgcctca actt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca gctacctgag cctcaccccc gagcagtgga accg gagctacagc 600 tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc 660 CR6260 Light Chain amino acid sequence (SEQ ID NO: 434) PPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVYKNTQRPSGVP ARFSGSKSGTSASLAHGLQSGDEADYYCVAWDDSVDGYVFGSGTKVTVLGAAAGQPKAAP SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE'I'ITPSKQSNNKY AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6260 VL amino acid sequence (SEQ I_I_Q,NO.; 432) SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVEWYQQVPEMAPKLLVYKNTQRPSGVP ARFSGSKSGTSASLAHGLQSGDEADYYCVAWDQSVDGYVFGSGTKVTVLG a“ .
The CR6261 PIA-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 PIA-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 aggg 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 ccca gcagcagcct ccag 600 acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag 660 agct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca tgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg agccacgagg aggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga gtac 900 aacagcacct accgggtggt gagcgtgctc accgtgctgc actg gctgaacggc 960 aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc 1020 agcaaggcca agggccagcc ccgggagccc taca cccc cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc gtga agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag cccgagaaqa actacaagac caccccccct 1200 gtgctggaca gcgacggcag cttcttcctg aaqc tcaccgtgga caagagccgg 1260 tggcagcagg tgtt cagctgcagc gtgatgcacg aggccctgca ctac 1320 acccagaaga gcctgagcct ’ gagccccggc aag 1353 CR6261 Heavy Chain amino acid sequence (SEQ ID NO: 437) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTlTADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS ‘ LSSVVTVPSSSLGTQTY[CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK CR6261 VH amino acid sequence (SEQ ID NO: 435) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGT'I‘VTVSS CR6261 Light Chain nucleotide sequence (SEQ ID NO: 439) cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 6O tcctgctctg gaagcagctc caacattggg aatgattatg ggta ccagcagctc 120 ccaggaacag cccccaaact cctcatttat gacaataapa agcgaccctc agggattcct 180 gaccgattct ctggctccaa gtctggcacg tcagccacéc tgggcatcac cggactccag 240 actggggacg acta ttactgcgca acatgggafc cgac tgcttatgtt 300 gtcttcggcg ccaa gctgaccgtc ctaggtgcgg gcca gcccaaggcc 360 gctcccagcg tgaccctgtt ccccccctcc tccgaggagc tgcaggccaa caaggccacc 420 ctggtgtgcc tcatcagcga cttctaccct ggchcgtga cctg gaaggccgac 480 agcagccccg tgaaggccgg cgtggagacc accaccccca gcaagcagag caacaacaag 540 tacgccgcca gcagctacct gagcctcacc cccgagcagt ggaagagcca ccggagctac 600 cagg tgacccacga gggcagcacc gtggagaaga ccgtggcccc caccgagtgc 660 2012/024971 CR6261 Light Chain amino acid sequence (SEQ ID NO: 440) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVLGAAAGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6261VL amino acid sequence (SEQ ID NO: 438) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLQTGDEANYYCATWDRRPTAXVVFGGGTKLTVLG .
The CR6262 HA-specific IgG antibody incliides y chain le 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 aagc cctc ggtgaaggtc 6O aagg tttccggagt cattttcagc ggcagtgcga tcagctgggt gcgacaggcc 120 cctggacaag gccttgagtg gatgggaggg atcagccctc tctttggcac aacaaattac 180 gcacaaaagt tccagggcag agtcacgatt accgcggacc cgaa 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 gaag gactacttcc ccgagcccgt gagc 480 tggaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600 tacatctgca acgtgaacca caagcccagc aagg tggacaaacg cgtggagccc 660 aagagctgcg acaagaccca cacctgcccc cctg cccccgagct gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacacpc teatgatcag ccggaccccc 780 acct gogtggtggt ggacgtgagc cacgaggabc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaégc cccgggagga gcagtacaac 900 tacc gggtggtgag cacc gtgctgcaec agéactggct gaacggcaag 960 gagtaqaagt gcaaggtgag caacaaggcc ctgcctggcc ccatcgagaa gaccatcagc 1020 aaggccaagg gccagccccg ggagccccag accc tgccccccag ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg gctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca tcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagagcc tgagcctgag ccccggcaag 1350 CR6262 Heavy Chain amino acid sequence (SEQ ID NO: 443) QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGGISPLFGTTNYAQ KFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGPKYYSEYMDVWGKG'I'I‘VTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPlEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTFPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK CR6262 VH amino acid sequence (SEQ ID NO: 441) QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGGISPLFGTTNYAQ KFQGRVTITADQSTNTI‘YMEVNSLRYEDTAVYFCARGPKYYSEYMDVWGKGTTVTVSS CR6262 Light Chain nucleotide sequence (SEQ ID NO: 445) gacatccaga agtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcgagtca gggcattagc agttatttag cctggtatca gcagaagcca 120 gggaaagttc ctacactcct gatctatgat gcatccactt tgcgatcagg atct 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 gqctgctgaa caacttctac 420 gagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct tgac ccaccagggc 600 agcc ccgtgaCcaa gagcttcaac gagt gt 642 CR6262 Light Chain amino acid sequence (SEQ ID NO: 446) SPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYDASTLRSGVPSRFSG 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) d 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. f3? CR6268 Heavy Chain nucleotide sequence? (SEQ ID No: 443) caggtccagc tggtacagtc tgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagt agttatgcta gggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagga atcatgggta tgtttggcac aactaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aattcacgag cgcagcctac 240 atggagctga ggagcctgag atctgaggac acggccgtct actactgtgc tagt 300 ggttattacc ccgaatactt ccaggactgg ggccagggca ccctggtcac gagt 360 gctagcacca agggccccag cgtgttcccc ccca gcagcaagag cggc 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 aagg tggacaaacg cgtggagccc 660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcag 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 aaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gtgg 1260 cagcagggca acgtgttcag cgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagagcc tgagcctgag ccccggcaag 1350 CR6268 Heavy Chain amino acid sequence (SEQ ID NO: 449) SGAEVKKPGSSVKVSCKASGGTFSSYAi§WVR'QAPGQGLEWMGGIMGMFGTTNY AQKFQGRVTITADEFI‘SAAYMELRSLRSEDTAVYYCARSSGYYPEYFQDWGQGTLVTVSSA VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYK'ITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK CR6268 VH amino acid sequence (SEQ ID NO: 447) SGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIMGMFGTTNY AQKFQGRVTITADEFI‘SAAYMELRSLRSEDTAVYYCARSSGYYPEYFQDWGQGTLVTVSS CR6268 Light Chain nucleotide sequenceISEQ ID NO: 451) cagtctgtgc tgactcagcc accctcagag tccgtgtccc caggacagac agccagcgtc 60 acctgctctg gacataaatt gggggataaa tcgt 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 ccca aggccgctcc cagcgtgacc 360 ‘ctgttccccc cctcctccga gcag gccaacaagg tggt gtgcctcatc 420 agcgacttct accctggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480 gtgg agaccaccac ccccagcaag cagagcaaqg acaagtacgc cgccagcagc 540 tacctgagcc tcacccccga gcagtggaag agccaccgga gctécagctg ccaggtgacc 600 cacgagggca tgga gaagaccgtg gcccccaccg agfigcagc 648 «x - 9‘ is? | WO 12489 2012/024971 CR6268 Light Chain amino acid sequenc§(SEQ‘ID NO: 452) QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQDNRRPSGIPERFIG SNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLGAAAGQPKAAPSVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET'ITPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6268 VL amino acid sequence (SEQ ID NO: 450) QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQDNRRPSGIPERFIG SNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLG The CR6272 HA-specific IgG dy includes a heavy chain variable region (SEQ lD 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 ce (SEQ ID NO: 454) cagatgcagc tggtgcagtc tggggctgag aagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttctcc gcta tcacctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcggta tgpttggttc aacaaactac 180 gcacagaact tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctcag ggac gpgt atbactgtgc gagaagtact 300 ggttattacc ctgcatacct ccaccactgg ggccagggcq 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 gcag cgtggtgacc agca 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 ctgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc gggtggtgag cgtgctcacc cacc aggactggct gaacggcaag 960 aagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag 1080 aaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt gtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca tcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagagcc tgagcctgag ccccggcaag 1350 CR6272 Heavy Chain amino acid sequence (SEQ ID NO: 455) QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGGIIGMFGSTNYA QNFQGRVTITADESTSTAYMELSSLRSEDTAVYYQARSTGYYPAYLHHWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV'I‘VSWNSGALTSGVHTFPAVLQSSGLYSLS SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK WO 12489 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK CR6272 VH amino acid sequence (SEQ ID NO: 453) QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGGIIGMFGSTNYA QNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTGYYPAYLHHWGQGTLVTVSS CR6272 Light Chain nucleotide ce (SEQ ID NO: 457) cagtctgccc tgactcagcc tcgctcagtg tctc ctggacagtc agtcaccatc 60 tcctgcactg gaaccagcag tgatgttggt ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa actcatgatt tatgatgtca gtaagcggcc ctcaggggtc 180 cgct tctctggctc caagtctggc aacacggcct ccctgaccat gctc 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 accg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt cacc acccccagca agcagagcaa caacaagtac 540 gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg cagc 600 tgccaggtga cccacgaggg cagcaccgtg gagaagageg tggcccccac cgagtgcagc 660 CR6272 Light Chain amino acid sequenfce(SEQ ID NO: 458) QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPD RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVLGAAAGQPKAAPSVTL ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETITPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAP’TECS CR6272 VL amino acid sequence (SEQ ID NO: 456) GSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPD RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVLG The CR696 PIA-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 PIA-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.
WO 12489 CR6296 Heavy Chain nucleotide sequence (SEQ ID NO: 460) gaggtgcagc agac cggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg gata caccttcacc agctactata tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180 gcacagaagt ttcagggcag ggtcaccatg gaca tcag cacagcctac 240 atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagagggg 300 aaatggggac ctcaagcggc ttttgatatc tggggccaag ggacaatggt caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc gcéc ccagcagcaa gagcaccagc 420 ggcggcacag tggg ctgcctggtg aaggactaét tcgccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccchccgt gctgcagagc 540 agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc agcaacaécq aggtggacaa acgcgtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgcq ccga gctgctgggc 720 ggaccctccq tgttcctgtt cccccccaag cccaaggaca tgat gacc 780 cccgaggtga cctgcgtggt cgtg agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca ggga ggagcagtac 900 aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960 aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc 1020 agcaaqgcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca ctac 1320 acccagaaga gcct gagccccggc aag 1353 CR6296 Heavy Chain amino acid sequence (SEQ ID NO: 461) EVQLVETGAEVKKPGASVKVSCKASGYTFI‘SYYMHWVRQAPGQGLEWMGWINPNSGGTN GRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGKWGPQAAFDIWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK CR6296 VH amino acid sequence (SEQ‘f‘I‘D‘N0g459) EVQLVETGAEVKKPGASVKVSCKASGYTFI‘SYYMHWVRQAPGQGLEWMGWINPNSGGTN YAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGKWGPQAAFDIWGQGTMVTV CR6296 Light Chain nucleotide sequence (SEQ ID NO: 463) gtga tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 6O ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct ctat gatgcatcca gcagggccac ccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 gatt 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 caac cggggcgagt gt 642 CR6296 Light Chain amino acid sequence (SEQ ID NO: 464) EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLA€WYQQKPGQAPRLLIYDASSRATDIPDRFS GSGSGTDEI‘LTISRLEPEDFAVYYCQQYGSSLWTFGQGTKVEIKRAAAPSVFIFPPSDEQLKSG 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 tide 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) d 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 aggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt tagc atctatgcca tgagctgggt ccgccaggca 120 aagg ggctggagtg ggtctcagct attagtagta atag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca acgccaggaa cacgctgtat 240 ctgcaaatga acagtctgag agccgaggac acggctgtgfi attactgtgc gagagcgtat 300 ggctacacgt tcgacccctg gggccaggga accctggtca ccgtptcgag 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 caag gtggacaaac gcgtggagcc caagagctgc 660 gacaagaccc acacctgccc cccctgccct gcccccgagc tgctgggcgg accctccgtg 720 ttcctgttcc cccccaagcc caaggacacc ctcatgatca gccggacccc gacc 780 tgcgtggtgg tggacgtgag ggac gtga agttcaactg ggac 840 gagg tgcacaacgc caagaccaag ccccgggagg agcagtacaa cagcacctac 900 cgggtggtga gcgtgctcac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 960 tgcaaggtga aggc cctgcctgcc cccatcgaga agaccatcag caaggccaag 1020 ggccagcccc gggagcccca ggtgtacacc ctgcccécca gccgggagga gatgaccaag 1080 aaccaggtgt ccctcacctg tctggtgaag ggcttctacc ccagcgacat cgccgtggag 1140 agca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1200 gacggcagct tcttcctgta cagcaagctc gaca agagccggtg gcagcagggc 1260 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 1320 ctgagcctga gccccggcaa g 1341 CR6301 Heavy Chain amino acid sequence (SEQ ID NO: 467) EVQLVESGGGLVQPGGSLRLSCAASGEFFSIYAMSWVRQAPGKGLEWVSAISSSGDSTYYAD SVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAYGYTFDPWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN LSLSPGK CR6301 VH amino acid sequence (SEQ ID NO: 465) EVQLVESGGGLVQPGGSLRLSCAASGFI‘FSIYAMS‘WVRQAPGKGLEWVSAISSSGDSTYYAD SVKGRFTISRDNARNTLYLQMNSLRAEDTAVYY_;CARAYGYTFDPWGQGTLVTVSS CR6301 Light Chain tide sequence (SEQ ID NO: 469) gaaattgtgc tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120 caga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tgca tgcaagctct acaaactccc 300 ctcactttcg gcggagggac caaggtggag atcaaacgtg cggccgcacc cagcgtgttc 360 atcttccccc cctccgacga gcagctgaag agcggcaccg ccagcgtggt gtgcctgctg 420 aacaacttct acccccggga ggtg cagtggaagg tggacaacgc cctgcagagc 480 ggcaacagcc aggagagcgt gaccgagcag gacagcaagg actccaccta gagc 540 agcaccctca ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 600 acccaccagg gcctgagcag ccccgtgacc ttca accggggcga gtgt 654 )486] CR6301 Light Chain amino acid sequence (SEQ ID NO: 470) EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKRAAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC CR6301 VL amino acid sequence (SEQ IDNO:468) SPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP DRFSGSGSGTDFFLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR The CR6307 ecific lgG antibody es 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 ggct 120 ccagggaagg ggctggagtg atcc 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 cgtg ttccccctgg cccccagcag caagagcacc 420 agcggcggca cagccgccct gggctgcctg‘gtgaaggaét acttccccga gcccgtgacc 480 gtgagctgga acagcggcgc cttgaccagc ggcgtgcaca ccttccccgc cgtgctgcag 540 agcagcggcc tgtacagcct gagcagcgtg gtgaccg.tgc gcag cctgggcacc 600 cagacctaca tctgcaacgt gaaccacaag cccagcaaca ccaaggtgga caaacgcgtg 660 gagcccaaga gctgcgacaa gacccacacc tgccccccct cccc 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 gtgc tgcaccagga ctggctgaac 960 ggcaaggagt acaagtgcaa caac aaggccctgc ctgcccccat cgagaagacc 1020 atcagcaagg ccaagggcca gccccgggag gtgt acaccctgcc ccccagccgg 1080 gaggagatga acca ggtgtccctc acctgtctgg tgaagggctt ctaccccagc 1140 gacatcgccg ggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1200 ctgg acagcgacgg cagcttcttc ctgtacagca agctcaccgt ggacaagagc 1260 cggtggcagc aggqcaacgt gttcagctgc atgc acgaggccct gcacaaccac 1320 tacacccaga agagcctgag cctgagcccc ggcaag 1356 CR6307 Heavy Chain amino acid sequence (SEQ ID NO: 473) QVQLVQSGGGLVKPGGSLRLSCAASGFI‘FSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYVD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGGSYGAYEGFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMlSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK w,- CR6307 VH amino acid sequence (SEQJD N03,” 471) QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYVD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCA’RGGGSYGAYEGFDYWGQGTLVTVSS CR6307 Light Chain nucleotide ce (SEQ ID NO: 475) gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 6O ctctcctgca gggccagtca gcgtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatggt gcatccacca gggccgctgg agac 180 aggttcagtg gcagtgggtc tgggacagac ctca ccatcagcag gcct 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 gcaa cagccaggag 480 agcgtgaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctg 540 gccg agaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600 agcagccccg tgaccaagag ccgg ggcgagtgt 639 1:»- CR6307 Light Chain amino acid sequence (SEQ ID NO: 476) 'EIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYGASTRAAGIPDRFSG FTLTISRLEPEDSAVYYCQQYGRTPLTFGGGTKVEIKRAAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC CR6307 VL amino acid ce (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 ce 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 gcba tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg gatgggaggg atcatcccfia tttttggtac aacaaaatac 180 gcaccgaagt tccagggcag gatt accgcggacg apptcgcggg cacagtttac 240 atggagctga gcagcctgcg atctgaggac acggccatgt accactgtgc tatg 300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc ccégcagcaa cagc 420 ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt acagcctgag cagcgtggtg ccca gcagcagcct gggcacccag 600 atct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag 660 cccaagagct gcgacaagac ctgc cccccctgcc ctgcccccga gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac 840 ‘tggtacgcgg tgga 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 gaca gcgacggcag cctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg gcaacgtgtt cagc cacg tgca ctac 1320 acccagaaga gcctgagcct gagccccggc aag 1353 CR6310 Heavy Chain amino acid sequence (SEQ ID NO: 479) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL -.i.
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 gggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca aggc 120 caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gccg actattactg tcaggtgtgg gatagtagta gtgatcatgc tgtgttcgga 300 ggaggcaccc agctgaccgt cctcggtgcg gccgcaggcc agcccaaggc cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc 420 agcg accc tggcgccgtg accgtggcct ggaaggccga cagcagcccc 480 gccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600 gtgacccacg agggcagcac cgtggagaag accgtggccc agtg cagc 654 CR6310 Light Chain amino acid sequence (SEQ ID NO: 482) SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG SNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLGAAAGQPKAAPSVTLF PPSSEELQANKATLVCLISDFYPGAVTVAWKADSISPVKAGVE'I'ITPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6310 VL amino acid sequence (SEQ ID NO: 480) SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG SNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLG The CR6314 PIA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 483) encoded by the heavy Chain nucleotide ce shown in SEQ ID NO: 484 and the heavy chain amino acid ce 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 andlthe light chain amino acid sequence shown in SEQ ID NO: 488. “1..
CR6314 Heavy Chain nucleotide sequence (SEQ ID NO: 484) gaggtgcagc tggtggagtc tggggctgag gtgaagaagc cctc ggtgaaagtc 60 aagg cttctggagg ccccttccgc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt tccagggcag agtcacgatt gacg atttcgcggg ttac 240 atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg 300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt caccgtctcg 360 agca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc 420 ggcggcacag tggg 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 agac ccacacctgc cccccctgcc ccga gctgctgggc 720 .ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca ggga ggagcagtac 900 aacagcacct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960 aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc 1020 gcca agggccagcc ccgggagccc caggtgtaca ccctgccccc cagccgggag 1080 acca aggt gtccctcacc tgtctggtga agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag cccgagaaFa actacaagac caccccccct 1200 gtgctggaca gcgacggcag cttcttcctg tacagcaadb tcaccgtgga caagagccgg 1260 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcaqg agchctgca caaccactac 1320 acccagaaga gcctgagcct gagccccggc ¥ aag 1353 CR6314 Heavy Chain amino acid sequence (SEQ ID NO: 485) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAP KFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP 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 ggta ccagcagctc 120 ccaggcacgg cccccaaact cctcatctat agggatggtc cctc aggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctcbc tggccatcag tggactccgg 240 tccgatgatg aggctgatta ttactgtgca acatgggaté agaacctgag tggtccagta 300 ggag ggaccaagct gaccgtccta ggtgcggccé caéficcagcc caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc ctgc aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga gcgt ggagaccacc acccccagca agcagagcaa caacaagtac 540 agca 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 QANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE'I'ITPSKQSNNKYAASSYLSLT PGQWKSHRSYSCQVTHEGSTVEKTVAPTECSG CR6314 VL amino acid sequence (SEQ ID'NO: 486) SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRDGQRPSGVPDRFS GSKSGTSASLAISGLRSDDEADYYCATWDDNLSGEVFGGGTKLTVLG 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 ce shown in SEQ ID NO: 491. The CR6323 HA—specific IgG antibody also es 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) cagc tggtggagtc tgag gtgaagaagc cagggtcctc ggtgaaggtc 60 tcctgtaagg cctctggagg caccttctcc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagac atcatcggta tgtttggttc aacaaactac 180 gcacagaact gcag actcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaagtagt 300 ggttdccacc acct cccccactgg ggccagggca ccttggtcac cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac cagcggcgtg ttcc ccgccgtgct gcagagcagc 540 ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600 tgca acgtgaacca caagcccagc aacaccaagg tggacaaacg gccc '660 aagagctgcg acaagaccca cacctgcccc ccctgccdfig ccéccgagct gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc cacgaggécc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaecaagq cccgggagga gcagtacaac 900 agcacctacc gggtggtgag cgtgctcacc gtgctgcacc'aggactggct caag 960 gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc agaa gaccatcagc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctge ctggtgaagg gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact ccac cccccctgtg 1200 ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa cacc 1320 cagaagagcc tgagcctgag ccccggcaag 1350 CR6323 Heavy Chain amino acid sequence (SEQ ID NO: 491) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGDIIGMFGSTNYA QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSSAST PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK' .
CR6323 VH amino acid sequence (SEQ ID N09 489) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGlSWVRQAPGQGLEWMGDIIGMFGSTNYA QNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSSGYYPAYLPHWGQGTLVTVSS CR6323 Light Chain nucleotide sequence (SEQ ID NO: 493) gaaattgtgt tgacccagtc tccaggcacc ttgt ctccagggga aagagccacc 60 ctctcctgca gtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 gatt ttgcagtgta ttactgtcag cagtatggta gctcacccag aactttcggc 300 ggagggacca aggtggagat caaacgtgcg gccgcaccca gcgtgttcat cttccccccc 360 tccgacgagc agctgaagag cggcaccgcc agcgtggtgt'gcctgctgaa caacttctac 420 gagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggqc 600 ctgagcagcc ccgtgaccaa caac cggggcgagt gt 642 CR6323 Light Chain amino acid sequence (SEQ ID NO: 494) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKRAAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH VTHQGLSSPVTKSFNRGEC CR6323 VL amino acid sequence (SEQ ID N05 492) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS DFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR The CR6325 HA—specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 495) encoded by the heavy chain nucleotide ce shown in SEQ ID NO: 496 and the heavy chain amino acid sequence shown in SEQ ID NO: 497. The CR6325 PIA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 498) encoded by the light chain tide sequence shown in SEQ ID NO: 499 and the light chain amino acid sequence shown in SEQ ID NO: 500.
CR6325 Heavy Chain nucleotide ce (SEQ ID NO: 496) gaggtgcagc tggtggagtc tggggctgag gtgaagaagc cggggtcctc ggtgaaggtc 60 aagg gagg caccttcagc ttctattcta tgagctgggt ggcc 120 cctggacaag agtg gatgggaggg atcatcccta tgtttggtac aacaaactac 180 gcacagaagt gcag agtcacgatt accgcggtcg aatccacgag ctac 240 atggaggtga gcagcctgag atctgaggac acggccgttt attactgtgc gagaggtgat 300 aagggtatct actactacta catggacgtc tggggcaaag ggaccacggt caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc cgagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt cggc gtgcacacbt tccccgccgt gctgcagagc 540 agcggcctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600 atct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat gacc 780 cccgaggtga tggt 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 cacc 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) EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFG'I'I‘NYA QKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGDKGIYYYYMDVWGKGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK - - CR6325 VH amino acid sequence (SEQ IDINO: 495) EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGGIIPMFG'ITNYA 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 ctga ctgc agctcatata caagcagcag cactcttgtc 300 ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc caaggccgct 360 cccagcgtga tccc cccctcctcc gaggagctgc acaa ggccaccctg 420 ctca tcagcgactt ctaccctggc gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt ggagaccacc acccccagca gcaa gtac 540 gccgccagca gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600 tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc 660 .....
CR6325 Light Chain amino acid ce" (SEQ ID NO: 500) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQ FSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLGAAAGQPKAAPSVTLF PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETITPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6325 VL amino acid sequence (SEQ ID NO: 498) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQ 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 dy 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 gtgaagaaqc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cctctggagg caccttcagg acccatgcta tcagttgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcgcta'tcttcggaac agcaaactac 180 gcacagaagt tccagggcag aatcacgatt accgcggacg aatccacgag ctac 240 atggagctga gcagcctgag ggac gtgt atttctgtgc gagaggcagt 300 ggttatcata cacc 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 gaca ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt cgtg agccacgagg aggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct accgggtggt gagcgtgctc ctgc accaggactg gctgaacggc 960 aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc 1020 agcaaggcca agggccagcc gccc caggtgtaca ccctgccccc cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag cccgagaaca agac caccccccct 1200 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 cagg tgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320 acccagaaga gcctgagcct gagccccggc aag 1353 CR6327 Heavy Chain amino acid sequence (SEQ ID NO: 503) EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISLWV,RQAPGQGLEWMGGIIAIFGTANYA ITITQDESTSTAYMELSSLRSEDTAVYFQARGSGYHISTPFDNWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV WO 12489 NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNl-IYTQKSLSLSPGK CR6327 VH amino acid sequence (SEQEI'DNO: 501) EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGGIIAIFGTANYA QKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGSGYHISTPFDNWGQGTLVTVSS CR6327 Light Chain nucleotide sequence (SEQ ID NO: 505) tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60 acctgtgggg gaaacaacat taaa ggtgtgcact ggtaccagca gaagcctggc 120 caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg ggcc accctgacca gggt cgaagccggg 240 gatgaggccg actg tcaggtgtgg gatagtagta gtgatcatgt ggtattcggc 300 ggagggacca agctgaccgt cctaggtgcg gccgcaggcc agcccaaggc cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag gcca acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc tcac ccccgagcag tggaagagcc accggagcta ccag 600 gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654 CR6327 Light Chain amino acid sequence (SEQ ID NO: 506) SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGAAAGQPKAAPSVTL FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET'I‘TPSKQSNNKYAASSYLSL 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 tide 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 gatt 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 agca agagcaccag cggcggcaca gccgccctgg gctgcctggt gaaggactac 480 ttccccgagc ccgtgaccgt gagctggaac agcggcgcct gcgg cgtgcacacc 540 ttccccgccg agag cctg tacagcctga gcagcgtggt gaccgtgccc 600 agcagcagcc tgggcaccca gacctacatc tgcaacgtga accacaagcc cagcaacacc 660 aaggtggaca aacgcgtgga gcccaagagc aaga cccacacctg ccccccctgc 720 cccg agctgctggg cggaccctcc gtgttcctgt tcccccccaa gcccaaggac 780 accctcatga tcagccggac ccccgaggtg acctgcgtgg tggtggacgt gagccacgag 840 gaccccgagg tgaagttcaa ctggtacgtg gacggcgtgg acaa cgccaagacc 900 aagccccggg agta caacagcacc gtgg 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 gcga catcgccgtg gagtgggaga gcaacggcca gcccgagaac 1200 aactacaaga ccaccccccc tgtgctggac agcgacggca gcttcttcct caag 1260 ctcaccgtgg acaagagccg gtggcagcag gtgt tcagctgcag cgtgatgcac 1320 ctgc acaaccacta cacccagaag agcctgagcc tgagccccgg caag 1374 CR6328 Heavy Chain amino acid sequence (SEQ ID NO: 509) EVQLVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGGHPIFGTFNYAQ KFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVKDGYCTLTSCPVGWYFDLWGRGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTI‘PPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK CR6328 VH amino acid sequence (SEQ ID NO: 507) EVQLVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGG[IPlFGTTNYAQ KFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVKDGYCTLTSCPVGWYFDLWGRGTL VTVSS CR6328 Light Chain nucleotide sequence-(SEQ ID NO: 511) gaaattgtga tgacgcagtc tccaggcacc ctgtctttéf ctqcagggga cacc 60' ctctcgtgca gggccagtca gagtgttagc tadt ggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatcttt ggtgcctéca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcaétc tcaccatcag ggag 240 cctgaagatt tgta ttactgtcag cagtatggta gctcactcac tttcggcgga 300 gggaccaagc tggagatcaa acgtgcggcc gcacccagcg tgttcatctt ccccccctcc 360 gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttbtacccc 420 cgggaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480 agcgtgaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctcaccctg 540 agcaaggccg agaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600 agcagccccg agag cttcaaccgg ggcgagtgt 639 CR6328 Light Chain amino acid sequence (SEQ ID NO: 512) EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASSRATGIPDRFS GSGSGTDFI‘LTISRLEPEDFAVYYCQQYGSSLTFGGGTKLEIKRAAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC CR6328 VL amino acid sequence (SEQ ID NO: 510) EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASSRATGIPDRFS GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGGGTKLEIKR The CR6329 HA-specific IgG dy includes a heavy chain variable region (SEQ ID NO: 513) encoded by the heavy chain tide ce shown in SEQ ID NO: 514 and the heavy chain amino acid ce 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) cagc tggtacagtc tggggctgag gttaagaagc ctdggtcctc 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 ggac acggccgttt attactgtgc gagaggcagt 300 ggctacacca cacgcaacta ctttgactac tggggccagg gcaccctggt caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc ccagcagcaa gagcaccagc 420 acag ccgccctggg ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt gaccagcggc gtgcacacct ccgt gctgcagagc 540 agcggcctgt tgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600 acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ccga gctgctgggc 720 tccg tgttcctgtt cccccccaag cccaaggaca ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg agccacgagg aggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagtac 900 acct accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960 aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc 1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc ggag 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 ctac 1320 acccagaaga gcct gagccccggc aag 1353 CR6329 Heavy Chain amino acid sequence (SEQ ID NO: 515) EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGGIFALFGTTDYAQ KFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGSGYTTRNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP V17" SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK CR6329 VH amino acid sequence (SEQ. ID NO: 513) SGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGGIFALFGTTDYAQ TITADESSTTVYLELSSLTSEDTAVYYCARGSGY'I‘TRNYFDYWGQGTLVTVSS 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 ccca 180 gacaggttca gtggcggtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacccct cactttcggc 300 ggagggacca aggtggagat caaacgtgcg ggcc agéccaaggc cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag gcca ccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg accgtggect ggaaggccga cccc 480 gtgaaggccg gcgtggagac caccaccccc agcaagcagé acaa gtacgccgcc 540 agcagctacc tgagcctcac ccccgagcag tggaagagcd accggagcta cagctgccag 600 gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654 CR6329 Light Chain amino acid sequence (SEQ ID NO: 518) EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWYQQKPGQAPRLLIYGASSRASGIPDRFS GGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRAAAGQPKAAPSVTLFPPSS EELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE'I'I'I‘PSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS CR6329 VL amino acid sequence (SEQ ID NO: 516) ElVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWYQQKPGQAPRLLIYGASSRASGIPDRFS GGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR The CR6331 PIA—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 lgG antibody also includes a light chain variable region (SEQ ID NO: 522) encoded by the light chain nucleotide sequence shown in SEQ ID,.V.N“O: 523 and the light chain amino acid i (Li sequence shown in SEQ ID NO: 524. ~lO3 PCT/U82012/024971 CR6331 Heavy Chain nucleotide sequence (SEQ ID NO: 520) gaggtgcagc tggtggagtc tggggctgag aagc Ctgggtcctc ggtgaaggtc 6O tcctgcaagg cttctggagg caccttcagc agctathta tcagctgggt ggcc 120 cctggacaag ggcttgagtg gatgggaggg géta 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 ccéa gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac cagcggcgtg ttcc ccgccgtgct gcagagcagc 540 ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600 tacatctgca acgtgaacca caagcccagc aagg tggacaaacg cgtggagccc 660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga' 720 ccctccgtgt tcctgttccc ccccaagccc accc tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc tgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct caag 960 gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa cagc 1020 aaggccaagg cccg ggagccccag gtgtacaccc ccag ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgaCatc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg gctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 ggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagagcc tgagcctgag caag 1350 CR6331 Heavy Chain amino acid sequence (SEQ ID NO: 521) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISVVVRQAPGQGLEWMGGIIGMFGTANYA QKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGNYYYESSLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK CR6331 VH amino acid sequence (SEQ ID NO: 519) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIGMFGTANYA QKFQGRVTITADEFFSTAYMELSSLRSEDTAVYYCARGNYYYESSLDYWGQGTLVTVSS 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 gcga 180 ttctctggct ctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatta tgtcttcgga 300 actgggacca ccgt cctaggtgcg gccgcaggcc agcccaaggc cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg accgtggcct ggaaggccga cagcagcccc 480 gccg gcgtggagac caccaccccc agcaagcag§ gcaacaacaa gtacgccgcc 540 agcagctacc tgagcctcac gcag tggaagagéc accggagcta cagctgccag 600 gtgacccacg agggcagcac cgtggagaag accgtggbcc ccéccgagtg cagc 654 CR6331 Light Chain amino acid sequence (SEQ ID NO: 524) QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGAAAGQPKAAPSVTL FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE'I'I‘TPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS ' CR6331 VL amino acid sequence (SEQ ID NO: 522) QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG The CR6332 ecific IgG dy 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 SEQID‘NQ: 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 tgag gtgaagaagc ctgggtcctc ggtaaaggtc 60 aagg cttctggagg ccccttccgc aattttgcta tcaactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcgctg tctttgggac gacaaagtac 180 gcacataagt tccagggcag catc accgcggacg actccacaaa tacagcttac 240 ctgg 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 gacc 600 tgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc 660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt gagc cacgaggacc tgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga caac 900 agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960 gagtacaagt gcaaggtgag caacaaggcc ctgcctgcdc ccatcgagaa gaccatcagc 1020 aaggccaagg gccagccccg ggagccccag gtgtacacéc tgqcccccag ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg thiCtaccc catc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact ccac cccccctgtg 1200 ctggacagcg acggcagctt cttcctgtac agcaagctca acaa gagccggtgg 1260 ggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagagcc tgagcctgag caag 1350 CR6332 Heavy Chain amino acid sequence (SEQ ID NO: 527) QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKYA HKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGPHYYSSYMDVWGEGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE TQKSLSLSPGK CR6332 VH amino acid sequence (SEQ ID NO: 525) QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGGIIAVFGTTKYA VTlTADDSTNTAYMELGSLKSEDTAVYYCARGPHYYSSYMDVWGEG'I'TVTVSS CR6332 Light Chain nucleotide sequence (SEQ ID NO: 529) gacatccagt tgacccagtc tccatcctcc ctgtctgcat Ctgtaggaga cagagtcacc 60 atcacttgcc gtca gggcattagc acttatttag cctggtatca gcagaaaccc 120 gggaaagttc ctaaactcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180 cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag gcct 240 gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccttcttt tggg 300 accaaagtgg atatcaaacg tgcggccgca cccagcgtgt tcatcttccc cccctccgac 360 gagcagctga agagcggcac cgccagcgtg gtgtgcctgc tgaacaactt ctacccccgg 420 gaggccaagg ggaa ggtggacaac gccctgcaga gcggcaacag ccaggagagc 480 gtgaccgagc aggacagcaa ggactccacc tacagcctga gcagcaccct caccctgagc 540 gact agca caaggtgtac gcctgcgagg tgacccacca gggcctgagc 600 agccccgtga ccaagagctt caaccggggc gagtgt 636 CR6332 Light Chain amino acid sequence-(SEQ‘ID NO: 530) DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWXQQKEGKVPKLLIYAASTLQSGVPSRFSG SGSGTDFTLTlSSLQPEDVATYYCQKYNSAPSFGPGTKVDIKRAAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC CR6332 VL amino acid sequence (SEQ ID NO: 528) DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSG SGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKR The CR6334 PIA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 531) encoded by the heavy chain nucleotide ce 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.
PCT/U82012/024971 CR6334 Heavy Chain nucleotide sequence (SEQ ID NO: 532) gaggtgcagc tggtggagac tggggctgag gtgaagaagc cctc ggtgaaggtc 60 ccctgcaaat cttctggaag ccccttcagg agtaatgctg tcagctgggt gcgacaggcc 120 cccggacaag ggcttgagtg ggtgggagga atcctcggtg tctttggttc ctac 180 gcacagaagt tccagggcag gatt accgcggacg aatccaccaa cacagtccac 240 ctga 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 tgac cgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600 tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc 660 aagagctgcg acaagaccca cacctgcccc cctg cccccgagct gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa ctgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga caac 900 agcacctacc gggtggtgag cgtgctcacc gtgctgcacc ggct gaacggcaag 960 'gagtacaagt tgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt cttcctgtac agcaagctéa ccgtggacaa gagccggtgg 1260 cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagagcc tgagcctgag ccccggcaag '- 1350 CR6334 Heavy Chain amino acid sequencé (SEQ ID NO: 533) EVQLVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGGILGVFGSPSYA QKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKG'I'I‘VTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYK'ITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK CR6334 VH amino acid sequence (SEQ ID NO: 531) EVQLVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGGILGVFGSPSYA QKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGPTYYYSYMDVWGKGTTVTVSS CR6334 Light Chain nucleotide sequence (SEQ ID NO: 535) tcctatgtgc tgactcagcc ggag tcagtggccc caggacagac ggccaggatt 6O gggg gaaataacat tggaagaaat agtgtgcact ggtatcagca gaagccaggc 120 caggcccctg tgctggtcgt gtatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttttctggct ccaagtctgg gaacacggcc accctgatta tcégcagggt cggg 240 gatgaggccg actactactg tcaggtgtgg catagtagta gtgatcatta tgtcttcgga 300 actgggacca aggtcaccgt cctaggtgcg gch agéccaaggc cgctcccagc 360 ctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac cctggtgtgc 420 ctcatcagcg accc 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 2012/024971 CR6334 Light Chain amino acid sequence (SEQ ID NO: 536) SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG SKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLGAAAGQPKAAPSVTLFP PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET’ITPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6334 VL amino acid ce (SEQ ID NO: 534) SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG SKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLG The CR6336 HA-specific lgG antibody es y 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 PIA-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 agtg gatgggaggg ggta tgtttgggac agcaaactac 180 gcgcagaagt tccagggcag agtcacgatt accgcggacg cgag ctac 240 atggagctga gcagcctggg atctgaggac acggccatgt attactgtgc gaggtctagt 300 ggttattacc cccaatactt ccaggactgg ggccagggca ccctggtcac cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ccca gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgt gagc 480 tqgaacagcg gcgccttgac cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600 tacatctgca acgtgaacca caagcccagc aacaceaagg tggacaaacg cgtggagccc 660 aagagctgcg acaagaccca cacctgcccc ccctgccceg cccccgagct gctgggcgga 720 ccctccgtgt tccc ccccaagccq aaggacanc tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc gaoc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaage ccégggagga gcagtacaac 900 agcacctacc gggtggtgag cgtgctcacc gtgctgciCc aggactggct gaacggcaag 960 gagtacaagt gcaaggtgag caacaaggcc gccc ccatcgagaa cagc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccd tgccccccag ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagd accc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200 agcg 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 STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVI-ITFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK CR6336 VH amino acid sequence (SEQ ID NO: 537) SGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIFGMFGTANY AQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSSGYYPQYFQDWGQGTLVTVSS CR6336 Light Chain nucleotide sequence (SEQ ID NO: 541) gtga tgacacagtc tccaggcacc ttgt ctccagggca aagagccacc 60 ctctcctgca gtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccagact cctcatgtat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtgg gtctgggaca gacttcactc tcaccatcag ggag 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 agga cagcaaggac tccacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagcttcaac cggggcgégt gt. 642 CR6336 Light Chain amino acid sequence (SEQ ID NO: 542) EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYGASSRATGIPDRF SGSGSGTDFTLTlSRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKRAAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC CR6336 VL amino acid sequence (SEQ ID NO: 540) EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYGASSRATGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKR The CR6339 cific 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 ce 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.
PCT/U82012/024971 CR6339 Heavy Chain nucleotide sequence (SEQ ID NO: 545) gaggtgcagc tggtggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 6O tcctgcaagg cttctggagg catcttcaac agttatgcta tcagctgggt gcgacaggcc 120 caag ggcttgagtg gatgggaggc'atcatcgcta atac accaaagtac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgaa cacagcctac 240 ctga gaagcctgaa atctgaggac acggccctgt attactgtgc gagagggtcc 300 acttacgatt tttcgagtgg ccttgactac tggggccagg gaaccctggt caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggqcc ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ggtg tact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 ctgt acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600 acctacatct tgaa ccacaagccc agcaacacca acaa acgcgtggag 660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag gaca ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga gtac 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 gtga agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg tgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320 acccagaaga gcctgagcct cggc aag 1353 CR6339 Heavy Chain amino acid sequence (SEQ ID NO: 546) EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGGIIAIFHTPKYAQ KFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGSTYDFSSGLDYWGQGTLVTVSSAST PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK L 1 CR6339 VH amino acid sequence (SEQ-ID NO: 543) EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYA!SWVRQAPGQGLEWMGGIIAIFHTPKYAQ KFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGSTYDFSSGLDYWGQGTLVTVSS CR6339 Light Chain nucleotide sequence (SEQ ID NO: 548) caggcagggc tgactcagcc accctcggtg gccc caggacagac gatt 60 acctgtgggg gaaacaacat taaa 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 aggc cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca acaaggccac gtgc 420 ctcatcagcg acttctaccc tggcgccgtg gcct 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 FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETI‘TPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6339 VL amino acid ce (SEQ ID NO: 547) QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPCQAPVLVVYDDSDRPSGIPERFS 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—speciflc IgG antibody also includes a light chain variable region (SEQ ID NO: 553) d by the light chain tide sequence shown in SEQ ID NO: 554 and the light chain amino acid ce shown in SEQ ID NO: 555.
CR6342 Heavy Chain tide sequence (SEQ ID NO: 551) caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg cagc agctatgcta tcagctgggt gcgccaggcc 120 caag gacttgagtg gatggggggg gtcatcccta tctttcgtac agcaaactac 180 gcacagaact tccagggcag agtcaccatt gacg aattcacatc gtatatggag 240 ctgagcagcc tgagatctga cgacacggcc gtgtattact gtgcgaggtt gaattaccat 300 gattcgggga cttattataa cgccccccgg ggctggttcg acccctgggg ccagggaacc 360 ctggtcaccg tctcgagtgc tagcaccaag ggccccagcg tgttccccct ggccbccagc 420 agcaagagca ccagcggcgg cacagccgcc ctgggctdgp tggtgaagga cccc 480 gagcccgtga ccgtgagctg cggc gccttgacca gcggcgtgca caccttcccc 540 gccgtgctgc agagcagcgg cagc ctgagcagcg tgdtgaccgt 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 gtggtggtég acgtgagcca cgaggacccc 840 gaggtgaagt tcaactggta cgtggacggc gtgc acaacgccaa gaccaagccc 900 cgggaggagc agtacaacag cacctaccgg gtggtgagcg ccgt gctgcaccag 960 gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct gcctgccccc 1020 atcgagaaga ccatcagcaa ggccaagggc cagccccggg agccccaggt gtacaccctg 1080 ccccccagcc gggaggagat gaccaagaac tccc tcacctgtct ggtgaagggc 1140 ttctacccca gcgacatcgc gtgg gagagcaacg gccagcccga ctac 1200 aagaccaccc cccctgtgct ggacagcgac ggcagcttct acag 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) QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAlSWVRQAPGQGLEWMGGVIPIFRTANYA QNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYHDSGTYYNAPRGWFDPWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS ‘T a”. g. ;av VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK'ITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS 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 cacc 60 atcaactgca agtccagcca gagtatttta tcca 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 ccga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 420 ctgaacaact cccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480 aaca gccaggagag cgtgaccgag agca aggactccac ctacagcctg - 540 agcagcaccc tcaccctgag caaggccgac aagc acaaggtgta cgcctgcgag 600 gtgacccacc agggcctgag cagccccgtg accaagagct gggg cgagtgt 657 CR6342 Light Chain amino acid sequence (SEQ ID NO: 555) DlQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPPKLLIYWASTRESG VPDRFSGSGSGTDFFLTISSLQAEDVAVYYCQQYYSSPPTFGQGTKVEIKRAAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC CR6342 VL amino acid sequence (SEQ ID NO: 553) DlQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPPKLLIYWASTRESG GSGSGTDFPLTISSLQAEDVAVYYCQQYYSSPPTFGQGTKVEIKR The CR6343 HA—specific IgG antibody es 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 SEQJD 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‘I‘NV‘O: 56D and the light chain amino acid V. , sequence shown in SEQ ID NO: 561.
PCT/U82012/024971 CR6343 Heavy Chain nucleotide sequence (SEQ ID NO: 557) caggtccagc tggtgcagtc tggagctgag gtgaagaagc ctgggtcctc ggtgaaggtc 6O tcctgcaagg cttctggagt caccttcagt tactatgcta tgagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagga atcagcccta tgtttgggac aacaacctac 180 gcacagaagt tccagggcag gatt 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 ttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac cagcggcgtg ttcc ccgccgtgct gcagagcagc -54O ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600 tgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc 660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tcatgatcag ccggaccccc 780 gaggtgacct tggt ggacgtgagc cacgaggacc tgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960 aagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag ccgggaggag 1080 atgaccaaga tgtc cctcacctgt ctggtgaagg gcttctaccc cagcgacatc 1140 gagt gcaa cggccagccc gagaacaact adaagaccac cccccctgtg 1200 ctggacagcg acggcagctt cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca acgtgttcag ctgcagcgtg gagg ccctgcacaa ccactacacc 1320 agcc tgagdctgag caag 1350 CR6343 Heavy Chain amino acid sequence (SEQ ID NO: 558) QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGGISPMFGTTTY AQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK CR6343 VH amino acid sequence (SEQ ID NO: 556) QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGGISPMFGI I IY AQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSSNYYDSVYDYWGQGTLVTVSS CR6343 Light Chain nucleotide sequence (SEQ ID NO: 560) cagtctgtcg tgacgcagcc gccctcggag tcagtggccc caggacagac ggccaggatt 60 acctgtgggg gacataacat tggaagtaat agtgtgcafit agca gaagccaggc 120 caggcccctg tgctggtcgt taat agcgaccqéc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cggg 240 gatgaggccg actattactg tcaggtgtgg ggtagtdéta gtgaccatta tgtcttcgga 300 actgggacca aggtcaccgt cctaggtgcg gccgcaégcc agcccaaggc cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag gcca acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc cgtg accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600 gtgacccacg agggcagcac gaag accgtggccc ccaccgagtg cagc 654 CR6343 Light Chain amino acid sequence (SEQ ID NO: 561) QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDNSDRPSGIPERFSG SNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLGAAAGQPKAAPSVTLF PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET’I‘TPSKQSNNKYAASSYLSL TPEQWKSIlRSYSCQVFllEGSTVEKTVAPFECS CR6343 VL amino acid sequence (SEQ ID NO: 559) QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDNSDRPSGIPERFSG SNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLG The CR6344 PIA-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 PIA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 565) d 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 tide sequence ‘(SEQ ID NO: 563) caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgagagtc 60 tcctgcaagg cttctggaag catcttcaga aactatgcta tgagctgggt gcgacaggcc 120 cctggacaag agtg gatgggaggg atcatcgcta tttttgggac accaaagtac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatcgacgag cactgtctac 240 atggaactga gcggactgag atctgaggac acggccatgt attactgtgc gaggattccc 300 Cactataatt ttqgtthgg gagttatttc gactactggg gccagggaac cctggtcacc 360 gtctcgagtg ctagcaccaa gggccccagc cccc tggcccccag cagcaagagc 420 ggcg gcacagccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg 480 accgtgagct ggaacagcgg cgccttgacc agcggcgtgc acaccttccc cgccgtgctg 540 agcg acag cctgagcagc gtggtgaccg gcag cagcctgggc 600 acct acatctgcaa cgtgaaccac aagcccagca acaccaaggt acgc 660 gtggagccca agagctgcga caagacccac acctgccccc cctgccctgc ccccgagctg 720 ctgggcggac cctccgtgtt cctgttcccc ccca aggacaccct catgatcagc 780 cggacccccg aggtgacctg cgtggtggtg gacgtgagcc acgaggaccc cgaggtgaag 840 ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc ccgggaggag 900 cagtacaaca gcacctaccg ggtggtgagc gtgctcaccg acca ggactggctg 960 aacggcaagg agtg caaggtgagc aacaaggccc cccc 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 ttcctgtacg gcaagctcac cgtggacaag 1260 agccggtggc agcagggcaa cagc tgcagcgtga tgcacgaggc cctgcacaac 1320 cactacaccc agaagagcct gagc cccggcaag‘ . 1359 CR6344 Heavy Chain amino acid sequence4(SEQ ID NO: 564) QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGGIIAIFGTPKYA QKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTI‘PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM 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 gggt caccatctcc 6O tgcactggga gcagctccaa catcggggca ggttatgatg tacactggta ccagcagctt 120 ccaggaacag cccccaaact cctcatctat ggtaacagea cctc aggggtccct 180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240 gacg aggccgatta ttactgcgga acatgggata gcagcctgag tgcttatgtc 300 actg ggaccaaggt caccgtccta ggtgcggccg agcc 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) TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDV SGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLGAAAGQPKAAPSVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE'I'TTPSKQSNNKYAASSYLS LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CR6344 VL amino acid ce (SEQ ID NO: 565) TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDV TSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLG HA antibody epitopes The invention relates to an isolated human HA antibody that is able to recognize and bind to an e in the HA2 subunit of the influenza haemagglutinin protein (HA) (also known as hemagglutinin(HA)), characterized in thatthe HA dy has neutralizing activity against an za virus 5 including of~ the H5 subtype. Examplesof influenza strains that contain such a HA of the H5 subtype andéthat are important strains in view of pandemic s are H5N1, H5N2, H5N8, and H5N9. Particularly preferred are HA antibodies that at least neutralize the HSNI influenza strain. Preferably, an HA antibody of the invention does not depend on an epitope in the HA1 subunit of the HA protein for g to said HA protein.
A number of the antibodies of the invention (such as CR6307 and CR6323) do not depend on conformational epitopes and ize the HA2 epitope even in a reduced form (when used in westem-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 on 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 ably wherein the HA antibody also has neutralizing activity against za 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 epitopeslpresent in the H5, H1, H2, H6, and H9 subtypes (see, International Patent Application PCTIEP2007/059356, published as WO 28946, 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 ion, 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 ce the binding of C I 79. Moreover, some HA antibodies, such as CR6307 and CR6323 are not even hampered by a escape mutant, as disclosed in Okuno et al. (1.993) with a valine -> glutamic acid mutation at position 6 (exemplified by GVTNKEHSIIDH'.(SEQ ID NO: 202)). This epitope is part of an extended alpha helix in the HA2 regignhkThe 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 e. Consistent with this notion the highlighted d) amino acids are absolutely conserved in identity and on in all the ces presented. This knowledge could be used to predict binding es in influenza es that do not carry the same sequence as above (i.e. H3, H7 and B strains).
HA Antibodies II ' '3“ The invention provides neutralizing human monoclonal antibodies that bind influenza A virus and inhibit the za A virus from ing a cell. Although neutralizing human monoclonal dies 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) n. 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 za hemagglutinin (HA) n is responsible for ng the virus to recognize target cells through g the ccharide 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 mal membrane with the viral membrane.
The Influenza hemagglutinin (HA) protein is'a hornotrim'eric 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 (HAO) in the endoplasmic reticulum, where it is also led 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 ed to the viral membrane whereas the HA1 subunit tops the HA2 subunit to form a large globule. The cleavage step, which converts the HAO precursor into the mature HA protein containing HA1 and HA2 ts, is essential for the viral pathogenicity of Influenza. urally, the mature HA protein contains a central ix 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, (x2-3 linkages as in avian serotypes versus 0t2-6 linléages as in human serotypes, not only determine species specificity of an Influenza virus,\but also ts cross-species infection.
However, within certain serotypes of HA, such as H1 andiH3, 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 ors present on the surface of the target cell. Consequently, the target cell ne endocytoses or engulfs the Influenza virus. Once inside theendosome, and upon the host cell’s acidification of that tment, the za HA4: 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: Hl-H16. Only HA serotypes Hl-H3 ly mediate human Influenza infection. r, Influenza s thought to infect only certain avian or mammalian species can mutate to infect . As described above, only a few amino acids need to change along the length of the entire protein to enable Influenza to cross a s 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 (HSNI). A pandemic arose when an Influenza strain common to swine species, became lethal to humans (HlNl). In contrast to Influenza A, Influenza B and C viruses each contaigfi‘only one form of HA protein.
Specifically, the invention provides an isolaied fully human monoclonal antibody, wherein said monoclonal antibody has the followingcharacteristics: a) binds to an influenza A virus; b) binds to a cell ted with influenza A, c) binds to an epitope of an influenza A viral n; 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 s, this antibody binds to a eukaryotic cell. Moreover, the cell is ally a human cell.
In another aspect, this antibody is ed from a B—cell from a human donor.
Isolation of a fully human monoclonal antibody of the ion 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 nza A viral hemagglutinin (HA) protein. Exemplary HA epitopes to which the antibodies of the invention bind include a hemagglutinin precursor peptide (HAO), a HA1 subunit, 3 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 hemalgglutinin precursor peptide (HAO), a HA1 subunit, a HA2 subunit, a mature n containing HA1 and HA2, or a recombinant HA polypeptide. Recombinant HA polypeptides are encoded;_f0r example, by the sequence of SEQ ID NO: 727, 728, 729, 730, 731, 732, 733, 734, 735736, 737, 738, 739, 740, 741, 742, 743, or 744.
Antibodies of the invention bind to an epitope that is linear or near. In certain aspects of the invention, a non-linear epitope is a discontinuous epitope.
An antibody of the invention is TCN—522 (3212_112), TCN-521 (3280_D18), TCN- 523 (5248_A17), TCN-563 (5237_B21), TCN-526 (5084_Cl7), TCN—527 (5086_C06), 8 (5087_P17), TCN-529 (5297_H01), TCN-53O (5248_H10a), TCN-531 (5091_Hl 3). TCN-532 (5262_H18), TCN-533 (5256_Al7), TCN-534 (5249_B02), TCN-535 (5246_Pl9), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538 (3206_Ol7), TCN—539 (5056_A08), TCN-S40 (5060_F05), TCN-541 _M11), TCN-542 (5079_A16), TCN- 543 (5081_023), 4 (5082_A19), TCN-545 (5082_115), TCN-546 L08), TCN-r 547 (5092_Fl 1), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN—550 (5096_FO6), TCN— 551 (5243_D01), TCN-552 (5249_123), TCN-553 (5261_C18), TCN-554 (5277_M05), S (5246_L16), TCN-556 (5089_K12), TCN—557 (5081_A04), TCN-558 H10b), TCN-559 (5097_GOS), TCN-560 (56§4_P10), or TCN-504 K17).
The invention further encompasses an antibody that binds the same epitope as TCN- 522 (3212_112), 1 (3280_D18), TCN-523 5(5248_A17), 3 (5237_BZI), TCN- 526 (5084_Cl7), TCN—527 (5086_C06), TCN-528 Pl7), TCN-529 (5297_H0|), TCN- 530 (5248_H10a), TCN-531 (509l_Hl3), TCN-532 (5262_Hl8), TCN-533 (5256_Al7), TCN—534 (5249_B02), TCN-535 (5246_Pl9), TCN-536 (5095_N01), TCN—537 (3194_D21), 8 (32061017), TCN-539 (5056_A08), 0 (5060_F05), TCN—541 (5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN—544 (5082_A19), TCN- 545 (5082_115), TCN-546 (5089_LO8), TCN-547 (5092_F1 l), TCN—S48 (5092_P01), TCN- 549 (5092_P04),TCN-550(5096_FO6),TCN-551 (5243_D01), TCN-552 (5249_123), TCN- 553 (5261_C18), TCN-554 (5277_M05), 5 (5246_L16), 6 (5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), 9 (5097_GOS), TCN—560 (5084_P10), or 4 K]7).
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 CDRl and CDR2, wherein the YH' region is encoded by a human IGHVl (or specifically, IGHVl-IS, IGHV1—2, IGHV1-69, IGHVl-S), IGHV2 (or specifically, IGHVZ- ) , IGHV3 (or specifically, IGHV3-30, IGHV3—33, IGHV3-49, IGHV3-53, 66, IGHV3-7), IGHV4 (or specifically, IGHV4-3l, IGHV4-34, IGHV4-39, IGHV4—S9, IGHV4-61), or IGHVS (or specifically, IGHVS-Sl) VH ne sequence or an allele thereof, or a nucleic acid sequence that is homologous to the IGHVI, IQHVZ, IGHV3, IGHV4, or IGHVS VH germline gene sequence or an allele thereof. In one: aspect, the nucleic acid sequence that is gous to the IGHVI, IGHV2, IGHV3, IGHVr4, or ICiHVS VH germline sequence is at least 75% homologous to the lGHVl, IGHV2,AIGl:lV3, lGHV4, or IGHVS VH germline sequence or an allele f. Exemplary alleles include, but are not limited to, IGHVl- 18*01, IGHV1-2*02, 2*04, IGHVl-69*Ol, 69*05, lGHVl-69*O6, IGHVl- 69* 12, IGHVl-8*Ol, IGHV2-5*10, IGHV33*Ol, 30*03, IGHV3—30*l8, IGHV3-33*05, IGHV3-49*04, IGHV3-53*01, IGHV3—66*03, IGHV3—7*Ol, IGHV4-31*03, IGHV4-31*06, 34*01, IGHV4—34*02, lGHV4—34*03, IGHV4-34*12, IGHV4— 39*Ol, IGHV4-59*Ol, 59*03, IGHV4-6l *Ol, 6l *08, and IGHV5-51*Ol.
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 es 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 tor. 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. Altematively, 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 ion provides a method for stimulating an immune response in a subject, including administering to the subject the pharmaceutical composition described herein. .45: i" > Moreover, the invention provides a method for the ent 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 ion also provides a method for the prevention of an Influenza virus infection in a subject, ing 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 ation.
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 suppressive y (for example, following organ transplant), those persons afflicted with human immunodeficiency syndrome (HIV) or ed immune deficiency syndrome A l ‘ (AIDS), certain forms of anemia that deplete or destroy lh'ite blood cells, those persons ing radiation or chemotherapy, or those persons ed with an atory 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 t 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 tor, a HA inhibitor, a sialic acid inhibitor and an M2 ion l. In one aspect of these methods, the M2 ion channel tor is amantadine or rimantadine. Alternatively, or in addition, the neuraminidase inhibitor is zanamivir or oseltamivir phosphate. ’5‘ These methods optionally include administeii‘inggai second anti-Influenza A antibody.
For example, the antibody is administered prior tdor after exposure to za virus. In certain aspects of these methods, the antibody is stered 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 ce of a Influenza virus infection in a subject, including the steps of: (in) contacting a biological sample obtained from the t 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 r.
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 HAO ns or fragments thereof derived from multiple strains of nza A (see, Table 9), or to several different epitopes of a molecule, e. g. a linear and a tinuous epitope of the same recombinant imeric HAO protein or fragment thereof derived from a single strain of Influenza A. Alternatively, or in addition, the term multivalent vaccine is meant fol describe the administration of a combination of human antibodies raised against more than one infectious agent, e.g. a combination of HuMHA antibodies raised againsfnreCombinant homotrimeric HAO proteins or fragments f derived from multiple s ofInfluenza A (see, Table 9).
The ion es a diagnostic kit including an antibody described .
The invention provides a prophylactic kit including an antibody bed herein or an epitope of an antibody described herein. Alternatively, or in addition, the invention provides a prophylactic kit including a vaccine composition bed herein.
In a preferred embodiment, the present invention provides fully human monoclonal dies specifically directed against the Influenza hemagglutinin glycoprotein, which lize 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 dy 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 imeric Influenza HAO protein or HA protein(s) isolated from 122 1 __., .44! multiple za 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 onal antibodies of the invention are anti-HA“, antibodies. .A monoclonal A antibody of the invention has one or more of the following .characteristics: a) binds to an epitope in an HA1 t 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 e of a inant homotrimeric Influenza HAO protein; 6) 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 ormed with a polynucleotide that encodes an Influenza HA n, or any nt thereof. The term “Influenza HA protein fragment” is meant to describe any portion of the n 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. Cellsjare iiifected with active or inactive virions. Exemplary ve 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. ically, 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 za hemagglutinin (HA) protein. HA proteins of the invention include hemagglutinin sor proteins (HAO), the HA1 subunit, the HA2 subunit, the mature n ning the HA1 and HA2 subunits, and a recombinant HA protein. Recombinant HA proteins n 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 ion are linear or non- linear. For instance, a non—linear epitope is discontinuous. Discontinuous epitopes are available for antibody binding only when the za 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, Le. the Influenza HA protein, upon which juxtaposed amino acids are alternatively exposed or masked. inant homotrimeric HAO proteins of the invention are encoded by, for instance, sequences bed 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 0r soluble inant homotrimeric Influenza HA proteins. Alternatively, the monoclonal anti-HA antibodies described herein bind membrane- bound and soluble recombinant imeric Influenza HA proteins. In certain ments of the ion, the monoclonal A antibodies described herein bind and neutralize Influenza virus subtypes H1, H2, and H3. In other embodiments of the invention, the monoclonal A 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 dies bind Influenza subtypes HlNl, H2N2, and H3N2, and neutralize HlNl.
In one aspect, the HA precursor polypeptide (HAO) of the soluble and recombinant homotrimeric Influenza HA protein contains a trimerization domain (foldon) encoded in the phage T4 fibritin. An exemplary trimerization domain ed 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: SGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFL_HH HHH (SEQ ID NO: 726). - @- As used herein, the term “neutralizing antibody” is meant to describe an antibody that inhibits or prevents influenza A infection, inhibits orj'prevents Influenza A viral entry into a cell, inhibits or prevents influenza ation, 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 ment, neutralizing antibodies of the invention prevent viral entry into the cytoplasmic compartment of host cells.
The present invention provides fully humahumono'clonal antibodies that bind influenza virus and neutralize ion. 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 glycdprotein found on the surface of the Influenza virus. To mimic the native conformation ' of this imeric protein, the methods of the invention provide an isolated HA protein precursor that is ly-linked to a trimerization or foldon domain from the phage T4 fibritin protein (SGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFL_HH HHH (SEQ ID NO: 726)).
The resultant recombinant homotrimeric foldon HA protein not only retains the native Influenza hemagglutinin homotrimeric mation, but also becomes soluble, i.e. the protein is no longer bound to a viral or cellular membrane, ically, these recombinant HA homotrimeric proteins lack an integral membrane or fransmembrane 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, , 100-150, 0, 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 n between 565-575 amino acids (aa). Recombinant HA homotrimeric proteins further include a signal cleavage site at the inus containing between 15-25 aa. Alternatively, or in addition, recombinant HA homotrimeric ns further include a transmembrane domain positioned n amino acids 525-535 of HA depending on the influenza A virus subtype. In a preferred embodiment, the HA n 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 théi‘iiiven‘tion include those signal sequences known in the art that allow ent secretion of profeinis, isuch as the signal ce of the immunoglobulin light kappa chain. Alternatively, recombinant HA homotrimeric ns, or the HAO precursors thereof, may have the native signal ces in the expression constructs used by Immune Technology Corp. //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 aisf'ubstituted HA1/HA2 protease cleavage site. For example, the recombinant HA protein d 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 ted to recombinant and e HA homotrimers of the invention in vitro, wherein the recombinant and soluble HA imers 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. yp‘eptide specifically expressed on the surface of cells infected with the infectious agentmbut infected cells. In particular embodiments, the serum or plasma samples are scieened by contacting the samples with a cell that has been transfected with an sion vectbr that expresses the polypeptide expressed on the surface of infected cells. In ular embodiments the serum or plasma samples are screened by ting the s with a recombinant protein which ents 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 ed 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 fectious agent such as, e.g. intact whole virions of the influenza A virus to determine the ce 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 ptide or virus of interest, mononuclear and/or B cells obtained from the same subject or patient are used to fy a cell or clone thereof that es 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 ted from} particular donor, Le. 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 on (RT-PCR) is applied to the B-cell lysatc to y the antibody variable regions and subsequently clone, express, and test for binding and function of the recombinant dy, Human dies, such as the MAbs listed in Table 10, which bind the recombinant and soluble HA homotrimer and/or bind whole s, and optionally inhibit or neutralize infection of live virus are recombinantly reproduced and formulated into a ceutical composition for administration to a subject at risk of contacting an Influenza virus.
Furthermore, recombinant and soluble HA homotrirriers are derived from multiple strains of Influenza viruses, including multiple strains of influenza A virus. Exemplary human antibodies specifically bind InfluenZa A, and mayi'fbe ed for an ity 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 dies 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. atively, or in addition, huMHA antibodies bind native HA, inant homotrimeric HA, purified virus, infected cells, linear e, synthetic HA 1 ~ 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 t novel specificity for a very broad range of Influenza A virus strains. These antibodies may be used prophylactically to t Influenza A ion, 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 t influenza A infection.
The huMHA antibodies of the invention has one or more of the following characteristics: a) binds to an e in an HA1 subunit of an Influenza lutinin (HA) protein; b) binds to an epitope in the HA2 t of Influenza hemagglutinin (HA) protein; c) binds to an epitope in the extracellular domain ofan Influenza hemagglutinin (HA) protein, consisting of an HAl subunit and an HA2 subunit; d) binds to an epitope of a recombinant homotrimeric Influenza HAOI proteinire) 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 tible eukaryotic cells. The huMHA antibodies of the invention eliminate Influenza infected cells through immune or mechanisms such as ADCC and/or CDC and promote direct viral clearance by binding to Influenza virions.
Exemplary Influenza A s used for screening human plasma samples, B Cell Culture supematants (BCC SN), and monoclonal transfection supematants (MN are shown in Table 8 below). Live s 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 ic HA binding assay. 2012/024971 Table 8 —_——— “——— _—-:I-—— _———— _———— —--n-—— _———_ —_——— —__—— _———— —_——— ————— _———— _-n-——— ————— __'-m-—— _“——— _———— Exemplary HA sequences include those sequences listed on Table 9 below.
Table 9 Type Gun Bank Subtype HA chucncc from Strain SEQ ID NO: Access ion No. alifomiaz‘Wlmll INI) SEQ ID NO: 727 —__— -__AmenhCarolina/m8(MINI) SEQ ID NO: 729 AA A43 185 305710577112m) SEQ ID NO: 730 AfWisconsin/(SWZOOSUHNZ) SEQ ID NO: 731 AiSwincl'Onlario/OlW 1-2/99‘UI4N6) SEQ ID NO: 732 Az’llong Kong/l 56:97 (“5N l) SEQ ID NO: 733 __sm.m.734 -——_smm~<:735 AlEgypt/BROO—NAMRIB/20080155!l) SEQ ID NO: 735 A/common magpicmong Kong/5052200701 5N!) SEQ [0 N0: 737 —_smm~o:238 Aiehicken/ViemarflNCND-Ol6120081“5N1) SEQ ID NO: 739 Ar’nonhcrn shovelcr/Calitbm iamxwm 15/2007mcnn -—mAr’duclo’\’angzhmi/O?JB(KJS(H8334) ongKong/l073/99(Hum) . SEQ IDNO: 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 ance with SEQ ID NO: 727-744.
Alternatively, the monoclonal dy is an antibody that binds to the same epitope as the mAbs listed in Table 10. .i ..
Table 10 BCCwelllD TheraclonelD BCCweIHD TheraclonelD BCCwelllD TheraclonelD BCCweIIID ‘IheraclonelD 3251-107 3280-018 3212_I12 5248_A17 5237-321 5086_c06 5087-217 5297_H01 szauma The antibodies of the invention are able to neutralize nza 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. r 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 alizing 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 ing to the invention is a novel monoclonal antibody referred to herein as TCN-522 (corresponding to BCC plate and well location 3212_Il2), TCN-521 D18), TCN-523 (5248_Al7), TCN-563 (5237_B21), TCN—526 (5084’C17), TCN-527 (5086_CO6), TCN-528 (5087_Pl7), TCN—529 (5297_HOll, TCN-53O (5248_H10a), TCN—531 H13), TCN-532 (5262_H18), TCN—533 Al7), TCN—534 (5249_BOZ), TCN-535 Pl9), TCN-536 (5095_N01), TON-537 (3194_D2‘l), TCN—538 (3206_Ol7), TCN-539 (5056_A08), 0 (5060_F05), TCN-541 (5062_Ml 1), TCN—542 (5079_A16), TCN— 543 (5081_GZ3), TCN-544 (5082_Al9), TCN-545 (5082_115), TCN-546 (5089_L08), TCN- 547 (5092_Fl 1), TCN-548 (5092_P01), TCN-549 (5092_PO4), TCN-550 (5096_F06), TCN— 551 (5243fD01), TCN—552 (5249_I23), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_Ll6), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN—558 (5248_H10b), TCN-559 G08), TCN-560 (5084_P10), and TCN-504 (3251_Kl7).
These antibodies were lly ed from human s and are produced by the B cell cultures referred to as 3212_112, 3280_D18, 5248_A17, 5237_B21, 5084_C17, 5086_C06, 5087_Pl7, 5297_H01, 5248_H10a, 509l_Hl3,‘5262_H18, l7, 5249_B02, 5246_P19, 5095_N01, 3194_D21, 3206_Ol7, 5056_A08, 05, 5062__Ml l, 5079fiA16, 5081_GZ3, 5082_Al9, 5082_115, 5089_L08, 5092_Fl l, 5092_P01, 5092_P04, 5096-1306, 5243_D01, 5249_123, 5261_Cl8, 5277_M05, 'l6, 12,‘ 5081_AO4, 5248_H10b, 5097_GOS, 5084_P10, and 325 l_Kl 7. 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 CDRH l, CDRHZ and CDRH3, respectively. Similarly, the CDRs of the antibody light chains are referred to as CDRLl, CDRL2 and CDRL3, tively. The on 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 ons 105 to 117. (Lefranc, M P. et al. 2003 IMGT unique numbering for immunoglobulin and T cell receptor variable regions and Ig amily V-like domains. Dev Comp Immunol. 27(1):55-77; Lefranc, M P. 1997. Unique database numbering system for genetic analysis. logy Today, 18509; Lefranc, M P. 1999. The IMGT unique numbering for Immunoglobulins, T cell receptors and e domains. The Immunologist, 7:132-136.) The sequences of the antibodies were determined, ing 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_112), 1 (3280_D] 8), 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—53O (5248_H10a), TCN- 531 (5091_Hl3), TCN-532 (5262_H18), TCN—533 (52564A17), TCN—534 (5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), 7 ’(3194_D21), TCN-538 (3206_Ol7), TCN-539 (5056_A08), TCN-540 (50604F05), TCN1541 (5062_Ml l), TCN-542 A16), TCN—543 (5081_G23), TCN-544 (5082;Al9), TCN-545 (5082_115), TCN-546 (5089_L08), TCN-547 (5092_Fl l), TCN-548 (5092;1301), 9 (5092__P04), 0 (5096_F06), TCN-SSI (5243_D01), TCN—552 123), TCN-553 (5261_C18), TCN—554 (5277_M05), TCN-SSS 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: CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCGGAGACCCTGTCCCTCACTTGCGCTGTCTCT GGTGTCTCCATCAGCAATATTGATTTCTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTAGAATGGATT GGCAATATCTATTATACGGGGATCACCTTCTACAACCCGTCCCTCAGCAGTCGAGTCGCCATATCCATTGACACC TCCAAGAACCAGTTCTCCCTGACTCTGACTTCTGTGACCGCCGCAGACACGGCTATGTATTACTGTGCGAGACAT TACGGTGACTCCGAGGCAATAAACGATGCCTTTGACATCTGGGGCCAAGGGACAATGCTCACCGTCTCGAGC (SEQ ID NO: 745) . \' TCN-504 (3251_K17) gamma heavy chain. variable region amino acid sequence: (Kabat CDRs in bold, a CDRs underlined) QVQLQESGPGLVKPSETLSLTCAVSGVSISNIDFYWGWIRQPPGKGLEWIGNIYYTGITFYNPSLSSRV AlSlDTSKNQFSLTLTSVTAADTAMYYCARHYGDSEAINDAFDIWGQGTMLTVSS (SEQ ID NO: 746) TCN-504 (3251_K17) gamma heavy chain Kabat CDRs: CDR l: NIDFYWG (SEQ ID NO: 747) ' CDR 2: NlYYTGITFYNPSLSS (SEQ ID NO: 748) CDR 3: HYGDSEAINDAFDI (SEQ ID NO: 749) TCN-504 (325 1_K17) gamma heavy chain‘Chothia CDRs CDR l: GVSISN (SEQ ID NO: 750) CDR 2: NIYYTGITF (SEQ ID NO: 751) CDR 3: HYGDSEAINDAFDI (SEQ ID NO: 749) TCN-504 (3251_K17) light chain variable region nucleotide ce: GAGATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTGTTGGCAATAGTTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGT GCATCCACCAGGGCCACTGGTATCCCACCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATC AGCAGCCTGCAGACTGAAGATTTTGCAGTTTATTACTGTCAACAATATATTAACTGGCGTCCGCTCAGTTTTGGC GGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 752) TCN-504 (3251_Kl7) light chain variable region amino acid sequence (KabatCDRs in bold, Chothia CDRS underlined) EIVMTQSPATLSVSPGERATLSCRASS2SVGNSLAWYQQRPGQAPRLLIYGASTRATGIPPRFSGSGSGT SSLQTEDFAVYYC!QQYINWRPLSFGGGTKVEIK (SEQ ID NO: 753) TCN-504 (3251_K17) light chain Kabat CDRs: CDR l: RASQSVGNSLA (SEQ ID NO: 754) CDR 2: GASTRAT (SEQIID NO: 755) CDR 3: RPLS (SEQ ID NO: 756) TCN-504 (325 1_K17) light chain Chothia'CDRsa CDR l: RASQSVGNSLA (SEQ ID NO: 754)‘ i” ‘ CDR 2: T (SEQ ID NO: 755) CDR 3: QQYINWRPLS (SEQ ID NO: 756) TCN-52l (3280_D18) heavy chain variable region nucleotide sequence: GAAGTGCAGTTGGTGCAGTCTGGAGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCGCCTGTGTAGTCTCT GGGTTCACCGTCACCAGCAATTATATAACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT ATTTATAGTCATGGTCGCGCATATTATTCAGCCTCCGTGAATGGCCGATTCACCATCTCCAGACACACTTCCAAG AACACAGTTTATCTTGAAATGAACAGCCTGAGACCTGAGGACACGGCCGTCTATTACTGTGCGGGCGGGGGCCTA GTCGGTGGCTACGACGAATATTTCTTTGACTATTGGGGCCAGGGAACCCTGGCCACCGTCTCCTCA (SEQ ID NO: 758) 1 (3280_D18) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) EVQLVQSGGGLVQPGGSLRLACVVSGFTVTSNYITWVRQAPGKGLEWVSVIYSHGRAYYSASVNGRF TISRHTSKNTVYLEMNSLRPEDTAVYYCAGGGLVGGYDEYFFDYWGQGTLATVSS (SEQ ID NO: 759) TCN—521 (3280_D18) gamma heavy chain Kabat CDRs: CDR l: SNYIT (SEQ ID NO: 760) CDR 2: VIYSHGRAYYSASVNG (SEQ ID NO: 761) CDR 3: GGLVGGYDEYFFDY (SEQ ID NO: 762) TCN-521 D18) gamma heavy chain Chothia CDRs: CDR 1: GFI‘VTS (SEQ ID NO: 763) CDR 2: VIYSHGRAY (SEQ ID NO: 764) CDR 3: GGLVGGYDEYFFDY (SEQ IDINO: 762) TCN-521 (3280_D18) light chain variable region nucleotide sequence: GAAACTGTCTTGACGCAATCTCCAGGCACCTTGTCTTTGACTCCAGGGGAAAGAGCCACCCTCTCCTGCAGAGTC GGTCAGAGTGTTAGCGGCAGCCACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCGGTGGCAGTGTGTCTGGGACAGACTTCACTCTCACC ATCAGCAGACTGGAGCCTGAAGATTCTGCAGTTTATTACTGTCAGCAGTATGGTGACTCACGATACACTTTTGGC CAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 765) TCN-521 (3280_D18) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) ETVLTQSPGTLSLTPGERATLSCRVG![SVSGSHLAWYQQKPGQAPRLLIYGASSRATGIPDRFGGSVSG TISRLEPEDSAVYYCS 2g QYGDSRYTFGQGTKLEIK (SEQ ID NO: 766) TCN-SZl (3280_D18) Light Chain Kabat CDRs: CDR l: SGSHLA (SEQ ID NO: 767) CDR 2: GASSRAT (SEQ ID NO: 768) CDR 3: QQYGDSRYT (SEQ ID NO: 769) TCN—521 (3280_D18) Light chain a CDRs: CDR 1: RVGQSVSGSHLA (SEQ ID NO: 767) CDR 2: GASSRAT (SEQ ID NO: 768) CDR 3: QQYGDSRYT (SEQ ID NO: 769) TCN-522 (3212_112) heavy chain variable region nucleotide sequence: CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTTTTGAAACCTTCGGAGACCCTGTCCCTCACCTGCACTGTGTCT GGGGGGTCCCTCACTGATTACTCTTGGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATCGGTGAC AccCTTCATAATGGCTACACCAACTACAACCCGTCCCTCAGGcGTCGAGTTTCCATCTCAATAGACACGTCCAAG AACCAGGTCTCACTCAGGCTGACCTCTGTGACCGCCGcGGACACGGCTCTTTATTACTGTGCGAGAGGCTCAGGT GGATATGGTGGCTTCGATTATTTTGGCAAGCTCCGGACATGGGACTTCTGGGGCCAGGGAACGCTGGTCACCGTC TCCTCA (SEQ ID NO: 770) TCN—522 (3212_112) gamma heavy ch‘ain‘va‘rfiable region amino acid sequence: (Kabat CDRs in bold, a CDRs underlined) QVQLQQWGAGLLKPSETLSLTCTVSGGSLTDYSWNWIRQPPGKGLEWIGDTLHNGYTNYNPSLRGR VSlSlDTSKNQVSLRLTSVTAADTALYYCARGSGGYGGFDYFGKLRTWDFWGQGTLVTVSS (SEQ ID NO: 77]) TCN—522 (3212_Il2) gamma heavy chain Kabat CDRs: CDR l: DYSWN (SEQ ID NO: 772) CDR 2: DTLHNGYTNYNPSLRG (SEQ ID NO: 773) CDR 3: GSGGYGGFDYFGKLRTWDF (SEQ ID NO: 774) TCN—522 (3212_112) gamma heavy chain Chothia CDRs: CDR l: GGSLTD (SEQ ID NO: 775) CDR 2: YTN (SEQ ID NO: 776) CDR 3: GSGGYGGFDYFGKLRTWDF (SEQ ID NO: 774) TCN-522 (3212_112) light chain variable region nucleotide sequence: GACATTCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCA AGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAAAAACCAGGGAACGCCCCTAAGCGCCTGATCTTTGGT GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACAATC AGCAGCCTGCAGCCTGAGGACTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGTACACTTTTGGCCAG AAGCTGGAGATCAAG (SEQ ID NO: 777) 2 (3212_112) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) DIQLTQSPSSLSASVGDRVTITCRASS[GIRNDLGWYQQKPGNAPKRLIFGASSLS[SGVPSRFSGSGSGT EFFLTISSLQPEDFATYYCL!QHNSYPYTFGQGTKLEIK (SEQ ID NO: 778) TCN-522 (3212_112) Light chain Kabat CDRS: CDR 1: RASQGIRNDLG (SEQ ID No: 779) CDR 2: GASSLQS (SEQ ID NO: 780) CDR 3; LQHNSYPYT (SEQ ID NO: 781) TCN-522 (3212_112)Light chain Chothia CDRS CDR 1: RASQGIRNDLG (SEQ ID NO: 779) CDR 2: S (SEQ ID NO: 780) CDR 3: LQHNSYPYT (SEQ ID NO: 781) TCN-523 (5248_Al7) heavy chain variable region nucleotide sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCAGCTTCAGCAACTATGCCTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ACCATCCCTCTACTTGGTACAACAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTTCCGCGGACCAATTC ACGAGCACAGCCTACATGGAGCTGGGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTACGAGACGGAAA ACGGCTTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 782) - TCN—523 (5248_Al7) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRS underlined) QVQLVQSGAEVKKPGSSVKVSCKASGGSFSNYAFSWVRQAPGQGLEWMGGTIPLLGTTNYAQKFQ GRVTISADQFI‘STAYMELGSLRSEDTAVYYCTRRKMTTAFDSWGQGTLVTVSS (SEQ ID NO: 783) TCN-523 (5248_Al7) gamma heavy chain Kabat CDRS: CDR 1: NYAFS (SEQ ID NO: 784) CDR 2: GTIPLLGTTNYAQKFQG (SEQ ID NO: 785) CDR 3. RKMTTAFDS (SEQ ID NO: 786) TCN—523 (5248_Al7)gamma heavy chainl'Chlothia CDRS: CDR l: GGSFSN (SEQ ID NO: 787) CDR 2: GTIPLLGTTN (SEQ ID NO: 788) CDR 3: RKMTTAFDS (SEQ ID NO: 786) TCN-523 Al7)light chain le region nucleotide sequence: CAGCCTGTTCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACCTGCACCCTGAGC AGCGCCTACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGCTTTGTGATGCGAGTG GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTTCTCAGTCTTGGGCTCAGGCCTGAAT CGGTACCTGACCATCAAGAACATCCAGGAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGG AGCAACTTCGTGTCCCCTTACGTATTCGGCGGAGGGACCAAGCTGACCGTTCTA (SEQ ID NO: 789) TCN-523 (5248_A17)light chain variable region amino acid ce (Kabat CDRs in bold, Chothia CDRs underlined) PPSASASLGASVTLTCTLSSAYSNYKVDWYQQRPGKGPRFVMRVGTGGIVGSKGDGIPDRF SVLGSGLNRYLTIKNIQEEDESDYHCGADHGSGSNFVSPYVFGGGTKLTVL (SEQ ID NO: 790) TCN-523 (5248_A17)Light Chain Kabat CDRs: CDR 1: TLSSAYSNYKVD (SEQ ID NO: 791) CDR 2: VGTGGIVGSKGD (SEQ ID NO: 792) CDR 3: GADHGSGSNFVSPYV (SEQ ID NO: 793) TCN-523 (5248_A17)Light chain Chothia CDRs: CDR 1: TLSSAYSNYKVD (SEQ ID NO: 791) CDR 2: VGTGGIVGSKGD (SEQ ID NO: 792) CDR 3: GSNFVSPYV (SEQ ID NO: 793) TCN—563 (5237_BZl) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGCGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTCGGTGAAAGTCTCATGCACGGCTTCT GGAGGCATCTTCAGGAAGAATGCAATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG ATCATCGCAGTCTTTAACACAGCAAATTACGCGCAGAAGTTTCAGGGCAGAGTCAAAATTACCGCAGACGAATCC ACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTCACCCA AAATATTTCTATGGTTCGGGGAGTTATCCGGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 794) ' 3 (5237,B21) gamma heavy chain-variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) A QVQLAQSGAEVKRPGSSVKVSCTASGGIFRKNAISWVRQAPGQGLEWMGGIIAVFNTANYAQKFQG RVKITADESGNTAYMELSSLRSDDTAVYYCASHPKYFYGSGSYPDFWGQGTLVTVSS (SEQ ID NO: 795) TCN-563 (5237_B21)gamma heavy chain Kabat CDRs: CDR 1: KNAIS (SEQ ID NO: 796) CDR 2: GIIAVFNTANYAQKFQG (SEQ ID NO: 797) CDR 3: HPKYFYGSGSYPDF (SEQ ID NO: 798) TCN-563 (5237_BZ] )gamma heavy chain Chothia CDRS: CDR 1: GGIFRK (SEQ ID NO: 799) CDR 2: GIIAVFNTAN (SEQ ID NO: 800) CDR 3: HPKYFYGSGSYPDF (SEQ ID NO: 798) TCN-563 (5237_B21)light chain variable region nucleotide sequence: CAATCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAAGC AGCAGTGATGTTGGTGCTTCTAACTCTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCGTTATT TATGATGTCACTGAGCGACCCTCAGGGGTCCCTCATCGCTIQTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACCGTCTCTGGGCTCCAGCCTGAGGACGAGGCTGATTATTTCTGCTGCGCATATGGAGGCAAATATCTTGTGGTC TTCGGCGGAGGGACCAAGGTGACCGTCCTC (SEQ ID NO: 801) TCN-563 (5237_B21)light chain le region amino acid sequence (Kabat CDRs in bold, Chothia CDRs ined) QSALTQPRSVSGSPGQSVTISCTGSSSDVGASNSVSWYQQHPGKAPKLVIYDVTERPSGVPHRFSGSKS GNTASLTVSGLQPEDEADYFCCAYGGKYLVVFGGGTKVTVL (SEQ ID NO: 802) TCN-563 (5237_B2l)light chain Kabat CDRS: CDR l: TGSSSDVGASNSVS (SEQ ID NO: 803) CDR 2: DVTERPS (SEQ ID NO: 804) CDR 3: CAYGGKYLVV (SEQ ID NO: 805) TCN-563 (5237_B2])light chain Chothia CDRS: CDR l: TGSSSDVGASNSVS (SEQ ID NO: 803) CDR 2: DVTERPS (SEQ ID NO: 804) CDR 3: CAYGGKYLVV (SEQ ID NO: 805) 6 (5084_C17)heavy chain variable region nucleotide sequence: GAGGTGCTGATGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCGTGAGACTCTCCTGTGTAGCCTCT GGATTCAGTTTCAGTAGTCATTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC ATAGAGGACGATGGAGGTGACAAGTACTATGTGGACTCTGTGAAGGGCCGATTCATTATCTCCAGAGACAACGCC TCAGTGTATCTGCAAATGAACAGCCTAAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGTTCG GGGAGCTCTGATAGAAGTGATTATGACCCCCACTACTACTACTACTTGGACGTCTGGGGCAAAGGGGCCACGGTC ACCGTCTCCTCA (SEQ ID NO: 806) 6 (5084_C17) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) EVLMVESGGGLVQPGGSVRLSCVASGFSFSSHWMTWVRQAPGKGLEWVANIEDDGGDKYYVDSVK GRFIISRDNAKNSVYLQMNSLRAEDTAVYFCARGSGSSDRSDYDPHYYYYLDVWGKGATVTVSS (SEQ ID NO: 807) TCN-526 (5084_C17) gamma heavy chaifi' Kabat CDRs: CDR 1: SHWMT (SEQ ID NO: 808) CDR 2: NIEDDGGDKYYVDSVKG (SEQ ID NO: 809) CDR 3: GSGSSDRSDYDPHYYYYLDV (SEQ ID NO: 810) TCN—526 (5084_C17) gamma heavy chain Chothia CDRs: CDR lz‘GFSFSS (SEQ ID NO: 811) CDR 2: NIEDDGGDKY (SEQ ID NO: 812) CDR 3: GSGSSDRSDYDPHYYYYLDV (SEQ ID NO: 810) TCN—526 (5084_C17) light chain variable region nucleotide sequence: GACATCCAGCTGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA AGTCAGAGCATTAGTAGGTATTTAAATTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGCTG'ITTGCT GCTTCTACTTTGCTAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACGGAATCACAGTCCCTCGTGGACGTTCGGCCAA GGGACCAGGGTGGAAATCAAA (SEQ ID NO: 813) ~54.
TCN-526 (5084_C17) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) DlQLTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLLFAASTLLDGVPSRFSGSGSGT ISSLQPEDFATYYCSQRNHSPSWTFGQGTRVEIK (SEQ ID NO: 814) 6 (5084_C17) Light chain Kabat CDRs: CDR 1: RASQSISRYLN (SEQ ID NO: 815) CDR 2: AASTLLD (SEQ ID NO: 816) CDR 3: SWT (SEQ ID NO: 817) TCN-526 (5084_C17) Light chain Chothia CDRs‘: ‘ CDR l: RASQSISRYLN (SEQ ID NO: 815) CDR 2; D (SEQ ID NO: 816) CDR 3: QRNHSPSWT (SEQ ID NO: 817) TCN-527 C06) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGCAAGAGTCGGGCCCGGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCAACTGCGCTGTCTCT GGAGGCTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGCTAT ATCTCTTACAATGGGAGGCCCAAGTACAACCCCTCCCTCACGAGTCGAGTCACCATATCCGTCGACACGTCCAAG GACCAGTTCTCCCTGGAGCTGCGCTCTGTGACCGCTGCGGACACGGCccTTTATTACTGTGCGAGAGAAACGCGG TTCGGGGAGTTATTATCTCCCTATGATGCTTTTGAAATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA (SEQ ID NO: 818) TCN-527 (5086_C06) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs ined) QVQLQESGPGLVKPSETLSLNCAVSGGSISNYYWSWIRQPPGKGLEWIGYISYNGRPKYNPSLTSRVTI SVDTSKDQFSLELRSVTAADTALYYCARETRFGELLSPYDAFEIWGQGTMVTVSS (SEQ ID NO: 819) TCN—527 (5086_C06) gamma heavy chain Kabat CDRs: CDR l: NYYWS (SEQ ID NO: 820) CDR 2: YISYNGRPKYNPSLTS (SEQ ID NO: 821) CDR 3: ETRFGELLSPYDAFEI (SEQ ID NO: 822) TCN—527 (5086_C06) gamma heavy chairi'I‘Chothia CDRs: CDR 1: GGSISN (SEQ ID NO: 824) , CDR 2: YISYNGRPK (SEQ ID NO: 823) CDR 3: ETRFGELLSPYDAFEI (SEQ ID NO: 822) TCN-527 (5086_C06) light chain variable region nucleotide sequence: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATGACTTGCCGGGCA AGTCAGAACATTAGAAGCTATTTAAATTGGTATCAGCAGAGACCAGGGACAGCCCCTAAACTCCTGATCTATGCT GCATCCACTTTACACAGTGGGGTCCCATCAAGGTTCAGIGGCGGTGGGTCTGGGACAGATTTCACTCTCACCATC AATAATCTGCAACCTGAAGATTTTGCATCTTACTACTGTCAACAGAGTTACGATAACCCTCAGACGTTCGGCCAA GGGACCAAGGTGGAAATCAAA (SEQ ID NO: 825) TCN-527 (5086‘C06) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) DIQMTQSPSSLSASVGDRVTMTCRASSQNIRSYLNWYQQRPGTAPKLLIYAASTLHSGVPSRFSGGGSG TDFTLTINNLQPEDFASYYCS 2! [SYDNPQTFGQGTKVEIK (SEQ ID NO: 826) WO 12489 TCN-527 (5086_C06) Light chain Kabat CDRS: CDR 1: RASQNIRSYLN (SEQ ID NO: 827) CDR 2: AASTLHS (SEQ ID NO: 828) CDR 3: QQSYDNPQT (SEQ ID NO: 829) TCN-527 (5086_C06) Light chain Chothia CDRS: CDR l: RASQNIRSYLN (SEQ ID NO: 827) CDR 2: AASTLHS (SEQ ID NO: 828) CDR 3: QQSYDNPQT (SEQ ID NO: 829) TCN-528 (5087_P17) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGATACACCTTCACCAATTATGACATCAACTGGATTCGACAGGCCCCTGGTCAAGGACTTGAGTGGATGGGCTGG ATAAATCCCAACAGTGGAACCACGGGCTCTGCACAGAGGTTCCAGGGCAGAGTCACCATAACCGTGGACACCTCC ATAACCACAGTCTACATGGAACTGAGCAGCCTGAGATCTGACGACACGGCCATTTACTACTGCGCGAGAGGCCGT CTCCGGCTTCAACATTTTTTGACTGACTCCCAGTCCGAGAGGAGGGACTGCTTCGACCCCTGGGGCCAG GGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 830) '_ TCN—528 (5087-P17) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRS underlined) QVQLVQSGSEVKKPGASVKVSCKASGYTEI‘NYDINWIRQAPGQGLEWMGWINPNSGTTGSAQRFQG RVTITVDTSITTVYMELSSLRSDDTAIYYCARGRELLRL! [HFLTDSS[SERRDCFDPWGQGTLVTVSS (SEQ ID NO: 83]) 8 P17) gamma heavy chain Kabat CDRS: CDR 1: NYDIN (SEQ ID NO: 832) CDR 2: WINPNSGTTGSAQRFQG (SEQ ID NO: 833) CDR 3: LQHFLTDSQSERRDCFDP (SEQ ID NO: 834) TCN—528 (5087_P17) gamma heavy chain a CDRS: CDR l: GYTFTN (SEQ ID NO: 835) CDR 2: WINPNSGTTG (SEQ ID NO: 836) CDR 3: GRELLRLQHFLTDSQSERRDCFDP (SEQ ID NO: 834) TCN-528 (5087_P17) light chain variable region nucleotide sequence: GATATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA AATCAAGACATTGGCATTTATTTAAATTGGTATCAACAGAATCCAGGGAAAGTCCCTAAACTCCTGCTCCATGGT GCGTCCAGTTTGCAGGGCGGGGTCCCATCAAGGTTCAGTGCCAGTGGATCTGGGACAGATTTCACTCTCACCATT CACAGTCTACAACCTGAAGATTTAGCAACCTACTACTGTCAACAGAGICGCCGTCTACCGTACACTTTTGGCCAG GGGACCAGGGTGGAACTCAAA (SEQ ID NO: 837) ' TCN-528 (5087_P17) light chain variable ’region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) DlQMTQSPSSLSASVGDRVTITCRANQQDIGIYLNWYQQNPGKVPKLLLHGASSLSZGGVPSRFSASGSG TDF'I‘LTIHSLQPEDLATYYCQSZSRRLPYTFGQGTRVELK (SEQ ID NO: 838) TCN-528 (5087_P17) Light chain Kabat CDRS: CDR 1; RANQDIGIYLN (SEQ ID NO: 839) CDR 2: GASSLQG (SEQ ID NO: 340) CDR 3: QQSRRLPYT (SEQ ID NO: 841) TCN—528 (5087_P17) Light chain Chothia CDRs: CDR 1; GIYLN (SEQ ID NO: 839) CDR 2: GASSLQG (SEQ ID NO: 840) CDR 3: PYT (SEQ ID NO: 841) TCN-529 H01) heavy chain variable region nucleotide sequence: CAGATCACCTTGAGGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCT GGGTTTTCACTCAGCACTAATGGAGTGAATGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT GCACTCATTTACTGGGATGATGATAAGCGCTACAGTCCGTCTCTGAAGAGAAGGCTCACCATCACCAAGGACACC TCCAAAAACCAAGTGGTCCTTACACTGACCAACATGGACCCTGTAGATACAGCCACATATTACTGTGCACACAGA CCCGACTTCTATGGTGACTTCGAGTACTGGGGCCCGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 842) TCN-529 (5297_H01) gamma heavy chain variable region aminoacid sequence: (Kabat CDRS in bold, Chothia CDRS underlined) QITLRESGPTLVKPTQTLTLTCTFSGFSLSTNGVNVGWIRQPPGKALEWLALIYWDDDKRYSPSLKRR LTITKDTSKNQVVLTLTNMDPVDTATYYCAHRPDFYGDFEYWGPGTLVTVSS (SEQ ID NO: 843) TCN-529 (5297_H01) gamma heavy chain Kabat CDRS: CDR l: TNGVNVG (SEQ ID NO: 844) = CDR 2: LIYWDDDKRYSPSLKR (SEQ ID NO: 845) CDR 3: RPDFYGDFEY (SEQ ID NO: 846) TCN-529 (5297__H01)gamma heavy chain Chothia CDRS: CDR 1: GFSLSTNG (SEQ ID NO: 847) CDR 2: LIYWDDDKR (SEQ ID NO: 848) CDR 3: RPDFYGDFEY (SEQ ID NO: 846) TCN-529 (5297_H01) light chain variable region nucleotide sequence: CAGTCTGCACTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCCGGACAGTCGATCACCATCTCCTGCACTGGAAGC AGCAGTGACATTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAGGCCCCCAAACTCATGATT TACGATGTCAATAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACTATCTCTGGGCTCCAGACTGACGACGAGGCTGATTATTACTGCGGCTCATATACAGGCAGTCCTCATTATGTC TTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 849) TCN-529 (5297_H01) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) QSALTQPASVSGSPGQSITISCTGSSSDIGGYNYVSWYQQ GNTASLTISGLQTDDEADYYCGSYTGSPHYVFGTGTKYTVL (SEQ ID NO: 850) TCN—529 H01) Light chain Kabat CDRS:i ;- CDR 1: IGGYNYVS (SEQ ID NO: 851), CDR 2: DVNNRPS (SEQ ID NO: 852) CDR 3: GSYTGSPHYV (SEQ ID NO: 853) TCN—529 H01)Light chain Chothia CDRS: CDR l: TGSSSDIGGYNYVS (SEQ ID NO: 851) CDR 2: DVNNRPS (SEQ ID NO: 852) CDR 3: GSYTGSPHYV (SEQ ID NO: 853) TCN-53O (5248_H10a) heavy chain variable'region nucleotide sequence: CAGGTCCAACTGGTGCAATCTGGGGcTGAGGTGAGGAAGCCIGGGchTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCCCCTTCATGAGTTATGCTATCGGCTGGGTGCGAQAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCAACCCTGTGTTTGGTAGACCGCACTACGCACAGAAGT'TCCAGGGCAGAGTCACCATCGCCACGGACGACTCC ACGAAGACATCGTACATGGAACTGAGTAGCCTGACGTCTGAGGACACGGGCATGTATTACTGTGCGAGTAGGTAT TCGTCCCCAGGGACCTTTGAGTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 854) TCN-530 (5248_H10a) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, a CDRS underlined) QVQLVQSGAEVRKPGSSVKVSCKASGGPFMSYAIGWVRQAPGQGLEWMGGINPVFGRPHYAQKFQ GRVTIATDDSTKTSYMELSSLTSEDTGMYYCASRYSRSSPGTFESWGQGTLVTVSS (SEQ ID NO: 855) 0 (5248_H10a) gamma heavy chain Kabat CDRS: CDR l: SYAIG (SEQ ID NO: 856) CDR 2: GINPVFGRPHYAQKFQG (SEQ ID NO: 857) CDR 3: RYSRSSPGTFES (SEQ ID NO: 858) TCN-530 (5248_H10a) gamma heavy chain Chothia CDRS: CDR l: GGPFMS (SEQ ID NO: 859) CDR 2: GINPVFGRPH (SEQ ID NO: 860) CDR 3: RYSRSSPGTFES (SEQ ID NO: 858) i . ., : TCN-530 (5248_H10a) light chain variable'region nucleotide sequence: GAAATAGTGATGACGCAGTTTCCAGCCACCCTGTCTGTGTPICCCGGGGAACGAGTCACCCTCTCCTGTAGGGCC AGTCAGAGTGTTAGCAACAATTTAGCCTGGTACCAGCAAAAACCTGGCCAGCCICCCAGGCTCCTCATCTATGAT GCATCTACCAGGGCCACGGGTGTCCCAGCCAAGTTCAGTGGCACTGGGTCTGGCACAGAGTTCACTCTCAGCATC AGCAGCCTGCAGTCCGAAGATTTTGCAGTTTATTACTGTCAGCAGTATCACAACTGGCCTCCCTCGTACAGTTTT GGCCTGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 861) TCN-530 H10a) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) FPATLSVSPGERVTLSCRASS[SVSNNLAWYQQKPGQPPRLLIYDASTRATGVPAKFSGTGSGT EFI‘LSISSLQSEDFAVYYCQSQYHNWPPSYSFGLGTKLEIK (SEQ ID NO: 862) TCN-530 (5248_H10a) Light chain Kabat CDRS: CDR 1: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT (SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865) TCN-53O (5248_H10a) Light chain Chothia CDRS: CDR 1: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT (SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865) - 141'_; TCN-531 (5091_H13) heavy chain variable region nucleotide sequence: GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTACAGCCAGGGCGGTCCCTGAAACTCTCCTGCACAGGTTCT ACCTTTGGTGATTATGGTGTGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTC ATTAGAACCAGACCTTGGGGTGGGACAGCAGATACCGCCGCGTCTGTGAAAGGCAG‘ATTCACTATTTCAAGAGAT GATTCCAAAAGTCTCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTTGTAGA GATGCCCCTCCAAATGTGGAAGTGGCTTCTATGACCAACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTC ACCGTCTCCTCA (SEQ ID NO: 866) TCN-531 H13) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) EVQLVESGGDLVQPGRSLKLSCTGSGEFFGDYGVTWVRQAPGKGLEWVGFIRTRPWGGTADTAASV KGRFI‘ISRDDSKSLAYLQMNSLKTEDTAVYYCCRDAPPNVEVASMTNWYFDLWGRGTLVTVSS (SEQ ID NO: 867) TCN~531 (5091_H13) gamma heavy chain Kabat CDRs: CDR l: DYGVT (SEQ ID NO: 868) CDR 2: FIRTRPWGGTADTAASVKG (SEQ ID NO: 869) CDR 3: EVASMTNWYFDL (SEQ ID NO: 870) TCN—531 (5091_H13) gamma heavy chain Chothia CDRS: CDR l: GFFFGD (SEQ ID NO: 871) CDR 2: FIRTRPWGGTAD (SEQ ID NO: 872) CDR 3: DAPPNVEVASMTNWYFDL (SEQ ID NO: 870) TCN-53l (5091_H13) light chain variable region nucleotide sequence: GACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCG AGTCAGGGCATTCTCAATTGTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGTTCCTAACCTCCTGATGTATGCT GCATCCACATTGCAGTCAGGGGTCCCATCTCGGTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATC AGCAGCCTGCAGCCTGAAGATGTTGCAACT'IA'ITACTGTCAAACGTATGGCGGTGTCTCTACTTTCGGCGGAGGG GTGGAGATCAGA (SEQ ID NO: 873) TCN-531 (5091_H13) light chain variable region amino acid sequence (Kabat CDRS in bold, a CDRs underlined) ,l: ‘ DIQLTQSPSSLSASVGDRVTITCRASS[GILNCLAWYQQKPGKYPNLLMYAASTLQQSGVPSRFSGSGFG TDFI‘LTISSLQPEDVATYYCSQTYGGVSTFGGGTKVEIR (SEQ ID. NO: 874) TCN-531 (5091_H13) Light chain Kabat CDRs: CDR l: RASQGILNCLA (SEQ ID NO: 875) CDR 2: AASTLQS (SEQ ID NO: 876) CDR 3: QTYGGVST (SEQ ID NO: 877) TCN—53l (5091_H13) Light chain Chothia CDRs: CDR 1: RASQGILNCLA (SEQ ID NO: 875) CDR 2: AASTLQS (SEQ ID NO: 876) CDR 3: QTYGGVST (SEQ ID NO: 877) 2_ a TCN—532 (5262_H18) heavy chain variable région nucleotide sequence: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCTTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCGTCAGCAGTGAGACTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTAGAGTGGATT GGATATATCTATTACATTGGGAACACCGACTACAGGCCCTCCCTCAAGAGTCGAGTCACCATATCACTGGACACG TCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGAGGC GCTTATTATGATAGTAGTGGTTACCCGGCTTTTTATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA (SEQ ID NO: 878) TCN-532 (5262_H18) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRS underlined) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSETYYWSWIRQPPGKGLEWIGYIYYIGNTDYRPSLKSR VTISLDTSKNQFSLKLSSVTAADTAVYYCARGAYYDSSGYPAFYIWGQGTMVTVSS (SEQ ID NO: 879) TCN-532 (5262_H18) gamma heavy chain Kabat CDRS: CDR l: SETYYWS (SEQ ID NO: 880) CDR 2: YIYYIGNTDYRPSLKS (SEQ ID NO: 881) CDR 3: GAYYDSSGYPAFYI (SEQ ID NO: 882) TCN-532 H18) gamma heavy chain Chothia CDRS: CDR l: ET (SEQ ID NO: 883) . ax CDR 2: YIYYIGNTD (SEQ ID NO: 884) CDR 3: SGYPAFYI (SEQ ID NO: 882) TCN—532 (5262_H18) light chain variable region nucleotide sequence: CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGC AGCTCCAACATCGGGTCAGATTATGATGTGCACTGGTACAAGCAACTTCCAGGAACAGCCCCCAAACTCCTCATC TTTGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTG GCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAATCCTATGACAGCAGCCTGAGTGGTTTT CATGTCTTCGGAAGTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 885) TCN-532 (5262_H18) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRs underlined) QSVLTQPPSVSGAPGQRVTISCT__—_GSSSNIGSDYDV GTSASLAITGLQAEDEADYYCSQSYDSSLSGFHVFGSGTKVTVL (SEQ ID NO: 886) TCN—532 (5262_H18) Light chain Kabat CDRS: CDR l: IGSDYDVH (SEQ ID NO: 887) CDR 2: GNSNRPS (SEQ ID NO: 888) CDR 3: QSYDSSLSGFHV (SEQ ID NO: 889) TCN-532 H18) Light chain a CDRS: CDR l: TGSSSNIGSDYDVH (SEQ ID NO: 887) ' CDR 2: GNSNRPS (SEQ ID NO: 888) CDR 3: QSYDSSLSGFHV (SEQ ID NO: 889) TCN-533 (5256_Al7) heavy chain variable region nucleotide sequence: CAGCTGGTGCAGTCTGGGGCTGACGTGAAGAAGCCTGGGTCCTCGGTGACGGTCTCCTGCAAGGCTTCT AGCTTCAGCAACTATGGAATCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGGGA ATCATCCCTCTCATTAATGCACCGAACTACGCACCGAAGTICCAGGGCAGAGTGACGATTACCGCGGACATGTTC TCGAATATAGTCTCCTTGCAGTTGACCAGCCTGAGAACTGACGACACGGCCGTGTATTATTGTGCGAGACGAAAA ATGACTACGGCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 890) TCN—533 (5256_Al7) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs ined) QVQLVQSGADVKKPGSSVTVSCKASGGSFSNYGINWVRQAPGQGLEWMGGIIPLINAPNYAPKFQG DMFSNIVSLQLTSLRTDDTAVYYCARRKMTTAIDYWGQGTLVTVSS (SEQ ID NO: 891) TCN-533 (5256_Al7) gamma heavy chain Kabat CDRs: CDR l: NYGIN (SEQ ID NO: 892) CDR 2: GIIPLINAPNYAPKFQG (SEQ ID NO: 893) CDR 3: RKMTTAIDY (SEQ ID NO: 894) TCN—533 (5256_Al7) gamma heavy chain Chothia CDRs: CDR l: GGSFSN (SEQ ID NO: 787) CDR 2: GIIPLINAPN (SEQ ID NO: 895) CDR_3: RKMTTAIDY (SEQ ID NO: 894) 3 (5256_Al7) light chain variable region nucleotide sequence: CAGCCTGTGTTGAGTCAGCCACCTTCTGCATCGGCCTCCCTGGGAGCCTCCGTCACACTCACCTGCAC CCTGAGTAGCGGCTTCGATAATTATCAAGTGGCCTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGCT TTGTGATGCGGGTGGGCAATGGTGGGAATGTGGCTTCCAAGGGGGATGGCATTCCTGATCGTTTCTCA GTCTCGGGCTCAGGCCTGAATCGGTACCTGACCATCAAGAACATCCAGGAAGACGATGAGAGTGACTA TTATTGTGGGGCAGACCATGGCAGTGGGAACAACTTCGTGTCCCCTTATGTGTTTGGCGGAGGGACCA AGCTGACCGTTCTA (SEQ ID NO: 896) TCN-533 (5256_Al7) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) QPVLSQPPSASASLGASVTLTCTLSSGFDNY!QVAWYQQRPGKGPRFVMRVGNGGNVASKG _I_)_GIPDRFSVSGSGLNRYLTIKNIQEDDESDYYCGADHGSGNNFVSPYVFGGGTKLTVL (SE ID NO: 897) , TCN-533 (5256~Al7) Light chain Kabat CDRs: CDR l: TLSSGFDNYQVA (SEQ ID NO: 898) CDR 2: VGNGGNVASKGD (SEQ ID NO: 899) CDR 3: GADHGSGNNFVSPYV (SEQ ID NO: 900) TCN-533 (5256_Al7) Light chain Chothia CDRs: CDR l: TLSSGFDNYQVA (SEQ ID NO: 898) CDR 2: VGNGGNVASKGD (SEQ ID NO: 899) CDR 3: GADHGSGNNFVSPYV (SEQ ID NO: 900) TCN-534 (5249_B02) heavy chain variable region nucleotide ce: CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCAGGGTCCTCGGTGAAGGTCTCCTGCAGGGAATCT GGAGGCACCTTCAACGGCTACACTATCACCTGGGTGCGACAGGCCCCTGGGCAAGGCCTTGAGTGGATGGGAGGG ATCATCCCTATGATGGGGACAGTCAACTACGCACAGAAGTTGCAGGGCAGAGTCACCATTACCACGGACTATTTC ACGAAAACAGCCTACATGGATCTGAACAATTTAAGATCTGAAGACACGGc'CATGTATTATTGTGTGAAAATCAGA TATACTGGGCAGCAGCTGCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 901) TCN-534 (5249_B02) gamma heavy chain le region amino acid sequence: (Kabat CDRs1n bold, Chothia CDRs underlined) QVQLVQSGAEVKKPGSSVKVSCRESGGTFNGYTITWVRQAPCQGLEWMGGIIPMMGTVNYAQKLQ GRVTITTDYFTKTAYMDLNNLRSEDTAMYYCVKIRYTGSZQLLWGQGTLVTVSS (SEQ ID NO: 902) TCN—534 (5249_B02) gamma heavy chain Kabat CDRs: CDR 1: GYTIT (SEQ ID NO: 903) CDR 2: GIIPMMGTVNYAQKLQG (SEQ ID NO: 904) CDR 3: IRYTGQQLL (SEQ ID NO: 905) TCN-534 (5249_802) gamma heavy chain Chothia CDRs: CDR l: GGTFNG (SEQ ID NO: 906) CDR 2: GIIPMMGTVN (SEQ ID NO: 907) CDR 3: IRYTGQQLL (SEQ ID NO: 905) TCN-S34 B02) light chain variable region nucleotide ce: GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCGGCATCTATAGGAGACAGAGTCACCATCACTTGCCGGGCC AGTCAGAGTATTGCAAGTTGGTTGGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAG GCAGTTAATTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGCCTGCAGCCCGATGATTTTGCAACTTATTTCTGCCAACATTATGGTACTATTTCTCAGACCTTCGGCGGA| AAGGTGGAGATCAAA (SEQ ID NO: 908) TCN-534 (5249_BOZ) light chain variable regionamino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) ‘- DlQMTQSPSTLSASlGDRVTITCRASQSIASWLAWYQQKPGKAPKLLIYEAVNLESGVPSRFSGSGSGT DFTLTISSLQPDDFATYFCSQHYGTISS[TFGGGTKYEIK(SEQ ID NO: 909) TCN-534 (5249_B02) Light chain Kabat CDRS: CDR l: RASQSIASWLA (SEQ ID NO: 910) CDR 2: EAVNLES (SEQ ID NO: 91 1) CDR 3: QHYGTISQT (SEQ ID NO: 912) TCN—534 (5249_B02) Light chain Chothia CDRs: CDR l: RASQSIASWLA (SEQ ID NO: 910) CDR 2: EAVNLES (SEQ ID NO: 911) CDR 3: QHYGTISQT (SEQ ID NO: 912) TCN-535 (5246_P19)_heavy chain variable region nucleotide sequence: CAGGTCCAGCTGGTGCAATCTGGGAGTGAGGTGAAGAAGCCTGGGACCTCGGTGAAGGTCTCCTGCACGGCCTCT GGAAGTGTCTTCACCAATTATGGAATTAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTCTCTTTGGCGCAGCCAAGTACGCACAGAAATTCCAGGGCAGAGTCACCATCACAGCGGACGAATCC ACGAAGACAGTCTACATGGAGCTGAGCAGGCTGACATCTAAAGACACGGCCATATATTTCTGTGCGAAGGCCCCC CGTGTCTACGAGTACTACTTTGATCAGTGGGGCCAGGGAACCCCAGTCACCGTCTCCTCA (SEQ ID NO: 913) TCN—535 P19) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRS underlined) QVQLVQSGSEVKKPGTSVKVSCTASGSVFI‘NYGISWVRQAPGQGLEWMGGIIPLFGAAKYAQKFQG RVTITADESTKTVYMELSRLTSKDTAIYFCAKAPRVYEYYFDS[WGQGTPVTVSS (SEQ ID NO: 914) TCN-535 (5246_Pl9) gamma heavy chain Kabat CDRS: CDR l: NYGIS (SEQ ID NO: 915) ' CDR 2: GIIPLFGAAKYAQKFQG (SEQ ID N02. 916) CDR 3: YYFDQ (SEQ ID NO: 917) x' TCN-535 (5246_P19) gamma heavy chain a CDRs: CDR l: GSVF'TN (SEQ ID NO: 918) CDR 2: GIIPLFGAAK (SEQ ID NO: 919) CDR 3: APRVYEYYFDQ (SEQ ID NO: 917) TCN—535 (5246_P19) light chain variable region nucleotide sequence: GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTAGCAGCAGTCAATTAGCCTGGTACCAGCAAAAACCTGGCCAGGCTCCCAGGCTCATCATCTAT GGTGCGTCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGAAGTGGGTCTGGGACAGACTTCACTCTCACC ATCGGCAGACTGGAGCCTGAAGATTTTGCAGTGTTTTTCTGTCAGCAGTATAGTACCTCACCTCCGACGTTCGGC CAAGGGACCAAGGTGGATTTCAAA (SEQ ID NO: 920) TCN-535 (5246_P19) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs ined) EIVLTQSPGTLSLSPGERATLSCRASQSVSSS! [LAWYQQKPGQAPRLIIYGASTRATGIPDRFSGSGSGT IGRLEPEDFAVFFCQ! QYSTSPPTFGQGTKVDFK (SEQ ID NO: 921) TCN-535 (5246_.P19) Light chain Kabat CDRS: CDR 1: RASQSVSSSQLA (SEQ ID NO: 922) 7 CDR 2: GASTRAT (SEQ ID NO: 755) i CDR 3: QQYSTSPPT (SEQ ID NO: 923) TCN-535 (5246319) Light chain Chothia CDRs: CDR 1: SSSQLA (SEQ ID NO: 922) CDR 2: GASTRAT (SEQ ID NO: 755) CDR 3: QQYSTSPPT (SEQ ID NO: 923) .s‘l TCN—536 (5095_N01) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT GGTGGGTCCTTCAGTGTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAGGGGGCTGGAGTGGATT GGGGAAATCAGTCATGGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTcGAGTCACCATATCAGTGGACACG AACCAGTTCTCCCTGAGACTGAGCTCTGTGACCGCCGCGGACACGGCCGTCTATTACTGTGCGAGAGGG ACAGACCCTGACACGGAAGTATATTGTCGTGTTGGTAACTGCGCGGCCTTTGACTACTGGGGCCAGGGAAGCCTG GTCACCGTCTCCTCA (SEQ ID NO: 924) TCN-536 (5095_N0]) gamma heavy chain le region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSVSGYYWSWIRQPPGRGLEWIGEISHGGSTNYNPSLKS RVTISVDTTKNQFSLRLSSVTAADTAVYYCARGTDPDTEVYCRVGNCAAFDYWGQGSLVTVSS (SEQ ID NO: 925) TCN-536 (5095_N01) gamma heavy chain Kabat CDRs: CDR 1: VSGYYWS (SEQ ID NO: 926) CDR 2: EISHGGSTNYNPSLKS (SEQ ID NO: 927) CDR 3: GTDPDTEVYCRVGNCAAFDY (SEQ ID NO: 928) TCN-536 (5095_N01) gamma heavy chain Chothia CDRs: CDR 1: GGSFSVSG (SEQ ID NO: 929) CDR 2. EISHGGSTN (SEQ ID NO: 930) CDR 3: GTDPDTEVYCRVGNCAAFDY (SEQ ID NO: 928) TCN—536 (5095_N01) light chain variable region nucleotide sequence: GAAATTATATTGGCGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAGAGAGCCACCCTCTCCTGCAGGGCC AGCCAGTTTGTTAGCACCAGATCccTGGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGACTCCTCATCTAT GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACGCTCACC ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTATGGTTACTCACCTAGGTACGCTTTT GGCCAGGGGTCCAAGGTTGAGATCAAA (SEQ ID NO: 931) TCN-536 (5095_N01) light chain le region amino acid sequence (Kabat CDRs in bold, a CDRs underlined) EIILAQSPGTLSLSPGERATLSCRASS SLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFI‘LTISRLEPEDFAVYYCSQHYGYSPRYAFGQGSKVEIK (SEQ ID NO: 932) TCN-536 (5095_NOl) Light chain Kabat CDRs: CDR l: RASQFVSTRSLA (SEQ ID NO: 933) CDR 2: GASSRAT (SEQ ID NO: 768) CDR 3: QHYGYSPRYA (SEQ ID NO: 934) TCN-536 (5095_N0]) Light chain Chothia CDRs: CDR l: RASQFVSTRSLA (SEQ ID NO: 933) CDR 2: T (SEQ ID NO: 768) CDR 3: QHYGYSPRYA (SEQ ID NO: 934) TCN—537 (3194_D21) heavy chain variable region nucleotide sequence: CAGGTGCAGCTCCAACAGTGGGGCTCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT GGTGGGTCCTTCAGAGATGACTACTGGACCTGGATTCGCCAGCCCCCAGGCAAGGGGCTGGAGTGGATTGGGGAA ATCAATCATAGTGGAAGAACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCCTG AAACAGTTCTCCTTGAAGGTGATTTCTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAGAGGGACGAGC CATGTTTCCCGGTATTTTGATTGGTTACCACCCACCAACTGGTTCGACCCCTGGGGCCAGGGAACCCAGGTCACC GTCTCGAGC (SEQ ID NO: 935) TCN—537 (3194_D21) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRS underlined) QVQLQQWGSGLLKPSETLSLTCAVYGGSFRDDYWTWIRQPPGKGLEWIGEINHSGRTNYNPSLKSRV TISVDTSLKQFSLKVISVTAADTAVYYCARGTSHVSRYFDWLPP’I‘NWFDPWGQGTQVTVSS (SEQ ID NO: 936) TCN-537 (3194_D21) gamma heavy chain Kabat CDRS: CDR l: DDYWT (SEQ ID NO: 937) CDR 2: EINHSGRTNYNPSLKS (SEQ ID NO: 938) CDR 3: GTSHVSRYFDWLPPTNWFDP (SEQ ID NO: 939) TCN-537 (3194_D21) gamma heavy chain Chothia CDRS: CDR l: GGSFRD (SEQ ID NO: 940) CDR 2: EINHSGRTN (SEQ ID NO: 941) CDR 3: RYFDWLPPTNWFDP (SEQ ID NO: 939) TCN-537 (3194_D21) light chain variable region nucleotide sequence: GACATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCACAAACCTGGCCAGGCTCCCAGGCTCGTCATGTAT GCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGCCAGACTTCACTCTCACC ATCAGCAGACTGGAGCCTGAAGATTTTGCAATGTATTAC126TCAGCAGTATGGTAACTCACCGATCACCTTCGGC CAAGGGACACGACTGGAGATCAAA (SEQ ID NO: 942) 'I‘CN-537 (3194_D21) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) DIVLTQSPGTLSLSPGERATLSCRASS[SVSSSYLAWYQQKPGQAPRLVMYGAATRATGIPDRFSGSGS GPDFTLTISRLEPEDFAMYYC!QQYGNSPITFGQGTRLEIK (SEQ ID NO: 943) ‘ TCN-537 (3194_D21) Light chain Kabat CDRS: CDR l: RASQSVSSSYLA (SEQ ID NO: 944) CDR 2: T (SEQ ID NO: 945) CDR 3: PIT (SEQ ID NO: 946) TCN—537 (3194_D21) Light chain Chothia CDRS: CDR 1: SSSYLA (SEQ ID NO: 944) CDR 2: GAATRAT (SEQ ID NO: 945) CDR 3: QQYGNSPIT (SEQ ID NO: 946) TCN-538 (3206_Ol7) heavy chain variable region nucleotide sequence: CAGATCACCTTGAAGGAGTCTGGTCCTACACTGGTGAAACCCACACAGACCCTCACACTGACCTGCGTCTTCTCT GGGTTCTCACTCAGCATTACTGGAGTGCGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT GCACTCATTTCTTGGGATGATGAAAAGCACTACAGCCCATCTCTGCAGAGTAGGCTCACCATCACCAAGGACACC TCCAAAAACCAGGTGGTCCTTACAATGACCAACCTGGACCCTGTCGACACAGCCACATATTACTGTGCACGGTCA ACCGACAGGGGCCACGTCTTACGATATTTTGGCTGGATGTTACCGGGTGATGCATTTGATGTCTGGGGCCAAGGG ACAATGGTCACCGTCTCGAGC (SEQ ID NO: 947) 8 (3206_Ol7) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRs underlined) QITLKESGPTLVKPTQTLTLTCVFSGFSLSITGVRVGWIRQPPQKALEWLALISWDDEKHYSPSLQSRL TITKDTSKNQVVLTMTNLDPVDTATYYCARSTDRGHVLRYFGWMLPGDAFDVWGQGTMVTVSS (SEQ ID NO: 948) .
TCN-538 (3206_Ol7) gamma heavy chain Kabat CDRS: CDR l: ITGVRVG (SEQ ID NO: 949) CDR 2: EKHYSPSLQS (SEQ ID NO: 950) CDR 3: VLRYFGWMLPGDAFDV (SEQ ID NO: 951) TCN-538 (3206_Ol7) gamma heavy chain Chothia CDRs: CDR l: GFSLSITG (SEQ ID NO: 952) CDR 2: LISWDDEKH (SEQ ID NO: 953) CDR 3: STDRGHVLRYFGWMLPGDAFDV (SEQ ID NO: 951) TCN—538 Ol7) light chain variable region nucleotide sequence: GACATCGTGATGACCCAGTCTCCAGACTTCCTGCCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCC AGCCAGAGAGTTTTATACAGCTCCAACAATAAAAACTACTTAGCTTGGTACCAGCTGAAACCAGGGCAGCCTCCT AAGTTGATCATTTATTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA ACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATAGTCGT CCGTACACTTTTGGCCAGGGGACCAAGCTCGAGATCAAA (SEQ ID NO: 954) TCN—538 (3206_017) light chain le region amino acid sequence (Kabat CDRS in bold Chothia CDRS underlined) DIVMTQSPDFLPVSLGERATINCKSSSZRVLYSSNNKNYLAWYQLKPGQPPKLIIYWASTRESGVPDRFS GSGSGTEFTLTISSLQAEDVAVYYCS)QYYSRPYTFGQGTKLEIK (SEQ ID NO: 955) TCN-538 (3206_Ol7) Light chain Kabat CDRs: CDR 1: KSSQRVLYSSNNKNYLA (SEQ ID NO: 956) CDR 2: WASTRES (SEQ ID NO: 957) CDR 3: QQYYSRPYT (SEQ ID NO: 958) TCN-538 (3206_Ol7) Light chain Chothia CDRS: CDR 1: KSSQRVLYSSNNKNYLA (SEQ ID NO: 956) CDR 2: WASTRES (SEQ ID NO: 957) CDR 3: QQYYSRPYT (SEQ ID NO: 958) TCN-539 (5056_A08) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT GAAATCACCTTCATTACCTATGCTATGCACTGGGTCCGCCAGGCCCCAGGCAAGGGGCTGGAGTGGGTGGCACTT ATATCAGATGATGGAAGCAATAAATTCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGCTTATTACTGTGCGAGAGAAGGG GTTTACTTTGATTCGGGGACTTATAGGGGCTACTTTGACTACTGGGGCCAGGAAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 959) TCN-539 (5056_A08) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) SGGGVVQPGRSLRLSCAASEITFITYAMHWYRQAPGKGLEWVALISDDGSNKFYADSVKG RDNSKNTLYLQMNSLRAEDTAAYYCAREGVYFDSGTYRGYFDYWGQETLVTVSS (SEQ ID NO: 960) - TCN-539 (5056_A08) gamma heavy chain Kabat CDRs: CDR l: TYAMH (SEQ ID NO: 96]) CDR 2: LISDDGSNKFYADSVKG (SEQ ID NO: 962) CDR 3: EGVYFDSGTYRGYFDY (SEQ ID NO: .963) TCN-539 (5056_A08) gamma heavy chain a CDRs: CDR l: EITFIT (SEQ ID NO: 964) - CDR 2: LISDDGSNKF (SEQ ID NO: 965) CDR 3: EGVYFDSGTYRGYFDY (SEQ ID NO: 963) TCN—539 (5056_A08) light chain le region nucleotide sequence: GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCCACTGGCCTCCGATCACCTTCGGC CAAGGGACACGACTGGAGATCAAA (SEQ ID NO: 966) TCN-539 (5056_A08) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) EIVLTQSPATLSLSPGERA'I‘LSCRAS! [SVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYC!QQRSHWPPITFGQGTRLEIK (SEQ ID NO: 967) TCN-539 (5056_A08) Light chain Kabat CDRs: CDR l: RASQSVSSYLA (SEQ ID NO: 968) CDR 2: T (SEQ ID NO: 969) CDR 3: QQRSHWPPIT (SEQ ID NO: 970) TCN-539 (5056_A08) Light chain Chothia CDRS: CDR l: RASQSVSSYLA (SEQ ID NO: 968) CDR 2: DASNRAT (SEQ ID NO: 969) .
CDR 3: QQRSHWPPIT (SEQ ID NO: 970) 2012/024971 0 (5060_F05) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTACAATCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTCAGTAGCTACGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCTATT ATATCATACGACGGAAATGATCAATACTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCTCC AAAGTGTATCTCCAAATGCACAGGCTGAGACCTGAGGACACGGCTGTTTATTACTGTGCGAAAGAATTTGAAACT AGTGGTTATTTTCATGGGAGTT-TTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCGAGC (SEQ ID NO: 971) TCN—54O (5060_F05) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLVQSGGGVVQPGRSLRLSCAASGFFFSSYAMHWVRQAPGKGLEWVAIISYDGND!QYYTDSVKG RFTISRDSSKVYLQMHRLRPEDTAVYYCAKEFETSGYFHGSFDYWGQGILVTVSS (SEQ ID NO: 972) TCN-54O (5060_F05) gamma heavy chain Kabat CDRs: ' CDR 1: SYAMH (SEQ ID NO: 973) CDR 2: IISYDGNDQYYTDSVKG (SEQ ID NO: 974) CDR 3: EFETSGYFHGSFDY (SEQ ID NO: 975) TCN—540 (5060_F05) gamma heavy chain a CDRs: CDR l: GFIFSS (SEQ ID NO: 976) CDR 2: IISYDGNDQY (SEQ ID NO: 977) CDR 3: EFETSGYFHGSFDY (SEQ ID NO: 975) TCN-54O (5060_F05) light chain variable region nucleotide sequence: CAGTCTGCCcTGACTCAGCCTGCCTCCGTGTCTGGGTCchTGGACAGTCGATCACCATCTCCTGCACTGGAACC AGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCTTGATT TATGAGGTCACTAATTGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG TCTGGGCTCCAGGCTGAGGACGAGGCTGACTATTACTGCAGCTCATATGCGGGCAGCAGCACTTGGGTG TTCGGCGGAGGGACCAGGGTGACCGTTCTA (SEQ ID NO: 978) TCN-540 (5060_F05) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLLIYEVTNWPSGVSNRFSGSK SGNTASLTISGLQAEDEADYYCSSYAGSSTWVFCGGTRVTVL (SEQ ID NO: 979) TCN-54O F05) Light chain Kabat CDRs: CDR l: TGTSSDVGGYNYVS (SEQ ID NO: 980) CDR 2: EVTNWPS (SEQ ID NO: 981) CDR 3: SSYAGSSTWV (SEQ ID NO: 982) TCN-540 (5060_F05) Light chain Chothia CDRs: CDR l: TGTSSDVGGYNYVS (SEQ ID NO: 980) CDR 2: EVTNWPS (SEQ ID NO: 981) CDR 3: SSYAGSSTWV (SEQ ID NO: 982) TCN—541 (5062_M11) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCATCAATAGTTACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGCTAT ATCTATCACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATTTCGGTAGACACGTCCAAG AACCAGTTCTCCCTGCAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGACTCCGGACG GACTACGGTGACCCCGACTCGGTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCG AGC (SEQ ID NO: 983) TCN-54l (5062_Ml l) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) ' SGPGLVKPSETLSLTCTVSGGSINSYYWNWIRQPPGKGLEWIGYIYHSGSTNYNPSLKSRVTI SVDTSKNQFSLQLSSVTAADTAVYYCARLRTDYGDPDSVYYYGMDVWGQGTTVTVSS (SEQ ID NO: 984) TCN—54l (5062_Ml l) gamma heavy chain Kabat CDRs: CDR 1: SYYWN (SEQ ID NO: 985) CDR 2: STNYNPSLKS (SEQ ID NO: 986) CDR 3: LRTDYGDPDSVYYYGMDV (SEQ ID NO: 987) TCN-54l (5062_Ml 1) gamma heavy chain Chothia CDRs: CDR l: GGSINS (SEQ ID NO: 988) CDR 2: YIYHSGSTN (SEQ ID NO: 989) CDR 3: LRTDYGDPDSVYYYGMDV (SEQ ID NO: 987) TCN-54l (5062_M11) light chain variable region nucleotide sequence: TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGAT CCAAAGCAAAATGCTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGCTGATATATAAAGAC AGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGCTCAGGGACAACAGTCACGTTGACCATCAGT‘ GGAGTCCAGGCAGAGGACGAGGCTGACTATTACTGTCAATCAGCAGACAGCAGTGGTACTTCTTGGGTGTTCGGC GGAGGGACCAAACTGACCGTTCTA (SEQ ID NO: 990) TCN—54l (5062_M1 1) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRs underlined) PPSVSVSPGQTARITCSGDALPKS QNAYWYQQKISGQAPVLLIYKDSERPSGIPERFSGSSSGTT VTLTISGVQAEDEADYYCgQSADSSGTSWVFGGGTKLTVL (SEQ ID NO: 991) TCN-541 (5062_M11) Light chain Kabat CDRs: CDR l: SGDALPKQNAY (SEQ ID NO: 994) CDR 2: KDSERPS (SEQ ID NO: 995) CDR 3: QSADSSGTSWV (SEQ ID NO: 996) TCN-54l (5062_M1 1) Light chain Chothia CDRs: CDR 1: SGDALPKQNAY (SEQ ID NO: 994) CDR 2: KDSERPS (SEQ ID NO: 995) CDR 3: QSADSSGTSWV (SEQ ID NO: 996) TCN-542 (5079_A16) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCATCAGCAGTGGTAATTACTACTGGAACTGGGTCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT GGGTACATCTATTACAGAGGGAGCACCTTCTACAACCCGTCCCTCAAGAGTCGAGTGACCATA’ICAATAGACACG TCTAAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACGGCCGCGGACACGGCCGTGTATTACTGTGCGAAGGAT ACAAGGTCGAGCCTAGACAATTACCAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC (SEQ ID NO: 992) TCN-542 (5079_A16) gamma heavy-chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGNYYWNWVRQHPGKGLEWIGYIYYRGSTFYNPSLKSR VTISIDTSKNQFSLRLSSVTAADTAVYYCAKDTRSSLDNY!QYGMDVWGQGTTVTVSS (SEQ ID NO: 993) 'I‘CN-542 (5079_A16) gamma heavy chain Kabat CDRS: CDR 1: SGNYYWN (SEQ ID NO: 997) CDR 2: YIYYRGSTFYNPSLKS (SEQ ID NO: 998) CDR 3: DTRSSLDNYQYGMDV (SEQ ID NO: 999) TCN-542 (5079_A16) gamma heavy chain Chothia CDRS: CDR 1: GGSISSGN (SEQ ID NO: 1000) CDR 2: YIYYRGSTF (SEQ ID NO: 1001) CDR 3: DTRSSLDNYQYGMDV (SEQ ID NO: 999) TCN-542 (5079_A16) light chain variable region nucleotide sequence: CAGACTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGCTTCCAGC GCAGTCACCAGTAGTTACTTTCCAAACTGGTTCCAGCAGAAACCTGGACAAGCGCCCAGGCCACTGATT TATAGTACAACTATCAGACACTCCTGGACCCCGGCCCGATTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTG ACACTGTCAGGTGTGCAGCCTGAGGACGAGGCTGACTATTACTGCCTGCTCTACTCTGGTGGTGATCCAGTGGCT TTCGGCGGAGGGACCAAACTGACCGTTCTA (SEQ ID NO: ' 1 1002) 2 A16) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) QTVVTQEPSLTVSPGGTVTLTCASSTGAVTSSYFPNWFQQKPGQAPRPLIYSTTIRHSWTPARFSGSLL GGKAALTLSGVQPEDEADYYCLLYSGGDPVAFGGGTKLTVL (SEQ ID NO: 1003) TCN-542 (5079_A16) Light Chain Kabat CDRS: i CDR l: ASSTGAVTSSYFPN (SEQ ID NO: 1004) CDR 2: STTIRHS (SEQ ID NO: 1005) CDR 3: DPVA (SEQ ID NO: 1006) TCN-542 (5079_A16) Light chain a CDRS: CDR l: ASSTGAVTSSYFPN (SEQ ID NO: 1004) CDR 2: STTIRHS (SEQ ID NO: 1005) CDR 3: LLYSGGDPVA (SEQ ID NO: 1006) 3 (5081_023) heavy chain variable region nucleotide sequence: CAGGTTCATCTGGTGCAGTCTGGAGCTGAGGTGAGGAAGCCTGGGGACTCAGTGAAGGTCTCCTGTAAGACTTCT ACCTTTTCCACCTATCCTGTCGCCTGGGTGCGACAGGTCCCCGGACAAGGGCTTGAGTGGATGGGATGG ATCAGCACTTACAATGGAAACACAAACTTTGCACAGAACTTCCAGGGCAGAGTCACCCTGACCACAGACACAACC ACGAACACAGCCTACATGGAAGTGAGGAGCCTGAAATTTGACGACACGGCCGTCTATTACTGTGCGAGAGTGGAA TACAGGGATTTTTGGAATAATCAAAACAGATTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCG AGC (SEQ ID NO: 1007) TCN—543 (5081_G23) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVHLVQSGAEVRKPGDSVKVSCKTSGYTFSTYPVAWVRQVPGQGLEWMGWISTYNGNTNFAQNFQ GRVTL'I'I‘DI I lNTAYMEVRSLKFDDTAVYYCARVEGSYRDFWNN![NRFDPWGQGTLVTVSS (SEQ ID NO: 1008) TCN-543 (5081_G23) gamma heavy chain Kabat CDRS: CDR 1: TYPVA (SEQ ID NO: 1009) CDR 2: WISTYNGNTNFAQNFQG (SEQ ID NO: 1010) CDR 3: VEGSYRDFWNNQNRFDP (SEQ ID 011) TCN-543 (5081_G23) gamma heavy chain Chothia CDRS: CDR 1: GYTFST (SEQ ID NO: 1012) CDR 2. WISTYNGNTN (SEQ ID NO: 1013) CDR 3: VEGSYRDFWNNQNRFDP (SEQ ID NO:1011) TCN—543 (5081_G23) light chain variable region nucleotide ce: TCCTATGTACTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTTCCTGTGGGGGAAGC AACATTGGAGGGAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGAT AGCGGCCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACACGGCCACCCTGACCATCAGC AGGGTCGAAGCCGGGGATGAGGCCGACTATTTCTGTCAGGTGTGGGATAATTTCGGGGGAGTCTTCGGAACTGGG ACCAAGGTCACCGTTCTA (SEQ ID NO: 1014) TCN-543 (5081 _GZ3) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) SYVLTQPPSVSVAPGQTARISCGGSNIGGKSVHWYQQKPGQAPVLVVYDDSGRPSGIPERFSGSNSGD TATLTISRVEAGDEADYFCS2VWDNFGGVFGTGTKVTVL (SEQ ID NO: 1015) TCN-543 (5081_G23) Light chain Kabat CDRS: CDR 1: GKSVH (SEQ ID NO: 1016) CDR 2: DDSGRPS (SEQ ID NO: 1017) CDR 3: QVWDNFGGV (SEQ ID NO: 1018) TCN-543 (5081_G23) Light chain Chothia CDRszj CDR 1: GGSNIGGKSVH (SEQ ID NO: 1016) CDR 2: DDSGRPS (SEQ ID NO: 1017) CDR 3: QVWDNFGGV (SEQ ID NO: 1018) TCN-544 (5082_A19) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGGCTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT CGTGGCTCCATCGGTCATTACTTCTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGTTAT ATCTCTTACAGTGGGAGCACCAAGTACAACCCCTCCCTCAGGAGTCGAGTCACCATATCAGTAGACACGTCCAAG TTCTCCCTGAATCTGAACTCTGTCACCGCTACGGACACGGCCCTATATTACTGTGCGAGAGAGGATTAC GATATTTTGACTGGGGCGGGACCCGGTATGGAGGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC (SEQ ID NO: 1019) TCN-544 (5082_A19) gamma heavy chain variable region amino acid sequence: (Kabat CDRsin bold, Chothia CDRs underlined) QVQLQESGPGLVKPSETLSLTCTVSRGSIGHYFWSWIRQPPGKGLEWIGYISYSGSTKYNPSLRSRVTIS VD’I‘SKNQFSLNLNSVTATDTALYYCAREDYDILTGAGPGMEVWGQG’I‘I‘VTVSS (SEQ ID NO: 1020) TCN—544 (5082_Al9) gamma heavy chain Kabat CDRs: CDR 1: HYFWS (SEQ ID NO: 1021) CDR 2: YISYSGSTKYNPSLRS (SEQ ID NO: 1022) CDR 3: EDYDILTGAGPGMEV (SEQ ID NO: 1023) TCN-544 Al9) gamma heavy chain Chothia CDRs: CDR 1: RGSIGH (SEQ ID NO: 1024) CDR 2: YISYSGSTK (SEQ ID N02. 1025) CDR 3: EDYDILTGAGPGMEV (SEQ ID NO: 1023) TCN—544 (5082_A19) light chain variable region nucleotide sequence: CAGTCTATGTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGGAGC AGCTCCAACATCGGAAGTAATACTGTCAACTGGTTCAAACATCTCCCAGGAACGGCCCCCAAACTCCTCATCTAC AGAAATGATCTGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAACATGGGATGACAGCCTGAATGGTTTTTAT GTCTTCGGAACTGGGACCAAAGTCACCGTTCTA (SEQ ID'No: 1026) TCN—544 (5082_A19) light chain variable iregion amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) QSMLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWFKHLPGTAPKLLIYRNDLRPSGVPDRFSGSKSGT SASLAISGLQSEDEADYYCATWDDSLNGFYVFGTGTKVTVL (SEQ ID NO: 1027) TCN—544 (5082_A19) Light Chain Kabat CDRs: CDR 1: IGSNTVN (SEQ ID NO: 1028) CDR 2: RNDLRPS (SEQ ID NO: 1029) CDR 3: ATWDDSLNGFYV (SEQ ID NO: 1030) 4 (5082_A19) Light chain Chothia CDRS: CDR 1: SGSSSNIGSNTVN (SEQ ID NO: 1028) ' CDR 2: RNDLRPS (SEQ ID NO: 1029) CDR 3: ATWDDSLNGFYV (SEQ ID NO: 1030) TCN—545 (5082_115) heavy chain variable region tide sequence: CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCTCCTGCGCTGTCTTT TCCTTCAGTGATTACTACTGGACCTGGATACGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGCGAA ATCAAACATAGTGGAAGAACCAACTACAACCCGTCCCTTGAGAGTCGAGTCACCATATCAGTGGACACGTCCAAG AACCAGTTTTCCCTGAAACTGAGTTCTGTGACCGCCGCGGACACGGCTATATATTATTGTGCGAGAGGGACAGAC CCTGACACGGAGGGATATTGTCGTAGTGGTAGCTGCTCGGCCTTTGACTTCTGGGGCCAGGGAACCCTGGTCACC GTCTCGAGC (SEQ ID NO: 1031) TCN—54S (5082_115) gamma heavy chain le region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLQQWGAGLLKPSETLSLSCAVFGGSFSDYYWTWIRQPPGKGLEWIGEIKHSGRTNYNPSLESRV TISVDTSKNQFSLKLSSVTAADTAIYYCARGTDPDTEGYCRSGSCSAFDFWGQGTLVTVSS (SEQ ID NO: 1032) TCN-545 (5082_115) gamma heavy chain Kabat CDRs: CDR 1: DYYWT (SEQ ID NO: 1033) CDR 2: EIKHSGRTNYNPSLES (SEQ ID NO: 1034) CDR 3: GTDPDTEGYCRSGSCSAFDF (SEQ ID NO: 1035) TCN-545 (5082_115) gamma heavy chain Chothia CDRs: CDR 1: GGSFSD (SEQ ID NO: 1036) CDR 2: EIKHSGRTN (SEQ ID NO: 1037) CDR 3: GTDPDTEGYCRSGSCSAFDF (SEQ ID NO: 1035) TCN-545 (5082_115) light chain variable region tide sequence: GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC TTTGTGAACTACAGGTCCTTAGCCTGGTACCAGCAGACACCTGGCCAGGTTCCCAGGCTCCTCATCTAT GGTGCGTCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACT'ICACTCTCACC ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTTCTGTCAGCAGTATGGTGGCTCACCTAGGTACACTTTT GGCCAGGGGACCAGGCTGGAGATCAAA (SEQ ID NO: 1038) TCN—545 (5082_I15) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) EIVLTQSPGTLSLSPGERATLSCRASHFVNYRSLAWYQQTPGQVPRLLIYGASTRATGIPDRFSGSGSGT DFI‘LTISRLEPEDFAVYFCgQQYGGSPRYTFGQGTRLEIK (SEQ ID NO: 1039) TCN-545 (5082_115) Light chain Kabat CDRs: CDR 1: RASHFVNYRSLA (SEQ ID NO: 1040) S CDR 2: GASTRAT (SEQ ID NO: 755) CDR 3: PRYT (SEQ ID NO: 1041) 5 (5082_115) Light chain Chothia CDRs: CDR 1: RASHFVNYRSLA (SEQ ID NO: 1040) CDR 2: GASTRAT (SEQ ID NO: 755) CDR 3: QQYGGSPRYT (SEQ ID NO: 1041) TCN-546 (5089_L08) heavy chain variable region nucleotide sequence: CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGGTGTCTAT GGTGGGTCCCTCAGTGATTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGAGAA ATCAATCATAGTGGAGGCACCAACTACAATCCGTCCCTCAAGAGACGAGTCACCATATCAGTAGACACGTCAAAG AAGCAATTCTCCCTGAAGATGAACTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGGGACAGAC CCTGACACGGAAGTATATTGTCGTGCTGGTAACTGCGCGGCCTTTGACTTCTGGGGCCAGGGAACCCTGGTCACC GTCTCGAGC (SEQ ID NO: 1042) '156 TCN-546 (5089_L08) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLQQWGAGLLKPSETLSLTCGVYGGSLSDYYWSWIRQPPGKGLEWIGEINHSGGTNYNPSLKRRV TISVDTSKKQFSLKMNSVTAADTAVYYCARGTDPDTEVYCRAGNCAAFDFWGQGTLVTVSS (SEQ ID NO: 1043) TCN-546 (5089_L08) gamma heavy chain Kabat CDRs: CDR l: DYYWS (SEQ ID NO: 1044) CDR 2: EINHSGGTNYNPSLKR (SEQ ID NO: 1045) CDR 3: GTDPDTEVYCRAGNCAAFDF (SEQ ID NO: 1046) TCN—546 (5089_L08) gamma heavy chain Chothia CDRs: CDR 1: GGSLSD (SEQ ID NO: 1047) ‘ CDR 2: GTN (SEQ ID NO: 1048) CDR 3: GTDPDTEVYCRAGNCAAFDF (SEQ ID NO: 1046) TCN-546 (5089_L08) light chain variable region nucleotide sequence: GTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAGAGAGCCACCCTCTCCTGCCGGGCC AGTCACTTTGTTATAGGCAGGGCTGTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAC GGTGCATCCAGCAGGGCCACTGGCATCCCGGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC ATCAGCAGACTGGAGACTGAAGATTTTGCTGTGTTTTACTGTCAGCACTATGGTAGCTCACCTAGGTACGCTTTT GGGACCAAGCTGGAGATCAAA (SEQ ID NO: 104 9) TCN-546 (5089_L08) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRs underlined) EIVLTQSPGTLSLSPGERATLSCRASHFVIGRAVAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT SRLETEDFAVFYC!QHYGSSPRYAFGQGTKLEIK (SEQ ID NO: 1050) TCN-546 (5089_L08) Light chain Kabat CDRs: CDR 1: RASHFVIGRAVA (SEQ ID NO: 1051) CDR 2: GASSRAT (SEQ ID NO: 768) CDR 3: QHYGSSPRYAF (SEQ ID NO: 1052) TCN-546 (5089_L08) Light chain Chothia CDRS: CDR 1: RASHFVIGRAVA (SEQ ID NO: 1051) CDR 2: GASSRAT (SEQ ID NO: 768) CDR 3: QHYGSSPRYAF (SEQ ID NO: 1052) TCN-547 (5092_F1 1) heavy chain variable region nucleotide sequence: CAGGIGCAGCIGCAGGAGTCGGGCcCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGACTCCATTAGTAGTGTTGATCACTACTGGAGCTGGATCCGCCAACACCCAGTGAAGGGCCTGGAGTGGATT GGGTTCATGTATTACAGTGCGAGCACCTATTACAACcCGTCCCTCAAGAGTCGAGTTACCATATCAACGGACACG AACCAGTTCTCCCTGAGGCTGAGTTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGAGAGGC ACTTGTGCTGGTGACTGCTCCCTTCACTACTACTACTACGGTTTGGACGTCTGGGGCCAAGGGAGGACGGTCACC GTCTCGAGC (SEQ ID NO: 1053) TCN—547 (5092_F11) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLQESGPGLVKPSQTLSLTCTVSGDSISSVDHYWSWIRQHPVKGLEWIGFMYYSASTYYNPSLKSR VTISTDTSKNQFSLRLSSVTAADTAVYYCARGTCAGDCSLHYYYYGLDVWGQGRTVTVSS (SEQ ID NO: 1054) TCN—547 (5092_F1 l) gamma heavy chain Kabat CDRs: CDR 1: SVDHYWS (SEQ ID NO: 1055) CDR 2: STYYNPSLKS (SEQ ID NO: 1056) CDR 3: GTCAGDCSLHYYYYGLDV (SEQ ID NO: 1057) TCN-547 (5092_Fl 1) gamma heavy chain Chothia CDRs: CDR l: GDSISSVD (SEQ ID NO: 1058) CDR 2: FMYYSASTY (SEQ ID NO: 1059) CDR~ 3: CSLHYYYYGLDV (SEQ ID NO: 1057) TCN-547 (5092_F1 1) light chain variable region nucleotide sequence: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA AGCATTAGCAGC'I‘ATTTAAATTGGTATCAGCACAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCT GTATCCATTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGGCAGATTTCACTCTCACCATC AGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTTCCCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA (SEQ ID NO: 1060) 7 (5092_F1 1) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) ' DIQMTQSPSSLSASVGDRVTITCRAS! QSISSYLNWYQHKPGKAPKVLMYAVSILS [SGVPSRFSGSGSGA SSLQPEDFATYYCS 2g[SYSSPLTFGGGTKVEIK (SEQ ID NO: 1061) TCN—547 Fl 1) Light chain Kabat CDRs: CDR l: RASQSISSYLN (SEQ ID NO: 1062) CDR 2: AVSILQS (SEQ ID NO: 1063) CDR 3: QQSYSSPLT (SEQ ID NO: 1064) TCN—547 (5092_Fl 1) Light chain Chothia CDRs: CDR l: RASQSISSYLN (SEQ ID NO: 1062) CDR 2: AVSILQS (SEQ ID NO: 1063) CDR 3: QQSYSSPLT (SEQ ID NO: 1064) TCN-548 (5092_P01) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT AGTGGCCCCATGAGTGATTATTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGCAT . GTCTCTGTCTCTCACGGAGGGAGGACCAAATCCAATCCCTCCGTCATGAGTCGAGTCACCATTTCAGTAGAAACG TCCAAGAACCAATTCTCCCTGAAACTGACCTCCGTGACCGCIGCGGACACGGCCGTTTATTACTGTGCGAGATTA AATTACTATGATAGAAGTGGTTATCATTCGCCTGACGGCCCCTCGAACAACTGGTTCGACCCCTGGGGCCAGGGA ACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1065) 8 (5092_P01) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRS underlined) QVQLQESGPGLVKPSETLSLTCTVSSGPMSDYYWSWIRQPPGKGLEWIGHVSVSHGGRTKSNPSVMS RVTISVETSKNQFSLKLTSVTAADTAVYYCARLNYYDRSGYHSPDGPSNNWFDPWGQGTLVTVSS (SEQ ID NO: 1066) TCN—548 (5092_P01) gamma heavy chain Kabat CDRS: CDR l: DYYWS (SEQ ID NO: 1044) CDR 2: HVSVSHGGRTKSNPSVMS (SEQ ID NO: 1067) CDR 3: LNYYDRSGYHSPDGPSNNWFDP (SEQ ID NO: 1068) TCN-548 (5092_P01) gamma heavy chain Chothia CDRS: CDR l: SGPMSD (SEQ ID NO: 1069) CDR 2: HVSVSHGGRTK (SEQ ID NO: 1070) CDR 3: LNYYDRSGYHSPDGPSNNWFDP (SEQ ID NO: 1068) TCN-548 (5092_P01) light chain variable region nucleotide ce: GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCC AGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGC'I‘GCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGAATCAGCGGCAGCGGGTCTGGGGCA GATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTATTTTGCTACT CCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 1071) - TCN-548 (5092_P01) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) DIVMTQSPDSLAVSLGERATINCKSS![SVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRI SGSGSGADFI‘LTISSLQAEDVAVYYC!QQYFATPRTFGQGTKVEIK (SEQ ID NO: 1072) TCN—548 (5092301) Light chain Kabat CDRS: CDR 1: KSSQSVLYSSNNKNYLA (SEQ ID NO: 1073) CDR 2; WASTRES (SEQ ID NO: 957) CDR 3: QQYFATPRT (SEQ ID NO: 1074) TCN-548 P01) Light chain Chothia CDRS: CDR 1: KSSQSVLYSSNNKNYLA (SEQ ID NO: 1073) CDR 2: WASTRES (SEQ ID NO: 957) CDR 3: QQYFATPRT (SEQ ID NO: 1074) TCN-549 (5092*P04) heavy chain variable region tide sequence: CAGGTACAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAACCCTAACAGTGGTGACACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC ATCACCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATTCC CCCTATAGCAGCAGCTGGTCCTTCTTTGACTACTGGGGCCAGGGACCCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1075) 9 (5092_P04) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGDTNYAQKF QGRVTMTRDTSIT’I‘AYMELSSLRSDDTAVYYCARDSPYSSSWSFFDYWGQGPLVTVSS (SEQ ID NO: 1076) TCN—549 (5092_P04) gamma heavy chain Kabat CDRs: CDR l: GYYMH (SEQ ID NO: 1077) CDR 2: WINPNSGDTNYAQKFQG (SEQ ID NO:.::1078) CDR 3: DSPYSSSWSFFDY (SEQ ID NO: 1079) ~ TCN-549 (5092_P04) gamma heavy chain Chothia CDRs: CDR 1: GYTFTG (SEQ ID NO: 1080) ‘ CDR 2: WINPNSGDTN (SEQ ID NO: 1081) CDR 3: DSPYSSSWSFFDY (SEQ ID NO: 1079) TCN-549 (5092_P04) light chain variable region nucleotide ce: GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCC AGCCAGAGTGTTTTATACAGCTCCAACAATAAGAGCCACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA GATTTCACCCTCATCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAATATTATTTTTCT CCCCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 1082) TCN-549 (5092_P04) light chain variable region amino acid ce (Kabat CDRS in bold, Chothia CDRs underlined) DIVMTQSPDSLAVSLGERATINCKSS!)SVLYSSNNKSHLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDEFLIISSLQAEDVAVYYC!QQYYFSPLTFGGGTKVEIK (SEQ ID NO: 1083) TCN-549 (5092_P04) Light chain Kabat CDRS: CDR 1: KSSQSVLYSSNNKSHLA (SEQ ID NO: 1084) CDR 2: WASTRES (SEQ ID NO:' 957) CDR 3: QQYYFSPLT (SEQ ID NO: 1085) TCN—549 (5092_P04) Light chain Chothia CDRs: CDR SVLYSSNNKSHLA (SEQ ID NO 1084) CDR 2. WASTRES (SEQ ID NO: 957) CDR 3: QQYYFSPLT (SEQ ID NO: 1085) TCDLSSO(5096_F06)heavychan1vanabbiegRNinudeofidesequenca CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGCCTCCATCAATAGTCACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT GTCTAPTACAGTGGGAGCACCACCTACAACCCCTCCCTCAAGAGTCGAGTCACCTTATCAGTAGATACGTCCAAG AACCAGTTCTCCCTGAACCTGAGCTCTGTGACCGCCGCAGACACGGCCTTCTATTACTGTGCGAGACATCCCTAC GATGTTTTGACTGGTTCCGGGGACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1086) TCN-SSO (5096_F06) gamma heavy chain le region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLQESGPGLVKPSETLSLTCTVSGASINSHYWSWIRQPPGKGLEWIGYVYYSGSTTYNPSLKSRVT LSVDTSKNQFSLNLSSVTAADTAFYYCARHPYDVLTGSGDWFDPWGQGTLVTVSS (SEQ ID NO: 1087) : TON-550 (5096_F06) gamma heavy chain Kabat CDRs: CDR 1: 51-1sz (SEQ ID NO: 1088) CDR 2: YVYYSGSTTYNPSLKS (SEQ ID NO: 1089) CDR 3: HPYDVLTGSGDWFDP (SEQ ID NO: 1090) TCN—550 (5096_F06) gamma heavy chain Chothia CDRs: CDR 1: GASINSH (SEQ ID NO: 1091) CDR 2: YVYYSGSTT (SEQ ID NO: 1092) CDR 3: HPYDVLTGSGDWFDP (SEQ ID NO: 1090) TCN-SSO (5096_F06) light chain variable-region nucleotide ce: GTTCTGACTCAGGCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAAT GGAAGTAAAAAAGTTCACTGGTACCAGCACAAGGCAGGCCAGGCCCCTGTACTCGTCGTCTATGATGAT ACAGACCGGCCCTCAGGGATCCCTGAGcGATTCTcTGGCTCCAACTcTTGGAGCACGGCCACCCTGACCATCAAC AGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATTTTACCATTGATCATGTGGTCTTCGGC GGAGGGACCAAGCTGACCGTTCTA (SEQ ID NO: 1093) TCN-SSO (5096_F06) light chain variable region amino acid ce (Kabat CDRs in bold, Chothia CDRs underlined) SYVLTQAPSVSVAPGQTARITCGGNAIGSKKVHWYQHKAGQAPVLVVYDDTDRPSGIPERFSGSNSW STATLTINRVEAGDEADYYCSQVWDFTIDHVVFGGGTKLTVL (SEQ ID NO: 1094) TCN-550 (5096_F06) Light chain Kabat CDRs: CDR l: GGNAIGSKKVH (SEQ ID NO: 1095) CDR 2: DDTDRPS (SEQ ID NO: 1096) CDR 3: QVWDFTIDHVV (SEQ ID NO: 1097) TCN—SSO (5096_F06) Light Chain Chothia CDRs: CDR l: GGNAIGSKKVH (SEQ ID NO: 1095) CDR 2: DDTDRPS (SEQ ID NO: 1096) CDR 3: QVWDFTIDHVV (SEQ ID NO: 1097) 14'}- TCN-551 D01) heavy chain variable region nucleotide sequence: GAGGTGCAACTGGTTCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCT GGCTACAGCTTTAGCAACTACTGGATCGGCTGGGTGCGCCACATGCCCGGGAAAGGCCTGGAATGGATGGGGATC ATTTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATGTCAGCCGACAAGTCC AGCAGCACCGTCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATTTATTATTGTGCGAGACGGGGC GGACATAGTTTTGGATATGGGTCGGGGGGGGACACGCACAGTGAATTCGACTCCTGGGGCCAGGGAACCCTGGTC ACCGTCTCGAGC (SEQ ID NO: 1098) TCN—551 (5243_D01) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRS underlined)ii}* SGSEVKKPGESLKISCKGSGYSFSNYWIGWVRHMPGKGLEWMGIIYPGDSDTRYSPSFQGQ VTMSADKSSSTVYLQWSSLKASDTAIYYCARRGGHSFGYGSGGDTHSEFDSWGQGTLVTVSS (SEQ ID NO: 1099) TCN-551 (5243_D01) gamma heavy chain Kabat CDRS: CDR 1: NYWIG (SEQ ID NO: 1100) CDR 2:11YPGDSDTRYSPSFQG (SEQ ID NO: 1 101) CDR 3: GYGSGGDTHSEFDS (SEQ ID NO: 1102) l (5243_D01) gamma heavy chain Chothia CDRS: CDR 1: GYSFSN (SEQ ID NO: 1103) CDR 2: IIYPGDSDTR (SEQ ID NO: 1 104) CDR 3: RGGHSFGYGSGGDTHSEFDS (SEQ ID NO: 1102) TCN—SSl (5243_D01) light chain le region nucleotide sequence: CAGTCTGTATTGACGCAGTCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGC GACTCCAACATTGGTGATTATTTTGTATGTTGGTACCAGCACCTCCCAGGAAAACCCCCCCAACTCCTCATCTAT GAAAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGC ATCACCGGAATCCAGACCGGGGACGAGGCCGATTACTACTGCGCAACTTGGGATGGCAGCCTGAGTGCT'I‘GGGTG TTCGGCGGAGGGACCAAGCTGACCGTTCTA (SEQ ID NO; 1105) TCN-551 (5243_D01) light chain variableZK—region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) QSVLTQSPSVSAAPGQKVTISCSGSDSNIGDYFVCWYQHNLPGKPPQLLIYENNKRPSGIPDRFSGSKSGT SATLGITGIQTGDEADYYCATWDGSLSAWVFGGGTKLTVL (SEQ ID NO: 1 106) TCN—551 (5243_D01) Light chain Kabat CDRS: CDR 1: SGSDSNIGDYFVC (SEQ ID NO: 1 107) CDR 2: ENNKRPS (SEQ ID NO: 1 108) CDR 3: ATWDGSLSAWV (SEQ ID NO: 1109) TCN—551 (5243_D01) Light chain Chothia CDRs: CDR l: SGSDSNIGDYFVC (SEQ ID NO: 1 107) CDR 2: ENNKRPS (SEQ ID NO: 1108) CDR 3: ATWDGSLSAWV (SEQ ID NO: 1109) TCN—552 (5249_123) heavy chain variable region nucleotide sequence: CAGGTCCAAGTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAGGGTCTCCTGCCAGGCTTCT ACCTTCATGAATTATGCTATCATTTGGGTGCGACGGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG ATCATCCCTGTCTTTCCTACACCAAACTACGCACAGATGTTCCAGGGCAGAGTCACGATTTCCACGGACGAATCC AGGAGCACA'I‘CC'ITCTTGGAACTGACCAACCTGAGATATGAGGACACGGCCGTTTATTACTGTGCGAGGCGAATT TATCACGGTGGTAACTCCGGCTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1 110) .
TCN-552 (5249_123) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) . QVQVVQSGAEVKKPGSSVRVSCQASGGTFMNYAIIWVRRAPGQGLEWMGGIIPVFP’I‘PNYAQMFQG RVTlSTDESRSTSFLELTNLRYEDTAVYYCARR]YHGGNSGFDFWGQGTLVTVSS (SEQ ID NO: 111 l) TCN-552 (5249_123) gamma heavy chain‘Kabat CDRS: CDR 1: NYAII (SEQ ID NO: 1112) ' CDR 2: GIIPVFPTPNYAQMFQG (SEQ ID NO:-'1 1 13) CDR 3: RIYHGGNSGFDF (SEQ ID NO: ' 11 14) TCN-552 (5249_123) gamma heavy chain Chothia CDRS: CDR 1: GGTFMN (SEQ ID NO: 1115) CDR 2: GIIPVFPTPN (SEQ ID NO: 11 16) CDR 3: RIYHGGNSGFDF (SEQ ID NO: 11 14) TCN-552 (5249_I23) light chain le region nucleotide ce: GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTGGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT AACAGGGCCCCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGCCTCGCGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAAGTGGCCTCCCATGTACAGTTTT GGCCATGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 1117) TCN—552 (5249_123) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRs underlined) EIVLTQSPATLSLSPGERATLSCRASS ESVGNYLAWYQQKPGQAPRLLIYDSSNRAPGlPARFSGSGSGT SSLAPEDFAVYYCSQQRSKWPPMYSFGHGTKLEIK (SEQ ID NO: I 1 18) TCN-552 (5249_123) Light chain Kabat CDRS: CDR 1: RASQSVGNYLA (SEQ ID NO: 1119) CDR 2: DSSNRAP (SEQ ID NO: 1120) CDR 3: QQRSKWPPMYS (SEQ ID NO: 1121) ' TCN-552 (5249-123) Light chain Chothia CDRS: CDR 1: RASQSVGNYLA (SEQ ID NO: 1119) CDR 2: DSSNRAP (SEQ ID NO: 1120) CDR 3: QQRSKWPPMYS (SEQ ID NO: 1121) TCN-553 (5261_C18) heavy chain variable region nucleotide sequence: CAGGTCCAGGTGGTGCAGTCTGGGACTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCCAGACTTCT GGAGGCAGGTTCATGAGTTATGCTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC ATCGTCCCTGTCTTCGGAACAGCAAACTACGCTCAGAAGTTCCAGGGCAGAGTCACGATCACCACGGACGATTCC ACGCGCACAGCCTATATGGAGTTGAGCAGCCTGAGAAGTGAGGACACGGCCGTTTATTACTGTGGGTTCCGATAC GGCTCTGGTTACGGGTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1122) TCN-553 C18) gamma heavy chain le region amino acid sequence: (Kabat CDRs in bold, Chothia CDRS underlined) QVQVVQSGTEVKKPGSSVKVSCQTSGGRFMSYAITWVRQAPGQGLEWMGGIVPVFGTANYAQKFQ GRVTITTDDSTRTAYMELSSLRSEDTAVYYCGFRYGSGYGFDSWGQGTLVTVSS (SEQ ID NO: 1 123) TCN-553 (5261_C18) gamma heavy chain Kabat CDRS: CDR 1: SYAIT (SEQ ID NO: 1124) - CDR 2: GIVPVFGTANYAQKFQG (SEQ ID NO: 1125) CDR 3: RYGSGYGFDS (SEQ ID NO: 1126) TCN—553 (5261_C18) gamma heavy chain a CDRS: CDR 1: GGRFMS (SEQ ID NO: 1127) ‘ CDR 2: GIVPVFGTAN (SEQ ID NO: 1 128) CDR 3: GFDS (SEQ ID NO: 1126) TCN-553 (5261_C18) light chain variable region nucleotide sequence: GAAATTGTATTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTAGTAGCAGCTACTTAGCCTGGTATCAGAAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCTTCCACTAGGGCCACTGGCATCCCGGACCGGTTCACTGGCAGTGGGTCTGGGACAGACTTCACTCTCAGC ATCAGTAGACTGGAGCCTGAAGA’I‘TTTGCAGTGTATTACTGTCAGCACTTTGGTACCTCAGTCTTCACTTTCGGC GGAGGGACCAAGGTTGAGATCAAA (SEQ ID NO: 1129) TCN—553 (5261_C18) light chain variable region amino acid sequence (Kabat CDRS in bold, Chothia CDRS underlined) EIV-LTQSPGTLSLSPGERATLSCRASS[SVSSSYLAWYQKKPGQAPRLLIYGASTRATGlPDRFl‘GSGSGT DFTLSISRLEPEDFAVYYCS [HFGTSVFTFGGGTKVEIK (SEQ ID NO: 1 130) TCN-553 (5261_C18) Light chain Kabat CDRs: CDR 1: SSSYLA (SEQ ID NO: 944) CDR 2: GASTRAT (SEQ ID NO: 755) CDR 3: QHFGTSVFT (SEQ ID NO: 1131) TCN—553 (5261_C18) Light chain Chothia CDRS: CDR 1: RASQSVSSSYLA (SEQ ID NO: 944) CDR 2: GASTRAT (SEQ ID NO: 755) CDR 3: QHFGTSVFT (SEQ ID NO: 1131) TCN-554 (5277_M05) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTGCAGTCTGGGGCTGATCTGAAGAAGCCfIGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGATACACCTTCACCGACTACTATATTCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAACCCTGAAAGTGGTGACACAAAGTATGCACAGAAGTTICAGGGCAGGGTCACCATGACCAGGGACACGTCC ACAGCCTACATGGAGCTGGGTAGGCTGAGATCCGACGACACGGCCGTGTATTACTGTGCGAGAGATGTA AGTACGACCTGGAGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1132) TCN—554 (5277_M05) gamma heavy chain variable region amino acid ce: (Kabat CDRS in bold, Chothia CDRS underlined) QVQLVQSGADLKKPGASVKVSCKASGYTFI‘DYYIHWVRQAPGQGLEWMGWINPESGDTKYAQKFQ GRVTMTRDTSITTAYMELGRLRSDDTAVYYCARDVS'I'I‘WSWFAPWGQGTLVTVSS (SEQ ID NO: 1 133) TCN-554 (5277_M05) gamma heavy chain Kabat CDRs: CDR l: DYYIH (SEQ ID NO: 1134) 'CDR 2: WINPESGDTKYAQKFQG (SEQ ID NO: 1 135) CDR 3: DVSTTWSWFAP (SEQ ID NO: 1 136) 4 (5277_M05) gamma heavy chain Chothia CDRs: CDR 1: GYTFTD (SEQ ID NO: 1137) CDR 2: GDTK (SEQ ID NO: 1138) CDR 3: DVSTTWSWFAP (SEQ ID NO: 1 136) TCN-554 (5277_M05) light chain le region nucleotide sequence: GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCAGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGGTCC AGCCAGAGTATTTTCCACAACTCCAACAATGAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA GATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCGGTTTATTTCTGTCAGCAATATTATAATGCT CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 1139) TCN-554 (5277_M05) light chain variable region amino acid ce (Kabat CDRs in bold, Chothia CDRs underlined) DIVMTQSPDSLAVSLGERATINCRSSQQSIFHNSNNENYLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFI‘LTISSLQAEDVAVYFC! 2! QYYNAPLTFGGGTKVEIK (SEQ ID NO: 1140) TCN—554 (5277M05) Light chain Kabat CDRs: CDR 1: RSSQSIFHNSNNENYLA (SEQ ID NO: 1 141) CDR 2: WASTRES (SEQ ID NO: 957) ‘ CDR 3: QQYYNAPLT (SEQ ID NO: 1 142) TCN-554 (5277_M05) Light chain Chothia CDRS: CDR 1: RSSQSIFHNSNNENYLA (SEQ ID NO: 1141) CDR 2: WASTRES (SEQ ID NO: 957) CDR 3: QQYYNAPLT (SEQ ID NO: 1142) TCN-555 (5246_L16) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTCGGTGAAGGTCTCATGCACGGCTTCT GGAGGCATCTTCAGGAAGAATGCAATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG ATCATCGCAGTCTTTAACACAGCAAATTACGCGCAGAAGTTCCAGAACAGAGTCAAAATTACCGCAGACGAGTCA GGCAATACGGCCTACATGGAGCTGAGCAGCCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGTCACCCA AAATATTTCTATGGTTCGGGGAGTTATCCGGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1143) 5 (5246_L16) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLVQSGAEVKRPGSSVKVSCTASGGIFRKNAISWVRQAPGQGLEWMGGIIAVFNTANYAQKFQN RVKITADESGNTAYMELSSLTSDDTAVYYCASHPKYFYGSGSYPDFWGQGTLVTVSS (SEQ ID NO: 1 I44) TCN-555 (5246_L16) gamma heavy chain Kabat CDRs: CDR 1: KNAIS (SEQ ID NO: 796) CDR 2: GIIAVFNTANYAQKFQN (SEQ ID NO: 797) CDR 3: HPKYFYGSGSYPDF (SEQ ID NO: 798) TCN-555 (5246_L16) gamma heavy chain Chothia CDRS: CDR 1: GGIFRK (SEQ ID NO: 799) CDR 2: GIIAVFNTAN (SEQ ID NO: 800) CDR 3: HPKYFYGSGSYPDF (SEQ ID NO: 798) ' 5 (5246_Ll6) light chain le region nucleotide sequence: GCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAATCACCATCTCCTGTACTGGTGGC AGCAGTGATATTGGTGCTTCTAACTCTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCGTTATT TTTGATGTCACTGAGCGACCCTCAGGGGTCCCGCATCGGTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACCGTCTCTGGGCTCCAGCCTGACGACGAGGCTGATTATTTCTGCTGCGCATATGGAGGCAAATATCTTGTGGTC TTCGGCGGAGGGACCAAGGTGACCGTTCTA (SEQ ID NO: 1145) TCN—555 (5246_L16) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) PRSVSGSPGQSITISCTGGSSDIGASNSVSWYQQHPGKAPKLVIFDVTERPSGVPHRFSGSKSG NTASLTVSGLQPDDEADYFCCAYGGKYLVVFGGGTKVTVL (SEQ ID NO: 1 146) TCN-555 (5246_L16) Light chain Kabat CDRS: CDR l: TGGSSDIGASNSVS (SEQ ID NO: 1147) CDR 2: DVTERPS (SEQ ID NO: 804) CDR 3: CAYGGKYLVV (SEQ ID NO: 805) 5 L16) Light Chain Chothia CDRS: CDR l: TGGSSDIGASNSVS (SEQ ID NO: 1 147) CDR 2. DVTERPS (SEQ ID NO: 804) CDR 3. CAYGGKYLVV (SEQ ID NO: 805) TCN-556 (5089_K12) heavy chain variable region nucleotide sequence: CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAACCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGATACACCTTCATCGGCTATGATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAACGCTAAAAGAGGTGGCACAAACTATGCACAAAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCT ATCAGCACAGCCTACATGGAGCTGAACAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGGGGTG GGGTCACGAACTACGATTTTTGGAGTTCTCAACCCGGAATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCGAGC (SEQ ID NO: 1148) TCN-556 (5089_K12) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRs underlined) QVQLVQSGAEVKKPGASVKVSCKAS_G—YTFIGYDMHWVRQAPGQGLEWMGWINAKRGGTNYAQKF QGRVTMTRDTSISTAYMELNSLRSDDTAVYYCARGVGSRTTIFGVLNPEFDYWGQGTLVTVSS (SEQ ID NO: 1149) TCN—556 (5089_K12) gamma heavy chain Kabat CDRS: CDR 1: GYDMH (SEQ ID NO: 1150) CDR 2: WINAKRGGTNYAQKFQG (SEQ ID NO: 1 151) CDR 3: GVGSRTTIFGVLNPEFDY (SEQ ID NO: 1152) TCN-556 (5089_K12) gamma heavy chain Chothia CDRS: CDR 1: GYTFIG (SEQ ID NO: 1153) CDR 2: WINAKRGGTN (SEQ ID NO: 1 154) .~ CDR 3: GVGSRTTIFGVLNPEFDY (SEQ ID NC: 1,152) TCN-556 (5089_K12) light chain variable region tide sequence: GCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGATCC AGCAGTGACGTTGGTGGTTATGACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCCTGATT TATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCGGGCAACTACAATCATGTC TTCGGACCTGGGACCAAGGTCACCGTTCTA (SEQ ID NO: 1155) TCN-556 (5089_K12) light chain variable region amino acid sequence (Kabat CDRs in bold, Chothia CDRs underlined) QSALTQPPSASGSPGQSVTISCTCSSSDVGGYDYVSWNQQHPGKAPKLLIYEVTKRPSGVPDRFSGSKS GNTASLTVSGLQAEDEADYYCSSYAGNYNHVFGPGTKVTVL (SEQ ID NO: 1 156) TCN-556 (5089_K12) Light chain Kabat CDRs: CDR 1: TGSSSDVGGYDYVS (SEQ ID NO: 1157) CDR 2: S (SEQ ID NO: 1158) CDR 3: SSYAGNYNHV (SEQ ID NO: 1 159) TCN-556 (5089_K12) Light chain Chothia CDRS: CDR l: VGGYDYVS (SEQ ID NO: 1157) CDR 2: EVTKRPS (SEQ ID NO: 1158) CDR 3: YNHV (SEQ ID NO: 1159) TCN-557 (5081_A04) heavy chain variable region nucleotide sequence: CAGGTGCAGC'IGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGACACACCTTCACCGGCTACTACATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAACCCTGACAGTGGTGCCACCAGTTCTGCACAGAACTTTCAGGGCAGGGTCACCATGACCGGGGACACGTCC TCTAGCACAGCCTACATGGAGCTGAGTAGGCTGAGTTTTGACGACACGGCCGTCTATTACTGTGCGAGAGTACTG TTTACCAGTCCTTTTGACTTCTGGGGTGAGGGAACCCTGGTCACCGTCTCGAGC (SEQ ID NO: 1160) TCN-557 (5081_A04) gamma heavy chain7Variable region amino acid ce: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLVQSGAEVKKPGASVKVSCKASGHTFI‘GYYIHWVRQAPGQGLEWMGWINPDSGATSSAQNFQ GRVTMTGDTSSSTAYMELSRLSFDDTAVYYCARVLFTSPFDFWGEGTLVTVSS (SEQ ID NO: 1 161) TCN-557 (5081_A04) gamma heavy chain Kabat CDRs: CDR 1: GYYIH (SEQ ID NO: 1162) CDR 2: WINPDSGATSSAQNFQG (SEQ ID NO: 1 163) 'CDR 3: VLFTSPFDF (SEQ ID NO: 1164) TCN-557 (5081_A04) gamma heavy chain Chothia CDRs: CDR 1: GHTFI‘G (SEQ ID NO: 1165) CDR 2: WINPDSGATS (SEQ ID NO: 1 166) CDR 3: VLFTSPFDF (SEQ ID NO: 1164) TCN-557 (5081_A04) light chain variable region nucleotide sequence: CAGGCTGTGGTGACTCAGGAGCCCTCACTGGCTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGC ACTGGAGCTGTCACCAGGGGTCATTATCCCTATTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGGCACTCATT TATGATAGTGCAGGCAACAGACACTCCTGGACTCCCGCCCGATTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCC CTGACCCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGTCTGGGTG TTCGGCGGAGGGACGAAGCTGACCGTTCTA (SEQ ID NO: 1167) TCN-557 (5081_A04) light chain variable region amino acid sequence (Kabat CDRs in bold, a CDRs underlined) QAVVTQEPSLAVSPGGTVTLTCGSSTGAVTRGHYPYWFQQKPGQAPRALIYDSAGNRHSWTPARFSG SLLGGKAALTLSGAQPEDEAEYYCLLSYSGVWVFGGGTKLTVL (SEQ ID NO: 1 168) 'TCN-557 (5081_A04) Light chain Kabat CDRs: CDR 1: GSSTGAVTRGHYPY (SEQ ID NO: 1169) CDR 2: HS (SEQ ID NO: 1170) CDR 3: LLSYSGVWV (SEQ ID NO: 1171) TCN-557 (5081_A04) Light chain Chothia CDRsf CDR l: GSSTGAVTRGHYPY (SEQ ID NO: 1169) CDR 2: DSAGNRHS (SEQ ID NO: 1 170) CDR 3: LLSYSGVWV (SEQ ID NO: 1171) TCN-SSS (5248_H10b) heavy chain le region nucleotide sequence: CAGGTCCAGCTGGTGCAATCTGGGAGTGAGGTGAAGAAGCCTGGGACCTCGGTGAAGGTCTCCTGCACGGCCTCT GGAAGTGTCTTCACCAATTATGGAATTAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTCTCTTTGGCGCAGCCAAGTACGCACAGAAATTCCAGGGCAGAGTCACCATCACAGCGGACGAATCC AcGAAGACAGTcTACATGGAGCTGAGCAGGCTGACATCTAAAGACACGGCCATATATTTCTGTGCGAAGGCCCCC CGTGTCTACGAGTACTACTTT‘GATCAGTGGGGCCAGGGAACCCCAGTCACCGTCTCCTCA (SEQ ID NO: 1172) TCN-558 (5248_H10b) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRs underlined) QVQLVQSGSEVKKPGTSVKVSCTASGSVFTNYGISWVRQAPGQGLEWMGGIIPLFGAAKYAQKFQG RVTITADESTKTVYMELSRLTSKDTAIYFCAKAPRVYEYYFDS[WGQGTPVTVSS (SEQ ID NO: 914) 8 (5248"H10b) gamma heavy chain Kabat CDRs: CDR l: NYGIS (SEQ ID NO: 915) CDR 2: GIIPLFGAAKYAQKFQG (SEQ ID NO: 916) CDR 3: APRVYEYYFDQ (SEQ ID NO: 917) TCN-SSS (5248_H10b) gamma heavy chain‘3Chothia CDRs: CDR 1: GSVFTN (SEQ ID NO: 918) CDR 2: GIIPLFGAAK (SEQ ID NO: 919) .
CDR 3: APRVYEYYFDQ (SEQ ID NO: 917) TCN-558 H10b) light Chain le region nucleotide sequence: GAAATAGTGATGACGCAGTTTCCAGCCACCCTGTCTGTGTCTCCCGGGGAACGAGTCACCCTCTCCTGTAGGGCC AGTCAGAGTGTTAGCAACAATTTAGCCTGGTACCAGCAAAAACCTGGCCAGCCTCCCAGGCTCCTCATCTATGAT GCATCTACCAGGGCCACGGGTGTCCCAGCCAAGTTCAGTGGCACTGGGTCTGGCACAGAGTTCACTCTCAGCATC AGCAGCCTGCAGTCCGAAGATTTTGCAGTTTATTACTGTCAGCAGTATCACAACTGGCCTCCCTCGTACAGTTTT GGCCTGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 1173) TCN-558 (5248_H10b) light chain variable region amino acid sequence (Kabat CDRS in bold, a CDRS underlined) ElVMTQFPATLSVSPGERVTLSCRAS!QSVSNNLAWYQQKPGQPPRLLIYDASTRATGVPAKFSGTGSGT EFTLSISSLQSEDFAVYYCSzflYHNWPPSYSFGLGTKLEIK (SEQ ID NO: 862) TCN-558 (5248_H10b) Light chain Kabat CDRS: CDR l: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT (SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865) TCN-558 H10b) Light chain Chothia CDRS: CDR 1: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT (SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865) TCN-559 (5097_008) heavy chain variable region nucleotide ce: CAAGAGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT TCCTTCATTGGCTACTATGTACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAGCCCTGACAGTGATGCCACAAAGTACGCACAGAAGTTTCAGGGCTCCGTCATCATGACCAGGGACACGTCC GTCAGCACAGTGTACATGGAGCTGAGTAGGCTGACATCTGAC‘GACACGGCccTTTATTACTGTCTCCTTTTTCGA GGTGGAAACTCCCTCTCCIGGGGCCAGGGAACCCTGGTCACQGTCTCGAGC (SEQ ID NO: 1174) TCN-559 (5097_GO8) gamma heavy chain variable region amino acid sequence: (Kabat CDRs in bold, Chothia CDRS underlined) QEQLVQSGAEVKKPGASVKVSCKASRKSFIGYYVHWVRQAPGQGLEWMGWISPDSDATKYAQKFQ GSVIMTRDTSVSTVYMELSRLTSDDTALYYCLLFRGGNSLSWGQGTLVTVSS (SEQ ID NO: 1 175) TCN-559 (5097_008) gamma heavy chain Kabat CDRS: CDR 1: GYYVH (SEQ ID NO: 1176) CDR 2: WISPDSDATKYAQKFQG (SEQ ID NO: 1 177) CDR 3: FRGGNSLS (SEQ ID NO: 1178) .
TCN-559 (5097~G08) gamma heavy chain Chothia CDRS: CDR 1: RKSFIG (SEQ ID NO: 1179) CDR 2: WISPDSDATK (SEQ ID NO: 1180) CDR 3: FRGGNSLS (SEQ ID NO: 1178) TCN-559 (5097_G08) light chain variable region tide sequence: , CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACCCTCACCTGTGGCTCCAGC ACTGGACCTGTCACCAGTGGTCATTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACATTGATT TCTGCTACATCCAACACACACTCCTGGACACCTGCCCGCTTCTCAGGCTCCCTCCTTGGGGGCAGAGCTGCCCTG ACCCT'l‘TCGGGTGCGCAGCCTGAGGATGAGGCTGACTATTAITGCTI‘TCTCTCCTACAGTGGTGCTTGGGTGTTC GGCGGAGGGACCACGCTGACCGTTCTA (SEQ ID No- '1181) TCN-559 (5097_GO8) light chain variable region amino acid ce (Kabat CDRS in bold, Chothia CDRS underlined) - QAVVTQEPSLTVSPGGTVTLTCGSSTGPVTSGHYPYWFQQKPGQAPRTLISATSNTHSWTPARFSGSL LGGRAALTLSGAQPEDEADYYCFLSYSGAWVFGGGT’I‘LTVL (SEQ ID NO: 1 182) TCN-559 (5097_GO8) Light chain Kabat CDRS: CDR 1: GSSTGPVTSGHYPY (SEQ ID NO: 1183) CDR 2: ATSNTHS (SEQ ID NO: 1184) CDR 3: FLSYSGAWV (SEQ ID NO: 1185) TCN-559 (5097_G08) Light chain Chothia CDRS: CDR 1: GSSTGPVTSGHYPY (SEQ ID NO: 1183) CDR 2: ATSNTHS (SEQ ID NO: 1184) ‘ CDR 3: FLSYSGAWV (SEQ ID NO: 1185) 1TCN-56O(5084_P10)heavychah1vafiabbiegkninudeohdcsequence GAGGTGCAGCTGGTGGAATCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTTATCTTTAGAAATTACTGGATGAGCTGGGTCCGGCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC ATAAAACAAGATGGAAGAGAGAAGTACTATGTGGACTCTCTGAGGGGCCGAGTCAACATCTCCAGAGACAACGCC GAGAACTCATTGTATCTGCACATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTTCTGTGCGAGAGCTCGG ATGGTGGTGGTTACTGGCGATGGTTTTGATGTCTGGGGCCATGGGACAATGGTCACCGTCTCGAGC (SEQ ID NO: 1186) TCN-560 (5084_P10) gamma heavy chain variable region amino acid sequence: (Kabat CDRS in bold, Chothia CDRS underlined) EVQLVESGGGLVQPGGSLRLSCAASGFIFRNYWMSWVRQAPGKGLEWVANIK!zDGREKYYVDSLR GRVNISRDNAENSLYLHMNSLRVEDTAVYFCARARMVVVTGDGFDVWGHGTMVTVSS (SEQ ID NO: 1187) TCN-56O P10) gamma heavy chain Kabat CDRS: CDR 1: NYWMS (SEQ ID NO: 1188) CDR 2: REKYYVDSLRG (SEQ ID NO: 1189) ' CDR 3: ARMVVVTGDGFDV (SEQ ID NO: 1 190) TCN-560 (5084_P10) gamma heavy chain"Chothia CDRS: CDR 1: GFIFRN (SEQ ID NO: 1191) CDR 2: NIKQDGREKY (SEQ ID NO: 1192) CDR 3: ARMVVVTGDGFDV (SEQ ID NO: 1 190) TCN-56O (5084_P10) light chain variable region nucleotide sequence: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCA AGTCAGAATATTAAGAGGTATTTCAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT GCATCCAATTTAGAAAATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGTCTGCAACCTGAGGATTTTGCGACTTATTACTGTCAGCAGAGTTTCAGTAAATCGTGGACATTCGGCCAA GGGACCAACGTGGACATCAAA (SEQ ID NO: 1193) TCN-560 (5084_P10) light chain variable region amino acid sequence (Kabat CDRS in bold; a CDRS ined) SPSSLSASVGDRVTITCRASS[NIKRYFNWYQQKPGKAPKLLIYAASNLENGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQ! QSFSKSWTFGQGTNVDIK (SEQ ID NO: 1 194) TCN-560 (5084_P10) Light chain Kabat CDRS: CDR 1: RASQNIKRYFN (SEQ ID NO: 1195) CDR 2: AASNLEN (SEQ ID NO: 1196) CDR 3: QQSFSKSWT (SEQ ID NO: 1197) 170‘ ‘ TCN-560 (5084_P10) Light chain Chothia CDRs: CDR l: RASQNIKRYFN (SEQ ID NO: 1 195) CDR 2: N (SEQ ID NO: 1196) CDR 3: QQSFSKSWT (SEQ ID NO: 1 197) 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 CDR] and CDRZ, wherein the VH regioh is encoded by a human IGHVl (or specifically, IGHVl-l8, IGHVl-Z, IGHV1-69, IGHVl-S), IGHV2 (or specifically, IGHV2- ) , 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 IGHVS (or specifically, 51)VH germline sequence or an allele thereof, or a nucleic acid sequence that is homologous to the IGHVl, IGHV2, IGHV3, IGHV4, or IGHVS VH germline gene sequence or an allele thereof. In one aspect, the nucleic acid sequence that is homologous to the IGHVl, IGHV2, IGHV3, IGHV4, or IGHVS VH germline sequence is at least 75% homologous to the IGHVl, IGHV2, IGHV3, IGHV4, or IGHVS VH ne sequence or an allele f. Exemplary alleles include, but are not limited to, IGHVl- 18*01, 2*02, IGHV1-2*04, IGHV1-69*01, IGHVl-69*05, 69*O6, IGHVI- 69* 12, IGHV1-8*01, 5*10, IGHV33*01, IGHV3-30*O3, IGHV3—30*l8, 33*05, IGHV3-49*04, IGHV3-53*01, IGHV3-66*03, IGHV3-7*01, IGHV4-31*O3, IGHV4-31*06, IGHV4-34*01, IGHV4-34*02, IGHV4-34*03, IGHV4-34* 12, IGHV4— 39*01, IGHV4-59*Ol, IGHV4-59*O3, IGHV4-61*Ol, IGHV4-61*08, and 51*01.
Exemplary sequences for each allele are provided below.‘ IGHVl-18*Ol nucleotide sequence (SEQ IDNO: 1198) CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCC ACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA IGHVl-2*02 nucleotide sequence (SEQ ID NO: 1 199) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC ATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA 2*O4 nucleotide sequence (SEQ ID NO: 1200) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCTGGGTCACCATGACCAGGGACACGTCC ATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA IGHV1-69*01 nucleotide ce (SEQ ID NO: 1201) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCC ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA IGHVl-69*05 nucleotide sequence (SEQ ID NO: 1202) CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCACGGACGAATCC ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA IGHV1—69*O6 nucleotide sequence (SEQ ID NO: 1203) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG CCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCC ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA IGHV1-69*12 tide ce (SEQ ID NO:' 1204) CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGAC‘AGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCT1“TGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCC ACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA lGHVl-8*01 nucleotide sequence (SEQ ID NO: 1205) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGG ATGAACCCTAACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCC ATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGG IGHV2—5*10 nucleotide sequence (SEQ ID NO: 1206) ACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCT GGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT GCACTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACC TCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGG IGHV33*01 nucleotide sequence (SEQ ID NO: 1207) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT ATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCIGAGGACACGGCTGTGTATTACTGTGCGAGA IGHV3-30*03 nucleotide sequence (SEQ ID NO: 1208) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCT ACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT ATATCATATGATGGAAGTAATAAAIACTATGCAGACTccGgfiGAAGGGcCGATTCACCATCTCCAGAGACAATTCC 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: 121 l) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTCCTGTACAGCTTCT GGATTCACCTTTGGTGATTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTC ATTAGAAGCAAAGCTTATGGTGGGACAACAGAATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGAT GATTCCAAAAGCATCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACTAGA IGHV3-53*01 nucleotide sequence (SEQ ID NO: 1212) GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGGTTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT ATTTATAGCGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAG CTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA IGHV3-66*03 nucleotide sequence (SEQ ID NO: 1213) CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGTTACAGTTTCACCACCTATGGTATGAATTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG TTCAACACCTACACTGGGAACCCAACATATGCCCAGGGCTTCACAGGACGGTTTGTCTTCTCCATGGACACCTCT GCCAGCACAGCATACCTGCAGATCAGCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATA IGHV3-7*01 nucleotide ce (SEQ ID NO: 1214) CAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC AIAAAGCAAGATGGAAGTGAGAAATACTATGIGGACTCTGTGAAGGGCCGATTCACCAICTCCAGAGACAACGCC AAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGA 4—31*O3 nucleotide sequence (SEQ ID NO: 1215) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCC'I‘CACCTGCACTGTCTCT GGTGGCTCCATCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT GGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACG TCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGA 31*O6 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 tide sequence (SEQ ID NO: 1218) CAGGTGCAGCTACAACAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT GGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA ATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG AACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGA’CACGGCTGTG'I‘ATTACTGTGCGAGAGG 34*03 tide sequence (SEQ ID NO: 1219) CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT GGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA CATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG AACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTG IGHV4-34*l2 nucleotide sequence (SEQ IDNO: 1220) CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCG‘GAGACCCTGTCCCTCACCTGCGCTGTCTAT GGTGGGTccTTCAGTGGTTACTACTGGAGCTGGATCCGcCAGCCCCcAGGGAAGGGGCTGGAGTGGATTGGGGAA ATCATTCATAGTGGAAGCACCAACTACAACCCGTcccTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG AACCAGTTCTCCCTGAAGCTGAGCTCTGTGACcGCCGCGGACACGGCTGTGTATTACTGTGCGAGA IGHV4—39*01 nucleotide sequence (SEQ ID NO: 1221) CAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT TcCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATT GGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACG TCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACA IGHV4-59*01 nucleotide sequence (SEQ ID NO: 1222) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTAAAGACTGGAGGGGTCTCTGAGACTCTCCTGTGCAGCCTC TGGATTCACCTTCAGTAGCTCTGCTATGCACTGGGTCCACCAGGCTCCAGGAAAGGGTTTGGAGTGGGTCTCAGT TATTAGTACAAGTGGTGATACCGTACTCTACACAGACTCTGTGAAGGGCTGATTCACCATCTCTAGAGACAATGC CCAGAATTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGACGACATGGCTGTGTATTACTGTGTGAAAGA IGHV4-59*03 nucleotide sequence (SEQ ID NO: 1223) CAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT ATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG AACCAATTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCG IGHV4—61*01 nucleotide sequence (SEQ ID NO:1224) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCGTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCGTCAGCAGTGGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATT GGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTC;CcTCAAGAGTCGAGTCACCATATCAGTAGACACG TCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGA IGHV4-6l*08 nucleotide ce (SEQ ID NO: 1225) CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCT GGTTACAGTTTCACCACCTATGGTATGAATTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG TTCAACACCTACACTGGGAACCCAACATATGCCCAGGGCTTCACAGGACGGTTTGTCTTCTCCATGGACACCTCT GCCAGCACAGCATACCTGCAGATCAGCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATA IGHV5-51*01 nucleotide sequence (SEQ ID NO: 1226) GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGITCT GGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATC ATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCC ATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACA In certain embodiments of the invention, the antibody further includes a variable light chain (VL) region encoded by a human IGKVl (or specifically, IGKVl-l7, IGKVl-27, IGKV1-39, IGKVlD-39, IGKVl—S), IGKV2 (or specifically, IGKV2-30), IGKV3 (or specifically, IGKV3-l 1, IGKV3-15, IGKV3-20), IGKV4 (or ically, IGKV4-l, IGKV4- 1), IGLV] (or specifically, IGLVl-40, IGLV1-44, IpLVl-SS), IGLV2 (or specifically, :1). \‘cl‘. 174' “,1, 2012/024971 IGLV2-11, l4, 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 sequencelGKVl, IGKVZ, IGKV3, IGKV4, IGLVl, IGLV2, IGLV3, IGLV7, or IGLV9 or an allele thereof, or a nucleotide acid ce that is homologous to the IGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLV2, IGLV3, IGLV7, or IGLV9 VL gennline gene sequence or an allele thereof. Furthermore, the nucleic acid sequence that is homologous to the IGKVl, IGKV2, IGKV3, IGKV4, IGLVI, IGLV2, IGLV3, IGLV7, or IGLV9 VL gennline sequence'oran allele thereof is at least 65% gous to the IGKVl, IGKV2, IGKV3, IGKV4, IGLVI, IGLV2, IGLV3, IGLV7, or IGLV9 VL germline sequence or an allele thereof.
IGKV1-17*O1 nucleotide sequence (SEQ ID NO: 1227) GACATCCAGATGACccAGTCTCCATCCTCCCTGTcTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGcA AGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCT GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATC AGCAGCCTGCAGCCTGAAGATTTTGCAACT'I‘ATTACTGTCTACAGCATAATAGTTACCCTCC 27*01 nucleotide sequence (SEQ ID NO: 1228) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCG AGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCT GCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCC IGKVl-39*01 tide sequence (SEQ ID NO: 1229) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA AGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGTC'I‘GCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCC IGKVlD-_39*Ol nucleotide ce (SEQ ID NO: 1230) GACATCCAGATGACCCAGTCTCCATCCTCCC'IGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA AGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGIACCCCTCC IGKV1-5*O3 nucleotide sequence (SEQ ID NO: 1231) GACATCCAGATGACCCAGICTCCTTCCACCCTGTCTGCATCTéTAGGAGACAGAGICACCATCACTTGCCGGGCC AGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAG GCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGA'I‘CTGGGACAGAATTCACTCTCACCATC AGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTCC IGKV2—30*02 nucleotide sequence (SEQ ID NO: 1232) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCT AGTCAAAGCCTCGTACACAGTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGG CGCCTAATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGAT TTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCT IGKV3-1 1*01 nucleotide sequence (SEQ ID NO: 1233) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCC IGKV3—15*01 nucleotide sequence (SEQID NO: 1234) GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT GCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATC AGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCC 20*01 nucleotide sequence (SEQ ID NO: 1235) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCC IGKV4-l*01 nucleotide sequence (SEQ ID NO: 1236) GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCICTGGGCGAGAGGGCCACCATCAACTGCAAGTCC AGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTAcT'T'AGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACA ACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACT CCTCC IGLV1—40*01 tide sequence (SEQ ID NO: 1237) CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGC AGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATC TATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTG GCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTTC IGLV1-44*01 nucleotide sequence (SEQ ID NO: 1238) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGC AGCTCCAACATCGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTCC IGLVl-51*02 nucleotide sequence (SEQ ID NO: 1239) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGC AGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTAT AATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGC ATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGAGTGCTGG IGLV2—11*01 nucleotide sequence (SEQ ID NO: 1240) CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACC AGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT TATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTTC IGLV2-14*01 nucleotide ce (SEQ ID NO: 1241) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACC AGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT TATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACTCTC IGLV2-8*Ol nucleotide sequence (SEQ ID NO: 1242) ‘ CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACC AGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACC5ACAGCACCCAGGCAAAGCCCCCAAACTCATGATT GTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGGCAGCAACAATTTC IGLV3-2l *02 nucleotide sequence (SEQ ID NO: 1243) TCCTATGAGCTGACACAGCTACCCTCGGTGTCAGTGTCCCCAGGACAGACAGCCAGGATCACCTGCTCTGGAGAT GTACTGGGGGAAAATTATGCTGACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGAGTTGGTGATATACGAAGAT AGTGAGCGGTACCCTGGAATCCCTGAACGATTCTCTGGGTCCACCTCAGGGAACACGACCACCCTGACCATCAGC AGGGTCCTGACCGAAGACGAGGCTGACTATTACTGTTTGTCTGGGGATGAGGACAATCC IGLV3—25*03 nucleotide sequence (SEQ ID NO: 1244) TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGAT GCATTGCCAAAGCAATATGCTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTGATATATAAAGAC AGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTcTGGCTCCAGCTCAGGGACAACAGTCACGTTGAcc-ATCAGT GGAGTCCAGGCAGAAGACGAGGCTGACTATTACTGTCAATCAGCAGACAGCAGTGG’I IGLV7-43*Ol tide sequence (SEQ ID NO: 1245) GTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGCTTCCAGC ACTGGAGCAGTCACCAGTGGTTACTATCCAAACTGGTTCCAGCAGAAACCTGGACAAGCACCCAGGGCACTGATT TATAGTACAAGCAACAAACACTCCTGGACCCCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTG ACACTGTCAGGTGTGCAGCCTGAGGACGAGGCTGAGTATTACTGCCTGCTCTACTATGGTGGTGCTCAG - IGLV7-46*01 nucleotide sequence (SEQ ID NO: 1246) CAGGCTGIGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGC ACTGGAGCTGTCACCAGTGGTCATTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATT TATGATACAAGCAACAAACACTCCTGGACACCTGCCCGGTTC-TCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTG ACCCT'I‘TCGGGTGCGCAGCCTGAGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGCTCGG IGLV7—46*02 nucleotide sequence (SEQ ID NO: 1247) CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGC ACTGGAGCTGTCACCAGTGGTCATTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATT ACAAGCAACAAACACchTGGACACCTGCCCGGTTCTCAGGCTcCCTCCITGGGGGCAAAGCTGCCCTG ACCCTTTTGGGTGCGCAGCCTGAGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGCTCGG IGLV9-49*Ol nucleotide sequence (SEQ ID NO: 1248) CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACCTGCACCCTGAGC AGCGGCTACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGGTTTGTGATGCGAGTG GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTTCTCAGTCTTGGGCTCAGGCCTGAAT CGGTACCTGACCATCAAGAACATCCAGGAAGAGGATGAGAGTGACTACCAC'IGTGGGGCAGACCATGGCAGTGGG TTCGTGTAACC - IGLV9-49*O3 nucleotide sequence (SEQ ID NO: 1249) CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACCTGCACCCTGAGC TACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGATTTGTGATGCGAGTG 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 IGHVl, IGHV2, IGHV3, IGHV4, or IGHV5 ne gene or an allele thereof.
The HA antibodies of the invention include a variable heavy chain (VH) region encoded by a human IGHVl, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele f. A IGHVl, 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 V” region that is encoded by a nucleic acid sequence that is at least 75% homologous to the IGHVl, 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 IGHVl, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele thereof, and more preferably, at least 98%, 99% homologous to the IGHVl, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele f. The VH region of the HA antibody is at least 75% homologous to the amino acid sequence of the VH region encoded by the IGHVl , 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 d by the 75%, 80%, 85%, 90%, 95%, 96%, 97% germline gene sequence or an allele thereof, and more ably, at least 98%, 99% gous to the sequence d 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 (V1) region encoded by a human IGKVl, IGKV2, IGKV3, roKK‘M, IbLVl, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof.‘ A htfman lGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLV2, IGLV3, IGLV7, or IGL$9 VL germline gene sequence, or an allele thereof is shown, e.g., at SEQ ID N03: 486 to 508. atively, the HA antibodies include a IGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLV2, IGLV3, IGLV7, or IGLV9 VL region that is encoded by a nucleic acid sequence that is at least 65% homologous to the IGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLV2, IGLV3, IGLV7, or IGLV9 ne 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 IGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence or an allele thereof, and more preferably, at least 98%, 99% gous to the IGKVl, IGKV2, IGKV3,‘IGKV4, IGLVl, 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 IGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLV2, IGLV3, IGLV7, or IGLV9 ne 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 d by the IGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLVZ, 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 IGKVl, IGKV2, IGKV3, IGKV4, IGLVl, IGLVZ, IGLV3, IGLV7, or IGLV9 gerrnline gene ce 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 t ion are capable of inducing dies against influenza A virus to prevent ion by the virus.
In one aspect the ion provides an immunogen having an epitope or epitope unit ized 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 onal antibody that competes with the binding of monoclonal antibody D7, D8, F10, Gl7, H40, A66, D80, E88, E90, or H98 to the HA protein. Preferably, the e is the F10 epitope.
In some embodiments the hemagglutinin protein is in the neutral pH conformation.
The immunogen is a peptide or a syntheticjpeptide: 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 ce 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 d 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 hemagglutinin1n the neutral pH conformation In some embodiments theimmunogen is a peptide having one or more of the ing amino acid sequences.
[Xaa0]m-Xaa.-Xaa2- [Xaao]p, wherein, preferably, Xaa, is S, T, F H or Y and Xaa 2 is H, Y, M, L or Q. Most preferably, Xaal is Y. Most preferably, Xaaz is H.
[Xaa0]m-Xaa.-Xaa2- [Xaa0]p_ wherein, preferably, Xaa, is H, Q, Y, S, D, N or T and Xaaz of is Q, E, K, I , V, M, E, R or T. Most preferably, Xaa. is H. Most preferably, Xaaz is [Xaao]m-Xaa.—Xaag-Xaa3-Xaa4- [Xaa0]p, wherein, preferably, Xaa. is I, V, M, or L; Xaaz is D, N, H, Y, D, A,S or E, Xaa; is G or A, and Xaa4 is W, R, or G. Most preferably, Xaa. is V; Xaaz is D, X2133 is G, and Xaa4 is W.
[Xaa0]m-Xaal--[Xaa0]q Xaag-Xaag- [Xaa0]q X524- [Xaao]r Xaas— [Xaa0]q——Xaa6 Xaa7 - [Xaao]q --Xaag-[Xaao]p, and [Xaao]m-Xaa1--[Xaao]q xaaz-xaag- [Xaao]q Xaa4- [Xaao]r Xaa5- [Xaao]q—Xaaé Xaa7- S —[Xaag],--[Xaa0]p, wherein, preferably Xaa, is K, Q, R, N, L, G, F, H or Y; XaagIS S or T, Xaa315 Q or P; Xaa415 F, V, I, M, L, or T; Xaa51s I, T, S, N, Q, D, or A; Xaaé, is I, V, M, or L; Xaa7 is N, S, T, or D and Xaag is 1, EV, A, or T. Most preferably, Xaa. is K; Xaaz is T, Xaa3 is Q; Xaa4 is I ; Xaas is T ; Xaa6 is V; Xaa7 is N, and Xaag is I.
For all of the preceding sequences, m, and p are independently O 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, sis 0 or 2, and t is 0 or 1, and Xaao, 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 r 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 ments the composition further comprises an anti—influenza antibody of antigen binding fragment f. Preferably, the aptibody'is monoclonal antibody D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98 or a oclonal antibody that competes with the binding of monoclonal antibody D7, D8, F10, G117, 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 ition comprising the nucleic acids.
The invention further comprises a method preventing a disease or er 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 inidase 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 zanarnivir, or oselta’mivir phosphate.
In another aspect the method includes further stering 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 za virus.
Administration is prior to or after re 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 ng a subject, the method comprising administering to the subject the stern region of influenza hemagglutinin in the neutral pH conformation in isolation from other ents of influenza virus, wherein the subject'iproduces an immune response to the stem region. For example, disclosed is a method of treating ect, the method comprising administering to the t the stem region of infliieiiza hemagglutinin in the neutral pH conformation in isolation from the head region of lutinin, wherein the subject produces an immune response to the stem region. For example, sed is a method of treating a subject, the method comprising administering to the subject influenza lutinin in the l 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 g to hemagglutinin immobilized on a surface, thereby identifying antibodies of interest. For example, disclosed is a method comprising ing 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, sed is a method comprismg screening antibodies reactive to hemagglutinin for binding to influenza 181 -; hemagglutinin in the neutral pH conformation in ion from other components of influenza virus, wherein the head region of the hemagglutinin is modified to reduce the antigenicity of the head region, thereby fying antibodies of interest.
In some forms, the head region of the lutinin can be modified by removing or replacing ylation 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 sed dies, 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 za viruses in each subtype within a group. In some forms, the immune response can be ve to influenza viruses within group 1.
In some forms, the disclosed methods can further comprise screening the dies of interest for competing with antibody F10 for binding to hemagglutinin, thereby identifying Flo-competing dies. 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 fied antibodies. Also disclosed are antibodies produced by the disclosed s. 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 Hl cluster influenza virus is an Hla r or an H lb cluster. The monoclonal antibody is fully human. In some forms, the monoclonal antibody can be a bivalent antibody, a lent 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 Hal 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 sed itions 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-glycansland uncharged es 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 enic 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 proteinj‘and isolating the antibody from the library. The fusion n can be immobilized on a solid e, 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 ed in a non-mammalian cell, such as an insect cell. The fusion protein can be, for e, a ic hemagglutinin (HA) protein.
Also disclosed is a method of vaccinating a subject against pathogenic ped 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 ed glycosylations compared to a wild type fusion protein.
Also disclosed is a ition comprising a monoclonal antibody as described herein and kits containing the ition in one or more containers and instructions for use.
The invention r 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 Jinventio'n 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. Sepcifically, a human Ab phage display library and H5 hemagglutinin (HA) main 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 HSNl 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 ered that nAbs targeting this pocket can e 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 es 18, 38, 39, 40 and 291 of HA] 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 ce analysis of all 16 HA subtypes points to the existence of only two variants of this epitope, ponding to the two phylogenetic groupings of HA (Groups 1 and 2). This ery 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 e the same VH germline gene, IGHVl -,69*01 and encode a CDR3 loop ning 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 toprevious 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 ered indicate that the precursor frequency of rearranged VH segments that could ize 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 sed simple solution to e universal tion against virus subtypes in both groups.
Three unique anti—HA—l scFvs were identified by sequencing analysis of the 58 HA-l 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 ve . 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 IGHVl—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 lizing influenza antibodies are ed below. Methods of making these antibodies are disclosed in PCT/USZOO9/054950 (Publication No. ), the entire contents of which are incorporated herein by nce.
Antibody 2A: Variable Region nucleic acid sequences , ‘ VH chain of 2A (SEQ ID NO: 1305) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGTGACAATGCTATCAGCTGGGTGCGACAGGCCCCAGGACAAGGGCTTGAGTGGATGGGGGGC ATCATTCCTATCTTTGGAAAACCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACTGCGGACGAATCC ACGAGCACAGCCTACATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAGATTCA TATTACTATGGTTCGGGGGGTATGGACGTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA VL chain of 2A (SEQ ID NO: 1306) CTGCCTGTGCTGACTCAATCATCCTCTGCCTCTGCTTCCCTGGGATCCTCGGTCAAGCTCACCTGCACTCTGAGC AGTGGGCATAGTAACTACATCATCGCATGGCATCAACAGCAGCCAGGGAAGGCCCCTCGGTACTTGATGAAGGTT AATAGTGATGGCAGCCACACCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGACCGC TACCTCACCATCTCCAACCTCCAGTCTGAGGATGAGGCTAGTTATTTCTGTGAGACCTGGGACACTAAGATTCAT GTCTICGGAACTGGGACCAAGGTCTCCGTCCTCAG Antibody 2A: Variable Region amino acid sequences VH chain of 2A (SEQ ID NO: 1307) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNAISWVRQAPGQGLEWMGGIIPIFGKPNYAQ KFQGRVTITADESTSTAYMDLRSLRSEDTAVYYCARDSDAYYYGSGGMDVWGQGTLVTVS VL chain of 2A (SEQ ID NO: 1308) l LPVLTQSSSASASLGSSVKLTCTLSSGHSNYIIAWHQQQPGKAPRYLMKVNSDGSHTKGDGI PDRFSGSSSGADRYLT ISNLQSEDEASYFCETWDTKSI HVFGTGTKVSVL Antibody D7: Variable Region nucleic acid sequences VH chain of D7 (SEQ ID NO: 1309) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTCCT GGAGGTATCTTCAACACCAATGCTTTCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGG GTCATCCCTTTGTTTCGAACAGpAAGCTACGCACAGAACGTCCAGGGCAGAGTCACCATTACCGCGGACGAATCC ACGAACACAGCCTACATGGAGCTTACCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGT GGTTACCATTTTAGGAGTCACTTTGACTCCTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA VL chain of D7 (SEQ ID NO: 1310) AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGCGTCTCCGGGGAAGACGGTGACCATCTCCTGCACCGGCAGC AGTGGCAACATTGCCGCCAACTATGTGCAGTGGTACCAACAACGTCCGGGCAGTGCCCCCACTACTGTGATCTAT GACCGAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGGTCCTCCAACTCTGCCTCC CTCACCATCTCAGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGACTTATGATACCAACAATCATGCT GTGTTCGGAGGAGGCACCCACCTGACCGTCCTC 2012/024971 Antibody H98: Variable Region nucleic acid sequences VH chain of H98 (SEQ ID NO: 1311) CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTCCT GGAGGTATCTTCAACACCAATGCTTTCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGG GTCATCCCTTTGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAGAGTCACCATTACCGCGGACGAATCC ACGAACACAGCCTACATGGAGCTTACCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGT GGTTACCATTTTAGGAGTCACTTTGACTCCTGGGGCCIGGGAACCCTGGTCACCGTCTCCTCA VL chain of H98 (SEQ ID NO: 1312) TCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGAAACACGGGCAGAGGGTCACCATCTCTTGTTCTGGAGGC ACCTCCAACATCGGACGTAATCATGTTAACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTAT GAACAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAATCTGGCACCTCCGCCTCCCTGGCC GTGAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCATCATGGGATGACAACTTGAGTGGTTGGGTG .TTCGGCGGAGGGACCAAGCTGACCGTCCTA ' Antibody D7 and H98: Variable Region chain ainino acid sequences VH chain of D7 and H98 (SEQ ID NO: 1313) QVQLVQSGAEVKKPGSSVKVSCKAPGGIFNTNAFSWVRQAPGQGLEWVGGVIPLFRTASYA QNVQGRVT I TADESTNTAYMELTSLRSADTAVYYCARSSGYH FRSHFDSWGLGTLVTVSS VL chain of D7 (SEQ ID NO: 1314) NFMLTQPHSVSASPGKTVTISCTGSSGNIAANYVQWYQQRPGSAPTTVIYEDDRRPSGVPDRF SGSIDRSSNSASLTISGLKTEDEAD'YYCQTYDTNNHAVFGGGTHLTVL VL chain of H98 (SEQ ID NO: 1315) SYELTQPPSASGKHGQRVTISCSGGTSNIGRNHVNWYQQLPGTAPKLLIYSNEQRPSGVPDRF TSASLAVSGLQSEDEADYYCASWDDNLSGWVFGGGTKLTVL Antibody D8: Variable Region nucleic acid sequences VH chain of D8 (SEQ ID NO: 1316) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCGCTTATGCTTTCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC ATCACCGGAATGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAACTC ACGAGCACAGCCTACATGGAGTTGAGCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTG TATTACTATGAGAGTAGTCTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG VL chain of D8 (SEQ ID NO: 1317) CAGTCTGTGCTGACTCAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACC AGCAGTGACGTTGGTGGTTATAACTCTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT TATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGCCTCCAAGTCTGGCAACACGGCCTCCCTG ACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTTCTGCTGCTCATATGCAGGCCACAGTGCTTATGTC ACTGGGACCAAGGTCACCGTCCTG Antibody D80: Variable Region nucleic acid sequences VH chain of D80 (SEQ ID NO: 1318) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAGGGCTTCT GGAGGCACCTTCAGCGCTTATGCTTTCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC ATCACCGGAATGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAACTC ACGAGCACAGCCTACATGGAGTTGAGCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTG TATTACTATGAGAGTAGTCTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG VK chain of D80 (SEQ ID NO: 1319) GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTCTTAGCAGCAAGTACTTAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACC ATCAGTAGACTGGAGCCTGAAGATTTTGCAGTGTATTCCTGTCAGCAGTATGATGGCGTACCTCGGACGTTCGGC CAAGGGACCACGGTGGAAATCAAA ' Antibody D8 and D80: Variable Region chain amino acid sequences VH chain of D8 and D80 (SEQ ID NO: 1320) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYAFTWVRQAPGQGLEWMGGITGMFGTANY AQKFQGRVTITADELTSTAYMELSSLTSEDTALYYCARGLYYYESSLDYWGQGTLVTVSS VL chain of D8 (SEQ ID NO: 1321) QSVLTQPPSASGSPGQSVTISCTGTSSDVGGYNSVSWYQQHPGKAPKLMIYEVTKRPSGVPD RFSASKSGNTASLTVSGLQAEDEADYFCCSYAGHSAYVFGTGTKVTVL VK Chain of D80 (SEQ ID NO: 1322) ' EIVLTQSPGTLSLSPGERATLSCRASQSLSSKYLAWYQQKPGQAPRLLIYGASSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVYSCQQYDGVPRTFGQGTTVEIK dy F10: Variable Region nucleic acid sequences VH chain of F10 (SEQ ID NO: 1323) CAGCTGGTGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCACGTCCTCT GAAGTCACCTTCAGIAGTTTTGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGG ATCAGCCCTATGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAGAGTCACCATTACCGCGGACCAGTCC ACGAGGACAGCCTACATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCT TCTTACATTTGTTCTGGTGGAACCTGCGTCTTTGACCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA VL chain of F10 (SEQ ID NO: 1324) CAGCCTGGGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGACCGCCACACTCACCTGCACTGGGAAC AGCAACAATGTTGGCAACCAAGGAGCAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTCCTATCCTAC AGGAATAATGACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAGGTCAGGAAACACAGCCTCCCTGACC GGACTCCAGCCTGAGGACGAGGCTGACTATTACTGCTCAACATGGGACAGCAGCCTCAGTGCTGTGGTA TTCGGCGGAGGGACCAAGCTGACCGTCCTA Antibody E90: Variable Region nucleic acid sequences .7; l VH chain of E90 (SEQ ID NO: 1325) "“ CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCACGTCCTCT GAAGTCACCTTCAGTAGTTTTGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGG CCTATGTTTGGAACACCTAATTACGCGCAGAAGTTCGAAGGCAGAGTCACCATTACCGCGGACCAGTCC ACGAGGACAGCCTACATGGACCTGAGGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCT TCTTACATTTGTTCTGGTGGAACCTGCGTCTTTGACCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA VL chain of E90 (SEQ ID NO: 1326) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA AGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT GCATCCAGTTTGCAAAGAGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATT AGCAGCCTGCAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGATAGTTCACCGTACACTTTTGGCCAG GGGACCAAGGTAGAGATCAAA Antibody F10 and E90 Variable Region amino acid ces VH chain of F10 and E90 (SEQ ID NO: 1327,) QVQLVQSGAEVKKPGS SVKVSCTSSEVTFSSFAISWVRQAPGQGLEWLGGISPMFGT PNYAQKFQGRVTITADQSTRTAYMDLRSLRSEDTAVYYCARSPSYICSGGTCVFDHWGQGT LVTVSS VL chain of F10 (SEQ ID NO: 1328) QPGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSYRNNDRPSGISER FSASRSGNTASLTITGLQPEDEADYYCSTWDSSLSAVVFGGGTKLTVL VL chain of E90 (SEQ ID NO: 1329)DIQMTQSPSSLSASVGDRVTlTCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQRGVPSRFSG SGSGTDFTLTISSLQPEDFAVYYCQQYDSSPYTFGQGTKVEIK Antibody G17: Variable Region nucleic acid sequences VH chain of 617 (SEQ ID NO: 1330) CAGGTGCAGCTGGTGCAATCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGACTTCT GGAGTCACCTTCAGCAGCTATGCTATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCGGTGTCTTTGGTGTACCAAAGTACGCGCAGAACTTCCAGGGCAGAGTCACAATTACCGCGGACAAACCG ACGAGTACAGTCTACATGGAGCTGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGCCC GGGTACTACGTAGGAAAGAATGGTTTTGATGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA VL chain of G17 (SEQ ID NO: 1331) TCCTATGAGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGACCGCCATACTCACCTGCACTGGAGAC AGCAACAATGTTGGCCACCAAGGTACAGCTTGGCTGCAACAACACCAGGGCCACCCTCCCAAACTCCTATCCTAC AGGAATGGCAACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAGGTCAGGAAATACAGCCTCCCTGACC ATTATTGGACTCCAGCCTGAGGACGAGGCTGACTACTACTGCTCAGTATGGGACAGCAGCCTCAGTGCCTGGGTG TTCGGCGGAGGGACCAAGCTGACCGTCCTA dy G17 Variable Region amino acid sequences QVQLVQSGAEVKKPGASVKVSCKTSGVTFSSYAISWVRQAPGQGLEWMGGIIGVFGVPKYA[1109] VH chain 0fG17 (SEQ ID NO: 1332) VTITADKPTSTVYMELNSLRAEDTAVYYCAREPGYYVGKNGFDVWGQGTMVTVS VL chain of 017 (SEQ ID NO: 1333) SYELTQPPSVSKGLRQTAILTCTGDSNNVG FSASRSGNTASLTIIGLQPEDEADYYCSVWDSSLSAWVFGGGTKLTVL Antibody H40: Variable Region nucleic acid sequences VH chain of H40 (SEQ ID NO: 1334) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGGCCTCAGTGAAGGTCTCATGTAAGGCTTCT GGATACACCTTCACCGGTTATTATATTCACTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGTTGG ATCAACCCTATGACTGGTGGCACAAACTATGCACAGAAGTTTCAGGTCTGGGTCACCATGACCCGGGACACGTCC ATCAACACAGCCTACATGGAGGTGAGCAGGCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGGGGGGCT TCCGTATTACGATATTTTGACTGGCAGCCCGAGGCTCTTGATATCTGGGGCCTCGGGACCACGGTCACCGTCTCC VL chain of H40 (SEQ ID NO: 1335) CAGCCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGCATTCCCTGTGGGGGGAAC AACATTGGAGGCTACAGTGTACACTGGTACCAACAAAAGCCGGGCCAGGCCCCCCTCTTGGTCATTTATGACGAT AAAGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCGCCAACTCTGGGAGCACGGCCACCCTGACAATCAGC AGGGTCGAAGCCGGGGATGAGGGCGACTACTACTGTCAGGTGTGGGATAGTGGTAATGATCGTCCGCTGTTCGGC GGAGGGACCAAGCTGACCGTCCTA Antibody H40: Variable Region amino acid sequences VH chain of H40 (SEQ ID NO: 1336) - QVQLVQSGAEVRKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPMTGGTN YAQKFQVWVTMTRDTSINTAYMEVSRLTSDDTAVYYCARGASVLRYFDWQPEALDIWGLG TTVTVSS VL Chain of H40 (SEQ ID N021337) PPSVSVAPGQTAS[PCGGNNlGGYSVHWYQQKPGQAPLLVlYDDKDRPSGIPERFSG ANSGSTATLTISRVEAGDEGDYYCQVWDSGNDRPLFGGGTKLTVL Antibody A66 Variable Region nucleic acid sequences VH chain of A66 (SEQ ID NO: 1338) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTCGGTGAAGGTTTCCTGCAAGGCTTCT GGAGGCCCCTTCAGCATGACTGCTTTCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGG ATCAGCCCTATCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCC ACGAACACAGCCAACATGGAGCTGACCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTT TACCAACCGAATAATGATGCTTTTGCTATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA VK chain of A66 (SEQ ID NO: 1339) GTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCC AGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGACTGGAGCCTGAAGATTTTGCAGTCTATTTCTGTCAGCAGTATGGTAGCTCACCTCAATTCGGCCIEAGGG ACACGACTGGAGATTAAA Antibody A66 Variable Region amino acid‘sequences VH Chain of A66 (SEQ ID NO:1340) QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGGISPIFRTPKYA VTITADESTNTANMELTSLKSEDTAVYYCARTLSSYQPNNDAFAIWGQGTMVTVS VK Chain of A66 (SEQ ID NO: 1341) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLT I SRLEPEDFAVYFCQQYGSSPQFGQGTRLEIK Antibody E88 Variable Region nucleic acid sequences VH chain of E88 (SEQ ID NO: 1342) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTCGGTGAAGGTTTCCTGCAAGGCTTCT GGAGGCCCCTTCAGCATGACTGCTTTCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGG ATCAGCCCTATCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCC ACGAACACAGCCAACATGGAGCTGACCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTT TCCTCCTACCAACCGAATAATGATGCTTTTGCTATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA VL chain of E88 (SEQ ID NO: 1343) CTGCCTGTGCTGACTCAGC‘CACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGC AGCTCCAACATCGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT AATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCAGGCACCTCAGCCTCCCTGGCC ATCATTGGACTCCGGCCTGAGGATGAAGCTGATTATTACTGTCAGTCGTATGACAGCAGGCTCAGTGCTTCTCTC TTCGGAACTGGGACCACGGTCACCGTCCTC Antibody E88 Variable Region amino acid sequences VH Chain of E88 (SEQ ID NO: 1344) QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFI‘WLRQAPGQGLEWMGGISPIFRTPKYA QKFQGRVTITADESTNTANMELTSLKSEDTAVYYCARTLSSYQPNNDAFAIWGQGTMVTVS S ~ VL Chain of E88 (SEQ ID NO: 1345) . .
LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFS GSRSGTSASLAIIGLRPEDEADYYCQSYDSRLSASLFGTGTTVTVL The amino acid sequences of the heavy and light chain complementary determining regions of the neutralizing za antibodies are shown below in Table 18. 2012/024971 Table 18 ANTIBODY CHAIN CDRl SEQ CDR2 SEQ CDR3 SEQ ID ID ID NO: ‘ NO: NO: CONSENSUS HEAVY SYAFS GIIPMFGTPNYAQKFQ 1263 SSGYYYGGGFDV D7/H98 HEAVY TNAFS 302 GVlPLFRTASYAQNV 1264 SSGYHFGRSHFD 1285 ON S D8/D80 HEAVY AYAFT 05 GITGMFGTANYAQKF 1265 GLYYYESSLDY 1286 F10/E90 HEAVY SFAIS m GISPMFGTPNYAQKF 1266 SPSYICSGGTCVF 1287 QG DH HEAVY SYAIS m GllGVFGVPKYAQKFQ 1267 EPGYYGKNGFDV 1288 H40 HEAVY GYYIH 630 WINPMTGGTNYAQKF 1268 YFDWQP 1289 QV EALDI HEAVY MTAFF GlSPlFRTPKYAQKFQ 1269 TLSSYQPNNDAF 1290 G AI HEAVY MTAFT GlSPlFRTPKYAQKFQ 1270 TLSSYQPNNDAF 1291 I G AI 2A HEAVY DNAIS 668 GIIPIFGKPNYAQKFQ 1271 DSDAYYYGSGG 1292 G MDV CONSENSUS LIGHT TGSSSMNIGN 757 S 1272 QSYDSLSAYV 1293 YVA : LIGHT TGSSSNIAAN 1252 EDDRRPS 1273 QTYDTNNHAV 1294 LIGHT TGTSSDVGGY 1253 EVTKRPS 1274 CSYAGHSAYV 1295 NSVS F 10 LIGHT TGNSNNVGN 1254 RNNDRPS 1275 STWDSSLSAVV 1296 O GAA ‘ TGNSNNVG OGTA H40 GGNNIGGYSV 1256 DDKDRPS -A66 LIGHT RASQSVSSYL 1257 DASNRAT -D80 LIGHT RASQ£LSSKY 1258 GASSRAT -E88 LIGHT SGSSSNIGSNT 1259 SNNQRPS LIGHT RAsoSISSYLN 1260 AASSLoR 1281m1302 LIGHT SGGTSNIGRN 1261 SNEQRPS 1282 ASWDDNLSGWV 1303 A LIGHT TLSSGHSNYII 1262 VNSDGSHTKGD 1283 ETWDTKIHV 1304 dies Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly tood by those of ordinary Skill in the art. Further, unless otherwise required by context, singular terms shall include ities and plural terms shall include the singular; Generally, nomenclatures utilized in connection with, and techniques of, cell and tissuc: culture; molecular biology, and protein and oligo- or cleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, loligonucleotide 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. lar 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); ucleotide Synthesis (N. Gait, ed., I984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and ation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); , A Practical Guide to Molecular Cloning (1984).
The nomenclatures utilized in tion with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic try, and nal and pharmaceutical chemistry bed herein are those well known and commonly used in the art. Standard ques are used for chemical syntheses, chemical es, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
The following definitions are useful in understanding the present ion: The term “antibody” (Ab) as used herein ihrc'ludes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e. g., bispecific ahtibodies), and dy 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 l environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, es, and other proteinaceous or nonproteinaceous solutes. In red 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 ; (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 SIDS-PAGE under reducing or non—reducing conditions using sie blue or, preferably, silver stain.
Isolated antibody includes the antibody in situ withiri, recOmbinant cells since at least one 192 2,7 . component of the antibody's natural nment 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) . An IgM antibody consists of S of the basic heterotetramer unit along with an additional polypeptide called I 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 aboutal50,0pO 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 0L and y chains and four CH domains for p. and e 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 d with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are ed 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 logy, 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 rate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda 0»), based on theaminq acid ces of their constant s (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 imrnunoglobulins: IgA, IgD, IgE, IgG, andiIgM, having heavy chains ated alpha (0;), delta (8), n (a), gamma ('y) and mu (u), respectively. The yand 0; classes are further divided into subclasses on the basis of relatively minor ences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgA], 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 dy for its ular n. However, the variability is not evenly distributed across the l lO-amino acid span of the variable domains. Instead, the V s consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by r 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 t uration, connected by three ariable regions, which form loops connecting, and in some cases forming part of, the B-sheet structure. The hypervariable s in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the ion 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. ). 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 es of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity 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- (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 ns 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. 6 al. Nucl. Acids Res. 282219—221 (2000)). Optionally the dy has symmetrical insertions at one or f the-following points 28, 36 (L1), 63, 74—75(L2) and 123 (L3) in the VL, and 28, 36 (H1),:63, 721175 (H2) and 123 (H3) in the VH when numbered in accordance with AHo; Honneg'erfA. ahd Plunkthun, A. J. Mol. Biol. 309:657-670 (2001 )).
By “germline nucleic acid residue” is meant the nucleic acid residue that lly 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, andv'vhen transmitted to offspring, such a mutation is incorporated in every cell of the body. Argermline mutation is in contrast to a c on which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence ng 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 le naturally occurringmutations 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 ed against different determinants (epitopes), each onal 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 er “monoclonal” is not to be construed as requiring production of the antibody by any particular . For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 2561495 (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 dies” may also be isolated from phage dy’libraries using the techniques described in Clackson et al., Nature, 352:624—628 (1991) and Marks er 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 gous to corresponding sequences in antibodies derived from a ular 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 d from another species or belonging to another dy class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see US. Pat. No. 4,816,567; and on 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 y interest herein include dies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more ces d from a non—human antibody, e.g., an FR or C region sequence. In addition, chimeric dies 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 ces 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 dies also include primatized and humanized antibodies. rmore, chimeric antibodiesmay 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 ; Riechmann et al., Nature 332:323-329 (1988); and , 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 man. These non- human amino acid residues are often ed to as “import” residues, which are lly taken from an “import” variable domain. Humanization is traditionally performed ing the method of Winter and co-workers (Jones et all" , 321:522—525 (1986); Reichmann et al., Nature, 332:323—327 (1988); yenlet a'l'., 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 (US.
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 lly made'to r 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 ““I‘ 196 a: 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 US. Pat. No. ,641,870; Zapata et al., Protein Eng. 8(10): 1057-l962 [1995]); single—chain antibody molecules; and pecific dies formed from antibody fragments.
The phrase “functional fragment or "; of an antibody is a nd having qualitative biological ty in common with a full-length antibody. For example, a functional fragment or analog of an gE 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, chRI.
Papain ion of antibodies produces two identical antigen-binding nts, called “Fab” fragments, and a residual “Fe” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the le 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 n— 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 CH1 domain including one or more cysteines from the antibody hinge regioni.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 dy fragments ally were produced as pairsof Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The “EC” fragment comprises the carboxy-terminal ns of both H chains held together by disulfides. The or 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 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 es 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 ize and bind antigen, gh at a lower affinity than the entire binding site.
“Single-chain Fv” also abbreviated as “st” or “scFv” are antibody fragments that comprise the VH and VL dy domains connected into a single polypeptide chain.
Preferably, the st polypeptide further comprises a polypeptide linker between the VH and VL s that enables the st to form the d. structure for n binding. For a review of st, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New-‘York, pp. 269—315 (1994); aeck 1995, infra.
The term “diabodies” refers to small antibody fragments prepared by constructing st fragments (see preceding paragraph) with short linkers (about 5-10 es) between the V“ 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 over” st fragments in which the VH and VL domains of the two antibodies are present on different ptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161; and Hollinger et 01., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993).
As used herein, an antibody that “intemalizes” 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 certjaingtherapeutic 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 ed cell.
Depending on the potency of the dy 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 “i'mmunospecific,n nspecific for” or to fically 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'l, or greater than or equal to about 105 M“, greater than or equal to about 106 M", r than or equal to about 107 M", or greater than or equal to 108 M". Affinity of an antibody for its cognate antigen is also commonly expressed as a iation constant KD, and in certain embodiments, HuM2e antibody specifically binds to M2e if it binds with a KB 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 e, those described by Scatchard et al. (Ann. N. Y. Acad.
Sci. USA 51:660 (1949)).
Binding ties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetectionzmethods including, for example, immunofluorescence-based assays, such as immuno—histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS). .. 3-; 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 ments, an antibody with a ical 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” dy is used in the broadest sense, and includes an antibody that lly or fully blocks, inhibits, or neutralizes a biological ty 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 ts the growth of infected cells” or a “growth inhibitory” antibody is one that binds to and results in able growth inhibition of infected cells _24971 expressing or capable of expressing an M2e epitope bound by an dy. Preferred growth inhibitory antibodies inhibit growth of ed cells by greater than 20%, preferably from about 20% to about 50%, and even more ably, 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 tration of about 0 l to 30 ug/ml or about 0 5 nM to 200 nM1n cell culture, where the growth inhibitionis determined l-lO days after exposure of the infected cells to the antibody. Growth inhibition of infected cells in viva can be determined in various ways known in the art. The antibody is growth tory in viva if administration of the antibody at about 1 ug/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 s 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 ed by annexin g; 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, ably about 5 to 50 fold, and most preferably about l0 to 50 fold, induction of annexin binding relative to untreated cell in an n binding assay.
Antibody “effector functions” refer to those ical activities utable to the Fc region (a native ce Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq 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 ed 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 uently 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 FcyRIII only, s monocytes express FcyRI, FcyRII and FcyRIII. 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 US. Pat. No. 5,500,362 or US. Pat. No. 337 may be performed.
Useful effector cells for such assays e 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 red FcR is one that binds an IgG dy (a gamma receptor) and es receptors of the FC’YRI, FcyRII, and I subclasses, including allelic variants and alternatively spliced forms of these receptors. FCyRII receptors include A (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ ily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. ting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its asmic domain. (see review M. in Daeron, Annu. Rev. l. 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 91457-92 (1991); Cape] 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 , 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. 1 17:587 (1976) and Kim et al., J. Immunol. 242249 (1994)).
“Human effector cells” are leukocytes that express one or more FcRs and perform or functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and phils; withiPBMCs and NK cells being red. 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 cal complement pathway is initiated by the binding of the first ent of the complement system (Clq) 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 yxoviridae 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 e is uncommon. Some isolates of influenza A virus cause severe disease both in ic poultry and, rarely, in humans. ‘ ,5: A “mammal" for purposes of treating n infection, refers to any mammal, including humans, domestic and farm animals, and zoo, sportshor pet s, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
“Treating” or ment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the ed pathologic condition or disorder. Those in need of treatment e 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 dy 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 , one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issuesi‘The above parameters for assessing successful treatment and improvement in the disease are readily able 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 ing.” ic” 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 ity) for an ed 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 r therapeutic agents includes aneous (concurrent) and consecutive administration in any order. ers” 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. es of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; idants 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, gine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose,_.mannose, or dextrins; chelating agents such as EDTA; sugar ls such as mannitol or sorbitol; salt—forming counterions such as ; 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., At”, 113', 1'25, Ygo, Re“, Re“, Sm153, Biz”, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin, vihca alkaloids (vincristine, stine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and nts thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, ing fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. 203 g.
WO 12489 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 tory 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 01 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 of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et at. (W B Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and xel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERETM, Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and xel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of s in cells.
“Label” as used herein refers to a detectable nd or composition that is conjugated directly or indirectly to the antibody so as to generate a “labeled” dy. The label may be detectable by itself (e. g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may ze al 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 ptides 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 - 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 ted 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 s. 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 t. Peptides, oligopeptides, and proteins are included within the tion of polypeptide, and such terms may be used interchangeably herein unless specifically ted 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 lly occurring and non-naturally'occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of st in the t of this invention are amino acid uences comprising CDRs and being capable of binding an n 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 ments, the isolated polypeptide will be purified (l) to greater than 95% by weight of ptide 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 sie blue or, preferably, silver stain. ed 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 cleotide derived from nature. A “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g., dy) derived from nature (e.g., from any s). Such native ce polynucleotides and polypeptides can be ed from nature or can be produced by inant or synthetic means.
A polynucleotide “variant,” as the term is used herein, is a polynucleotide that typically s 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 ting one or more biological activities of the d 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 usedtherein,_is a polypeptide that typically differs from a ptide specifically disclosed herein ingone or more substitutions, deletions, ons and/0r insertions. Such variants rnay 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 t 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 ce.
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 ptides (e. g., antigens) or cells. Since it is the binding ty and nature of a protein that defines that protein's biological functional activity, certain amino acidsequence substitutions can be made in a protein sequence, and, of course, its underlying QNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the e sequences of the disclosed compositions, or ponding DNA sequences that encode said peptides t appreciable loss of their biological utility or activity.
In many instances, a ptide 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 ohe skilled in the art of peptide chemistry would expect the secondary ure 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 ter of the amino acid contributes to the secondary ure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for e, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a athic 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); threoniine' (—O.7); serine (~08); tryptophan (—0.9); tyrosine (—l.3); proline (—l.6); histidine (~32); glutamate (—3.5); glutamine (—3.5); aspartate (—3.5); asparagine ; 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 onally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within i2 is red, those within 1‘] are particularly preferred, and those within i0.5 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. Patent 4,554,101 states that the greatest local average hydrophilicity of a n, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
. . As ed 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 i l); glutamate (+3.0 1' l); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (—0.5 i 1); alanine (—0.5); histidine (—0.5); cysteine (—1.0); methionine (—1.3); valine ; leucine (—1.8); isoleucine (—1.8); tyrosine ; 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 logically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within i2 is red, those within :1 are particularly preferred, and those within 1:05 are even more particularly preferred.
As outlined above, amino acid tutions 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 e: 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, lity, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For e, negatively charged amino acids e aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hilicity values e leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, alanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, ash, 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 l influence on the genicity, secondary structure and hydropathic nature of the polypeptide.
Polypeptides may comprise a signal (or leader) sequence at the N—terminal end of the n, which nslationally or post—translationally directs transfer of the protein. The ptide may also be conjugated to a linker or other sequence for ease of sis, purification or fication 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 ces is the same when aligned for maximum correspondence, as described below. isons 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 ons, usually 30 to about 75, 40 to about 50, iniwhichHa sequence may be ed to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of ces for comparison may be conducted using the Megalign m in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: f, MO. (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 DC 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, CA; Higgins, DC. and Sharp, PM. (1989) CABIOS 52151-153; Myers, E.W. and Muller W.
-CABIOS 4: 1 1-17; Robinson, ED. (1971) Comb. Theor 112105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 42406—425; Sneath, P.H.A. and Sokal, RR. (1973) Numerical Taxonomy — the Principles and Practice ofNumerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, DJ. (1983) Proic. Natl. Acad, Sci. USA 80:726— 730. atively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (198]) Add. APL. Math 2:482, by the 2012/024971 identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 482443, 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, WI), or byinspection.
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. M01. 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 d score for a pair of ng 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 ed value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring,res'idue alignments; or the end of either sequence is reached. The BLAST algon’thm parameters W, T and X ine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1, and expectation (E) of 10, and the BLOSUM62 scoring l(see Henikoff and ff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) ents, (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 ce is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
In one approach, the “percentage of ce identity” is determined by comparing two optimally d sequences over a window parison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sEquence in the comparison window may comprise additions or ons (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or IO to 12 t, 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 ces to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference ce (i.e., the window size) and multiplying tljéresuits 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 m 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 .
“Vector” includes shuttle and expression vectors. Typically, the plasmid construct will also include an origin of replication (e.g., the ColEl origin of replication) and a selectable marker (e. g., ampicillin or ycline resistance), for replication and selection, respectively, of the plasmids in bacteria. An “expression vector” refers to a vector that contains the necessary control ces or regulatory elements for expression of the dies including antibody fragment of the invention, in ial or eukaryotic cells.
Suitable vectors are disclosed below.
As used in this specification and the appen‘déd claims, the singular forms “a,” “an” and “the” include plural references unless the content y dictates ise.
The present invention includes human hionocl‘l‘onalv anti—influenza. antibodies comprising a ptide of the present invention, as well as fragments and variants thereof.
In one embodiment, the antibody is an antibody designated herein as TCN-032 (8110), 21B15, TCN-031 (23K12), 3241_G23, 3244_110, 3243_J07, 3259_J21, 3245_Ol9, 3244_H04, 3136_G05, 3252_C13, O6, 3420_123, 3139_P23, 3248_P18, 3253_P10, 19, 3362_B11, 3242_P05, TCN-522 (3212__112), TCN-521 (3280_D18), TCN-523 (5248_A17), 3 (5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN—528 (5087_P17), TCN-529 (5297_H01), TCN—53O 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_Ol7), TCN-539 (5056_A08), TCN- 540 (5060~F05), TCN-541 (5062__Ml 1), TCN-542 (5079_A16), TCN-543 (5081_G23), 4 (5082_A19), TCN-545 (5082_115), 6 (5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), 9 (5092_P04), TCN-550 (5096_F06), TCN-551 D01), TCN-552 (5249_123), TCN—553 (5261_C18), TCN-554 (5277_M05), TCN-555 (5246_Ll6), TCN-556 (5089_K12), TCN—557 (508l_A04), TCN 558 (5248_H10b), TCN-559 (5097_GOS), TCN—560 (5084_P10), TCN—504 (325]_Kl7), SC06-l4l, SCO6—255, SC06-257.
SC06-260, SC06-26l, SC06-262, SC06—268, SCO6—272, SCO6-296, SC06—301, SCO6-307, SC06-310, SCO6-3 l4, SCO6-323, SC06—325, SCO6—327, SCO6-328, SCO6-329, SC06—33l.
'SC06—332, SC06-334, SC06-336, SCO6-339, SC06-342, SCO6-343, SCO6-344, CR6141, CR6255, , CR6260, CR6261, CR6262,‘CR6268, CR6272, CR6296, CR6301, CR6307, CR6310, CR6314, CR6323, CR6325, , CR6328, CR6329, CR6331.
CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, or CR6344. These dies preferentially bind to or specifically bind to influenza A infected cells as compared to uninfected l cells of the same cell type. ’ In ular 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 ed 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 n, 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, l description of antibodies herein and methods of preparing and using the same also! apply to individual dy 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 dies, Methods of producing polyclonal and monoclonal antibodies are known in the art and described lly, e. g., in US. Patent No. 780. Typically, the antibodies of the t invention are produced recombinantly, using vectors and s availablefin the art, as bed r below.
Human antibodies may also be generated by in vitro activated B cells (see US. Pat. Nos. ,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 gous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in te inhibition of endogenous antibody production. Transfer of the human germ-line globulin 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, 9022551 (1993); Jakobovits et al., Nature, 362:255—258 ; mann et al., Year in Immuno., 7:33 (1993); US Pat. Nos. 5,545,806, 5,569,825, ,591,669 (all of rm); US. Pat. No. 5,545,807; and WO 97/17852. Such s 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 es 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 ces, 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 ant that chimeric antibodies retain high binding affinity-for the antigen and other favorable biological properties. To achieve this goal, ing to a preferred , chimeric antibodies are prepared by a process of analysis of the parental sequences and various conceptual chimeric products using 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 ate globulin 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 ed from the recipient and import sequences so that the desired antibody teristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in cing 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 . 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 serologieal means (i. e. by the use of antibodies directed to these ences). 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 mayg'ybe alpha 1 or alpha 2.
In a preferred embodiment, an antibody of the t invention, or fragment f, is an IgG. IgG is ered 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. hages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fe region of IgG. Not all subclasses bind equally well; IgGZ and IgG4 do not bind to Fe 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 ytosis. Binding of IgG to Fe 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 r preferred embodiment, an dy, 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 tic 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 thegpresent invention, and fragments f, comprise a variable light chain that is either kappa or lambda. The lamba chain may be any of subtype, ing, e.g., lambda l, lambda 2, lambda 3, and lambda 4.
As noted above, the present invention further provides dy nts 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 dies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-1 17 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by inant host cells. Fab, Fv and Selfv antibody fragments can all be expressed in and secreted from E. coli, thus ng the facile production of large amounts of these fragments. Fab'-SH fragments can be directly recovered from E. coli and ally d to form F(ab')2 fragments (Carter et al., Bio/Technology -167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell e. Fab and F(ab')2 fragment with increased in viva half-life comprising a salvage receptor binding epitope residues are described in US. Pat. No. 5,869,046. Other ques for the production of antibody fragments will be nt to the skilled practitioner. 2012/024971 In other embodiments, the dy of choice is a single chain Fv fragment (scFv).
See WO 93/16185; US. Pat. Nos. 5,571,894; and 5,587,458. Fv and st 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. st fusion proteins may be constructed to yield fusion of an effector n at either the amino or the carboxy terminus of an st. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody", e.g., as described in US. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
In certain embodiments, antibodies of the t ion are bispecific or multi- specific. Bispecific antibodies are antibodies that have binding icities 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 ntigen binding site with a g site for a second antigen. Alternatively, an 2e 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 lgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FCYRIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize xic agents to infected cells. These antibodies possess an M2e-binding arm and an arm that binds the cytotoxic agent (e. g., saporin, anti- interferon-0t, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten).
Bispecific antibodies can be prepared as full length dies or antibody fragments (e. g., F(ab')2 bispecific antibodies). WO 73 describes a bispecific anti-ErbBZ/anti-FcyRIII antibody and US. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcyRI antibody.
A bispecific anti-ErbBZ/Fctx antibody is shown in WO98/02463. US. Pat. No. 5,821,337 teaches a bispecific anti—ErbBZ/anti—CD3 antibody.
Methods for making bispecific antibodies are known in the art. Traditional tion of full length bispecific antibddies is based on the co-expression of two immunoglobulin heavy chain—light chain pairs, where the two chains have different icities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) e a potential e 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 .
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin nt domain sequences. Preferably, the fusion is with an Ig heavy chain nt domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain nt 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 ments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired ific 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 ific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid globulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure tates the tion of the desired ific 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, 1212210 (1986).
According to another approach described in US. Pat. No. 5,731,168, the ace between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred ace comprises at least a part of the CH 3 domain. In method, one or more small amino acid side chains from the interface of the first antibody “molecule are ed 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 r ones (e. g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other ed end— ts such as homodimers.
Bispecific antibodies include‘cross—linked orv-“heteroconjugate” antibodies. For example, one of the antibodies in the conjugate can. be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed ”to target immune system cells to unwanted cells (US Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking s. Suitable linking agents are well known in the art, and are disclosed in US. Pat. No. 4,676,980, along with a number of cross-linking techniques.
Techniques for ting bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using - al 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 te, to stabilizevicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatiyes. 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 deriyatiye to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the ive immobilization of enzymes.
Recent progress has facilitated the direct recovery of H 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 tion of a fully humanized bispecific antibody 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, ific dies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). The leucine zipper es from the F05 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 r fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (st) dimers has also been reported. See Gruber et al., J. Immunol., 15225368 . dies 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 ion can be multivalent antibodies with three or more antigen binding sites (e. g., tetravalent antibodies), which can be readily produced by recombinant expression of c acid ng the polypeptide chains of the antibody. The multivalent antibody can se a zation domain and three or more antigen binding sites. The- red 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 VDl-(Xl)n X2)n -Fc, n VD] is a first variable , VD2 is a second variable domain, Fe is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is O or 1. For ce, the polypeptide chain(s) may se: VH-CH l-llexible linker-VH-CHl-Fc region chain; or VH-CHl—VH—CHl-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, r comprise a CL domain.
Antibodies of the present ion r include single chain antibodies.
In particular ments, antibodies ofthe 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 irito a polynucleotide that encodes the antibody, or a chain thereof, or by peptide Synthesis. Such modifications e, 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, ion, and substitution may be made to arrive at the final dy, 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 t invention may be included in antibodies of the present ion.
A useful method for identification of certain residues or regions of an antibody that are preferred ons for nesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells in e, 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 e or polyalanine) to affect the interaction of the aminoacids with PSCA antigen. Those amino acid locations demonstrating functional sensitivitY‘to-zthe 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 ning a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions e an antibody with an N-terminal methionyl residue or the dy fused to a cytotoxic polypeptide. Other insertional ts 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. r 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 nservative substitutions are contemplated.
Substantial modifications in the biological properties of the antibody are lished 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 l conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
Any ne residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with , to improve the oxidative stability of the molecule and prevent aberrant crosslinking. sely, 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 es tuting 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 ting 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; andnosubstitutions at each site. The 1 f; 221‘ . 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 ed for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning nesis can be performed to identify hypervariable region residues contributing significantly to antigen g. atively, 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 ing 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 ties in one or more relevant assays may be selected for further development. r type of amino acid variant of the antibody alters the al 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. ylation 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 gine side chain. Thus the presence of either of these tripeptide sequences in a polypeptide s a potential g-iycosylation site. O-linked glycosylation refers to the attachment of one of the stigars lgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5— hydroxyproline or 5—hydroxylysine may also be used.
Addition of ylation 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 ed 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). -' '4“ The antibody of the invention is modified with t 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 ed by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or onally, 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. 176:1191-1 195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with ed anti-infection activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 532256012565 (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 nti-Cancer Drug Design 3:219-230 (1989).
To increase the serum half-life of the dy, one may incorporate a salvage. receptor g epitope into the antibody (especially an antibody fragment) as described in US. 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., IgG., Ing, 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 ns to therapy with immunoconjugates comprising an dy conjugated to an anti-cancer agent such as a cytotoxic agent or a growth inhibitory agent. herapeutic agents useful in the generation of such conjugates have been described above.
Conjugates of an dy and one or more small le 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 nts) 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 n shrub Maytenus serrata (US. Pat. No. 3,896,l l 1). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as, maytansinol and C-3 maytansinol esters (US. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in US. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 814; 4,294,757; 4,307,016; 4,308,268; 269; 4,309,428; 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 eutic index, maytansine and maytansinoids have been conjugated to antibodies ically binding to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for e, in US Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 13]. Liu et (11., Proc. Natl.
Acad. Sci. USA 8-8623 (1996) described immunoconjugates comprising a maytansinoid designated DMl 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 bye-chemically linking an antibody to a maytansinoid molecule without significantly diminishing the ical activity of either the dy or the maytansinoid molecule. An average of 3—4 maytansinoid les conjugated per antibody molecule has shown cy 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 sized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in US. Pat. No. 5,208,020 and in the other patents and nonpatent publications ed to hereinabove. red maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as s maytansinol esters.
There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in US. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research 52: 127-131‘ (1992)“. The g groups include disufide groups, thioether groups, acid labile groups, photblabile 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 n coupling agents such as N-succinimidy1(2-pyridyldithio)propionate , succinimidyl(N- maleimidomethyl)cyclohexane—1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl idate 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-diazoniumbenzoy1)— ethylenediamine), diisocyanates (such as toluene 2,6—diisocyanate), and tive fluorine compounds (such as l,5—difluoro-2,4-dinitrobenzene). Particularly red 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., e 238: 1098 (1987). Carbon-14—labeled 1-isothiocyanatobenzylmethyldiethylene nepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/1 1026. The linker may be a able linker" facilitating e of the cytotoxic drug in the cell. For example, an acid-labile linker, Cancer Research 52; 127—131 (1992); US. Pat. No. 5,208,020) may be used.
Another immunoconjugate of st ses an antibody conjugated to one or more calicheamicin molecules. The calicheamiciniifamily 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, ,739,] 16, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company). r 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 alization 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 US. 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, ding 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 tor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, llin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for e, WO 93/21232.
The present invention further includes an irri'r'nunoconjugate formed between an antibody and a compound with nucleolytic activity (e. g., éi 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 dies. es include my ', 1m, 1'25, Y90, Rem’, Rc'gs, Sm'53, Biz”, P32, Pb“ and radioactive isotopes of Lu. When the ate is used for diagnosis, it may . . . . . . . 99m :23 comprise a radioactive atom for scmtigraphic studies, for example to or I or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine—131, indium-1 l l, fluorine-19,’carbon-l3, nitrogen-15, oxygen-l7, nium, ese or iron.
The radio- or other label is incorporated in the conjugate in known ways. For e, 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 1'23, Rel“, Relgsgand Inl can be attached via a cysteine residue in the peptide. Yttrium-90 can be edu‘via a lySine residue. The IODOGEN method r et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate -123. “Monoclonal Antibodies inlinmunoscintigraphy” (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 sis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either nt one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate. ' 226 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 prodmg (e. g., a peptidyl herapeutic agent, see WOSl/Ol 145) to an active anti-cancer drug (see, e.g., WO 88/07378 and US. Pat. No. 4,975,278).
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable ofacting on a prodrug in such a way so as to covert it into its more active, cytotoxicform. 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; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non—toxic 5-fluorocytosine into the anti-cancer drug, S-fluorouracil; proteases, such as serratia protease, lysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing gs ‘into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid tuents; carbohydrate-cleaving enzymes such as B-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; B-lactamase useful for converting drugs tized with B-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 , respectively, into free drugs. Alternatively, antibodies with enzymatic ty, also known in the art as “abzymes”, can be used to convert the prodrugs of the ion 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 dies by techniques well known in the art such as the use of the heterobifunctional inking reagents discussed above. atively, fusion ns comprising at least the antigen binding region of an antibodyof the invention linked to at least a onally active portion of an enzyme of the invention can be constructedusing 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 2012/024971 glycol, opylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in microcapsules prepared, for example, by coacervation ques or by interfaéial 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 ated 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, 8 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 7724030 (1980); US. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in US. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising atidylcholine, cholesterol and PEG- derivatized atidylethanolamjne (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 bed 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 er al., J.
National Cancer Inst. 81(19)l484 (1989).
Antibodies of the present ion, 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 ic antibodies, indicating that they specifically bind to or entially bind to Influenza A or the M2 protein f, respectively, as compared to a normal control cell. In particular emdodiments, the antibodies are I-IuMZe antibodies, indicating that they ically bind to ls'protein, ably to an epitope of the M2e domain that is only t 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 dy 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. Inanother embodiment, an antibody of the t invention induces apoptosis. In yet r embodiment, an dy of the present invention induces or promotes antibody-dependent cell-mediated cytotoxicity or ment dependent cytotoxicity.
Methods of Identifying and Producing Antibodies Specific for Influenza Virus The present invention provides novel methods for the identification of human anti- nza 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 y adapted to identify antibodies specific for other polypeptides expressed on the cell surface by infectious agents, or even polypeptides expressed on the surface of ectious agent itself.
In general, the methods include obtaining serum s 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., ayftpolypeptide or protein specifically expressed on the surface of cells infected with the infectious agent, but not uninfected cells.
In particular embodiments, the serum samples arecscreened by contacting the samples with a cell that has been transfected with an expression vector that ses 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 es the antibody, using any of the methods described herein or available in the art. Once a B cell that es the dy is identified, cDNAs encoding the variable regions or fragments thereof of the antibody may be cloned using standard RT—PCR vectors and primers ic for conserved antibody sequences, and subcloned in to expression s used for the recombinant production of monoclonal antibodies specific for the infectious agent polypeptide of interest. w; 1.“ » .
In one embodiment, the present invention provides a method of identifying an dy that specifically binds influenza A—infected cells, comprising: contacting an za 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 l value, wherein if the value determined is at least two-fold greater than the control value, an antibody that ically 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 es the M2e domain and enough additional M2 sequence that the n 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 sion vector and ecting an appropriate cell, including those described , may be y accomplished, in View of the M2 and HA sequences being publicly available.
See, for example, the Influenza ce Database (ISD) (flu.lan1.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., aus & 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 bed 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 cellidetected, 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 ihvention may be practiced using high throughput techniques.
Identified human antibodies may then be Characterized further. For example the particular conformational epitopes with in the M26 or HA protein that are necessary or sufficient for binding of the antibody may be determined, e.g., using site-directed mutagenesis of expressed M26 or HA polypeptides. These smay be readily adapted to identify human antibodies that bind any protein expressed on a cell surface. Furthermore, these methods may be adapted to ine binding of the antibody to the virus itself, as opposed to a cell expressing recombinant M26 or HA, or ed 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 ning the identified human monoclonal anti-M2e or anti-HA dy was obtained; producing B cell clones from the mononuclear cells; inducing the B cells to become antibody-producing plasma cells; and screening the supematants produced by the plasma cells to determine if it contains the human onal anti-M26 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 on (RT-PCR) is performed to clone the 'DNAs encoding the variable regions or portions thereof of the human monoclonal anti-M26 or anti— HA antibody These sequences are then subcloned into expression vectors suitable for the recombinant production of human monoclonal anti-M215 or anti-HA antibodies. The g specificity may be confirmed by ining the recombinant antibody’s ability to bind cells sing M2e or HA polypeptide or protein.
In particular embodiments of the methods described herein, B cells isolated from peripheral blood or lymph nodes are soned, 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 supematants 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 f, are subcloned into human antibody expression vectors for recombinant expression.
The resulting recombinant antibodies are then tested-‘to confirm their original g specificity and may be r tested for ecificity across various strains of isolates of the infectious agent.
Thus, in one embodiment, a method of fying human monoclonal anti-M2e or anti-HA dies is ced as follows. First, full length or approximately full length M2 2012/024971 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“.i These methods may be practiced to identify a y of ent HuM2e dies, including antibodies ic 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 le 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 icity is perhaps specific for all A strains of influenza.
Polynucleotides that encode the human monoclonal anti—M2e 0r anti-HA dies or portions thereof of the present invention may be isdiated from cells sing human monoclonal anti-M2e or anti-HA antibodies, according tormethods available in the art and described , including ication 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 ing to molecular biology techniques described in WO 92/02551; US. Patent No. 5,627,052; or Babcook et al., Proc.
Natl. Acad. Sci. USA 93:7843-48 (1996). In certain ments, 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 dy are subcloned and sequenced. The ce of the encoded polypeptide may be readily determined from the polynucleotide sequence.
Isolated polynucleotides encoding a ptide of the present invention may be subcloned into an expression vector to recombinantly e antibodies and polypeptides of the present invention, using procedures known in the art and described herein. 232 :1.
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 e controls and procedures to determine r 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 ular disease or infection, or the patient or t 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 ns 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 ion. However, it is understood that any biological sample sing B cells may be used for any of the ments of the present invention. .[1282] Once isolated, the B cells are induced to produce antibodies, e.g., by culturing the B cells under conditions that support B cell eration or development into a plasmacyte, plasmablast, or plasma cell. The antibodies are then screened, typically using high throughput techniques, to identify an antibody that ically 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 dy is not known, while in other embodiments, the antigen ically bound by the antibody is known.
According to the present invention, B cells may be isolated from a ical 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. r, other methods known in the art may be ed, such as, e.g., column purification using CD19 magnetic beads or IgG-specific magnetic beads, followed by elution from the column. 2012/024971 However, magnetic isolation of B cells utilizing any marker may result in loss of certain B cells. Therefore, in certain ments, 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 ing an antigen-specific antibody, until a single cell specificity per well is ed, thereby facilitating the identification of the B cell that produces the antigen—specific antibody. Cell supematants or a portion thereof and/or cells may be frozen and stored for future testing and later ry of antibody polynucleotides.
In certain embodiments, the B cells are cultured under conditions that favor the production of antibodies by the B cells. For e, the B cells may be cultured under conditions ble for B cell proliferation and differentiation to yield antibody—producing blast, plasmacytes, or plasma cells. In particular ments, 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 supematants or antibodies obtained therefrom may be tested for their y to bind to a target antigen, using routine methods ble in the art, including those described herein. In particular embodiments, culture supematants 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 itre dishes, such that robotic plate handlers may be used to simultaneously sample multiple cell supematants 'and test for the presence of dies 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 g is determined using FMATTM analysis and mentation (Applied Biosystems, Foster City, CA). FMATTM is a fluorescence macro—confocal platform for high-thro‘ughput screening, which mix-and-read, non-radioactive assays using live cells or beads.
In the context of comparing the binding of'vzan’antibody to a ular 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 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 s 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 ures 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 ing 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 e ures in the art (see, e.g., US Patent No. 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, on, transformation, and transfection. Thus, in certain ments expression of an antibody fragment may be preferred in a prokaryotic host cell, such as Escherichia coli (see, e. g., Pluckthun et a1., Methods l. 178:497-515 (1989)). In certain other embodiments, expression of the antibody or an antigen-binding fragment thereof may be preferred in a otic host cell, including yeast (e. g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia is); animal cells (including mammalian cells),;’_,0r plant cells. es of suitable animal cells e, but are not d 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 2012/024971 vector may be designed for expressing n sequences in a particular host system, and then cleotide ces 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 oducing myeloma cell line, such as a mouse NSO line or a ium, such as Ecoli, 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 er 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 tory, New York, 1989; see also Sambrook et al., 3rd ed., Cold Spring Harbor Laboratory, New York, ). 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 7425463 (1977)) and the Amersham International plc sequencing handbook and including improvements thereto.
In particular embodiments, the resulting inant 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 ed 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 itions. In preferred embodiments, these polynucleotides encode a polypeptide-of the ion, 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 nse) 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 manner, and mRNA molecules, which do not contain introns.
Alternatively, or in addition, coding or non-coding sequences are present within a 236 .z‘d‘ WO 12489 ‘ 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 ization assays to detect the presence of an Influenza A dy 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 red embodiments, the cleotide sequences set forth herein encode polypeptides capable of preferentially binding a Influenza A-infected cell as compared to a normal control cted 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 cleotide variants having substantial identity to the sequences set forth herein, for e 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 ion, 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 tide 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 theiiimmunogenic 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 t invention provides polynucleotide nts comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences sed herein. For example, cleotides are provided by this invention that se 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 ediate 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 rs through 200-500; 500-1,000, and the like.
In another embodiment of the invention, polynucleotide compositions are provided that are capable of izing 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 y.
For purposes of ration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS. One skilled in the art will understand that the ency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization on 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 60-650C or 65—700C.
In preferred embodiments, the polypeptide encoded by the cleotide t or nt 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 t invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as ers, polyadenylation signals, onal restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary erably. A nucleic acid fragment of almost any length is ed, with the total length preferably being limited by the ease of preparation and use in the iQteiided recombinant DNA protocol. For example, illustrative polynucleotide segments with tdtal lengths of about , about 5000, about 3000, about 2,000, about 1,000, about 500, abdut 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 ing 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 n may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database ce ison).
In certain embodiments of the present invention, mutagenesis of the sed cleotide 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 ts of both polypeptides and polynucleotides. A mutagenesis approach, such as site-specific mutagenesis, is employed for the ation of variants and/or derivatives of the polypeptides described herein. By this approach, specific cations in a polypeptide sequence are made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to e and test sequence variants, for example, incorporating one or more of the ing 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 sed. Mutations are employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the cleotide itself, and/or alter the properties, activity, composition, stability, or primary ce of the encoded polypeptide.
In other embodiments of the present invention, the polynucleotide sequences provided herein are used as probes or primers for nucleic acids hybridization, e.g., as PCR primers. The y of such nucleic acid probes to ically hybridize to a sequence of interest enable 239 7 , . 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 ce that has the same ce as, or is mentary to, a 15 nucleotide long contiguous sequence disclosed herein is particularly useful. Longer contiguous identical or complementary ces, 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 sed herein, are particularly contemplated as hybridization probes for use in, e. g., Southern and Northern ng, 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 mentary 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 le that is both stable and selective. Molecules having contiguous complementary sequences over stretchesigreiatier than 12 bases in length are generally preferred, , in order to increase stability and selectivity of the hybrid, and thereby improve the y and degree of ic hybrid molecules obtained. Nucleic acid molecules having omplementary stretches of 15 to 25 uous nucleotides, or even longer where desired, nerally preferred.
Hybridization probes are selected from any portion of any of the sequences disclosed . 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 e 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. . 2012/024971 Polynucleotide of the present invention, or fragments or variants thereof, are readily prepared by, for example, directly synthesizing the nt 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 logy of U. S. Patent 4,683,202, by ucing ed 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 c acid of the t 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,icosmids, and mini chromosomes. In Various embodiments, vectors sing a polynucleotide of the present invention are vectors suitable for propagation or replication of the cleotide, or vectors le for sing a ptide 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 s 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 génes. i In order to s a polypeptide of the present iniiention, the nucleotide ces 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 ed 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, forexample, in Sambrook, J., et al. (1989) Molecular Cloning, A tory Manual, Cold Spring Harbor Press, Plainview, N.Y, and Ausubel, F. M. et a1. (1989) Current Protocols1n Molecular Biology, John Wiley & Sons, New York. NY.
A variety of expression vector/host systems are utilized to contain and express polynucleotide sequences. These e, but are not limited to, microorganisms such as bacteria transformed with inant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems ed 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, California), which sells primers for amplifying mouse and human variable s. The primiars are used to amplify heavy or light chain variable regions, which are then efd into s such as ZAPTM H or ImmunoZAPTM L agene), respectively. These vectors are then introduced into E. coli, yeast, or mammalian-based systems for expression. Large s of a single-chain protein containing a fusion of the VH and VL domains are ed using these methods (see Bird 6! 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 ation.
Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable ription and translation elements, including constitutive and inducible promoters, is used.
Examples of promoters suitable for use with prokaryotic hosts include the phoa promoter, [5-lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid ers such as the tac promoter r, other known bacterial promoters are suitable. Promoters for use in bacterial systems also y contain a Shine--Dalgamo sequence operably linked to the DNA encoding the polypeptide. Inducible promoters such as the hybrid lacZ er of the PBLUESCRIPT WO 12489 phagemid (Stratagene, La Jolla, Calif.) or l 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 imately 25 to 30 bases upstream from the site where transcription is initiated. r 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 ian 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, x 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 ISV40), 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 ker. One example of a suitable expression vector is pcDNA—3.l (Invitrogen, Carlsbad, CA), which includes a CMV promoter.
A number of viral-based expression s 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 ting of the late promoter and tripartite leader sequence. Insertion in a sential E1 or E3 region of the viral genome may be used to obtain a viable virus that is capable of expressing the ptide 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 ed ing upon the use intended for the expressed polypeptide; For example, when large quantities are 243 -:,.' 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 d into the vector in frame with ces for the amino- terrninal Met and the subsequent 7 residues of B-galactosidase, so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 26425503- 5509); and the like. pGEX s (Promega, n, WI) are also used to express foreign ptides as fusion ns with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by tion to glutathione— agarose beads followed by elution1n the presenceiof free hione. Proteins madein 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 cérevisiae, 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 dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucdse—6—phosphate isomerase, 3—phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. For s, 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 ions include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase degradative enzymes associated with-'nitrogen metabolism, metallothionein, aldehyde-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable s and promoters for use in yeast expression are further described in EP 73,657. Yeast ers also are advantageously used with yeast promoters.
In cases where plant sion vectors are used, the expression of sequences encoding polypeptides are driven by any of a number of promoters. For e, viral promoters such as the 358 andl9S promoters of CaMV are used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 62307-31 1.
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 . 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 californicq nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in tera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide are cloned into a sential 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 inant 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 st. Such signals include the ATG initiation codon and adjacent sequences. In cases where ces encoding the polypeptide, its tion codon, and upstream ces 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 ed, exogenous translational control signals including the ATG initiation codon are provided. rmore, 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 . Many enhancer sequences are known, including, e. g., those identified in genes encoding globin, elastase, n, oprotein, and insulin. Typically, however, an enhancer i'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 polyoriia enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on ing elements for activation of eukaryotic promoters. The er 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 ces necessary for the termination of transcription and for stabilizing the mRNA. Such ces are commonly available from the Sand: occasionally 3', untranslated regions of otic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the slated n of the mRNA encoding anti-PSCA antibody. One useful ription termination component is the bovine growth hormone polyadenylation . 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, Enrerobacteriaceae 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 omyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. £21“ B, E. coli X1776 (ATCC ), 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, s, and strains are commonly available and used herein, such as saccharomyces 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 (B? 402,226); Pichia pastoris. (EP 183,070); Candida; Trichoderma reesia (EP 244,234); pora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicilliam, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
In certain ments, a host cell strain is chosen for its ability to modulate the expression of the inserted sequences or to process the sed protein in the d fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidatiomfland 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, , and W138, which have specific ar machinery and characteristic mechanisms for such post-translational activities, are chosen to ensure the correct modification and processing of the foreign protein. s and reagents specifically adapted for the expression of antibodies or fragments thereof are also known and available inthe art, including those described, e.g., in US. Patent Nos. 4,816,567 and 6,331,415. In various embodiments, antibody heavy and light chains, or fragments thereof, are sed-ffom the same or separate expression vectors. In one ment, both chains are expressed in the same cell, thereby facilitating the formation of a onal antibody or fragment f.
Full length antibody, antibody fragments, and antibody fusion proteins are produced in bacteria, in particular when glycosylatio'n and PC effector function are not needed, such as when the eutic dy is conjugated to a cytotoxic agent (e. g., a toxin) and the immunoconjugate by itself shows iveness in infected cell destruction. For expression of antibody fragments and ptides in bacteria, see, e.g., US. Pat. Nos. 5,648,237, ,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 r 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. es of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants alrid‘corresPonding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti ito), 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-l 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 Spodopt'erafrugiperda cells. Plant cell cultures of cotton, corn, potato, n, petunia, tomato, and tobacco are also ed 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 sion culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/—DHFR (Cl-IO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. . 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); n green monkey kidney cells 76, ATCC CRL—1587); human al oma 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); TRl cells (Mather et al., Annals NY. Acad. Sci. 383:44—68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells are transformed with the described expression or cloning vectors for polypeptide production'and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transforrnants, 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 ts and a selectable marker gene on the same or on a separate vector. Following the uction of the vector, cells are allowed to grow for 1-2 days in an enriched media before they are ed to selective media. The purpose of the able 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 td recover ormed cell lines. These include, but are not limited to, the herpes simplex,,vims thymidine kinase (Wigler, M. et al. (1977) Cell 112223-32) 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. 7723567— 70); npt, which confers ance to the aminoglycosides, neomycin and 0-418 (Colbere- Garapin, F. et 81) J. M01. Biol. 150:1-14); and als or' pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry; supra). Additional 2012/024971 able genes have been bed. 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. an (1988) Proc. Natl. Acad. Sci. 85:8047—51). The use of visible markers has gained popularity with such s as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transforrnants, 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 M01. 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 e 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 avdesired 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 izations 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 ing 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 -linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two— site, onal-based immunoassay utilizing onal 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 a1. (1990; gical s, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et ail... (1983; J. Exp. Med. 15821211-1216).
Various labels and conjugation techniques;are known by those skilled in the art and are used in various c acid and amino acid . Means for producing labeled hybridization or PCR probes for detecting sequences-related to polynucleotides include oligolabeling, nick translation, beling 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 cially available kits. Suitable er les or labels, which are used include, but are not limited to, uclides', s, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic les, and the like.
The polypeptide ed by a recombinant cell is ed 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 otic cell membrane.
In certain embodiments, a polypeptide of the invention is produced as a fusion ptide 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 allowigurification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Amgen, Seattle, WA). The ion of cleaVable'linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, CA) n the purification domain and the encoded ptide 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. 32263-281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors used for ing fusion proteins is providedin Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).
In certain ments, 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 tliat is recognized and processed (i.e., 250 . cleaved by a signal ase) by the host cell. For prokaryotic host cells, the signal sequence is selected, for example, from the group of the alkaline phOSphatase, penicillinase, lpp, or heat-stable toxin II leaders. For yeast secretion, the signal sequence is selected from, e.g., the yeast invertase leader, 0L factor leader (including Saccharomyces and Kluyveromyces on factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in W0 90/] 3646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are ble.
When using recombinant techniques, the polypeptide or dy 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 6! al., Bio/Technology 10: 163-167 (1992) be 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 methylsulforiylfluoride (PMSF) over about 30 min. Cell debris is removed by centrifugation. Where the polypeptide or dy is ed into the medium, supematants from such expression systems are generally first concentrated using a commercially ble 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 itious contaminants.
The polypeptide or antibody composition prepared from the cells are purified using, for e, hydroxylapatite chromatography, gel-electrophoresis, dialysis, and affinity tography, with affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the species and e 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 y], 72, or 7.; heavy chains (Lindmark et al., J. Immunol. Meth. 3 (1983)). n G is recommended for all mouse isotypes and for human Y3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is ed 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, NJ.) is useful for purification. Other techniques for n 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 ing on the polypeptide or antibody to be recovered.
Following any preliminary purification step(s), the mixture comprising the polypeptide or antibody of interest and inants are ted 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 ations including a polypeptide, antibody, or tor of the present invention, at a desired degree of purity, and a pharmaceutically acceptable carrier, excipient, or stabilizer (Remingion's Pharmaceutical Sciences 16th edition, 0501, A. Ed. (1980)). In certain embodiments, pharmaceutical formulations are prepared to e the stability of the polypeptide or antibody during storage, e. g., in the form of lyophilized formulations or aqueous ons.
Acceptable carriers, excipients, or stabilizers are nontoxic to ents at the dosages and concentrations employed, and include, e. g., s such as acetate, Tris, phosphate, e, and other organic acids; antioxidants including ic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium de; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl ns such as methyl or propyl n; 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 glycinefglutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbbhydrates including glucose, mannose, or dextrins; chelating agents such as ESTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, lose or sorbitol; surfactant such as polysorbate; salt—forming counter-ions such as sodium; metal complexes (6. g. Zn—protein complexes); and/0r non—ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). In certain embodiments, the therapeutic formulation preferably

Claims (6)

What we claim is:
1. A ition coinprising: (a) an isolated human monoclonal antibody, wherein said antibody comprises a heavy chain variable regio~i comprising the amino acid sequence ofSEQ ID . and a light chain variable region comprising the amino acid sequence ofSEQ ID NO:778;and (b) an isolated human il~tonoclonal antibody, wherein said antibody comprises: a VH CDR1 r°egion comprising the amino acid ce ofNYYWS(SEQ ID NO:72);a VH CDR2 region rising the amino acid ce ofFIYYGGNTKYNPSLKS{SEQ ILA NO:74);a VH LDR3 region comprising the amino acid sequence ofASCSGGYCILD(SEQ ID NO:7b);a VLCDR1 region comprising the amino acid sequence NIYKYLN(SEQ ID NO:59); a VL CDR2region comprising the amino acid sequence ofAASGLQS(SEQ ID NO:61);and a VLCDR3 region comprising the amino acid sequence ofand QQSYSPPLT(SEQ ID , n at least one ofthe antibody of(a)and(h)is directly conjugated to either a detectable label or a therapeutic agent.
2. A passive immunization composition comprising the composition ofclair7~ 1.
3. A pharmaceutical composition sing the composition ofclaim 1 and a pharmaceutical carrier.
4. A composition as defined in claim l and as substantially described herein with reference to one or more ofthe accompanying examples.
5. A passive immunization composition.comprising the composition ofclaim 4.
6. A pharmaceutical composition comprising the composition ofclaim 4 and a pharmaceutical carrier. Theraclone Sciences,Inc. by the patent attorneys for the applicant
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