U.S. patent application number 14/578777 was filed with the patent office on 2015-04-16 for compositions and methods for the therapy and diagnosis of influenza.
The applicant listed for this patent is Theraclone Sciences, Inc.. Invention is credited to Thomas C. Cox, Andres G. Grandea, III, Phil Hammond, Gordon King, Jennifer Mitcham, Matthew Moyle, Ole Olsen.
Application Number | 20150104459 14/578777 |
Document ID | / |
Family ID | 46637044 |
Filed Date | 2015-04-16 |
United States Patent
Application |
20150104459 |
Kind Code |
A1 |
Grandea, III; Andres G. ; et
al. |
April 16, 2015 |
Compositions and Methods for the Therapy and Diagnosis of
Influenza
Abstract
The present invention provides compositions, vaccines, and
methods for diagnosing, treating, and preventing influenza
infection using a combination of antibodies raised against the
influenza hemagglutinin and the matrix 2 ectodomain
polypeptides.
Inventors: |
Grandea, III; Andres G.;
(Shoreline, WA) ; King; Gordon; (Shoreline,
WA) ; Cox; Thomas C.; (Redmond, WA) ; Olsen;
Ole; (Everettt, WA) ; Mitcham; Jennifer;
(Redmond, WA) ; Moyle; Matthew; (Newtown, CT)
; Hammond; Phil; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Theraclone Sciences, Inc. |
Seattle |
WA |
US |
|
|
Family ID: |
46637044 |
Appl. No.: |
14/578777 |
Filed: |
December 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13372870 |
Feb 14, 2012 |
8916160 |
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14578777 |
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61442733 |
Feb 14, 2011 |
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Current U.S.
Class: |
424/139.1 ;
424/142.1; 435/5 |
Current CPC
Class: |
C07K 2317/565 20130101;
C07K 2317/51 20130101; C07K 2317/515 20130101; A61K 2039/505
20130101; A61P 31/16 20180101; A61P 31/00 20180101; A61K 39/42
20130101; G01N 2333/11 20130101; C07K 2317/34 20130101; A61K
2039/507 20130101; A61K 45/06 20130101; C07K 2317/21 20130101; C07K
16/1018 20130101; C07K 2317/76 20130101; G01N 33/54306 20130101;
G01N 33/56983 20130101 |
Class at
Publication: |
424/139.1 ;
424/142.1; 435/5 |
International
Class: |
C07K 16/10 20060101
C07K016/10; A61K 45/06 20060101 A61K045/06; G01N 33/543 20060101
G01N033/543; A61K 39/42 20060101 A61K039/42 |
Claims
1. A composition comprising: (a) an isolated human antibody or
antigen-binding fragment thereof that specifically binds to an
epitope of the hemagglutinin (HA) glycoprotein of an influenza
virus; and (b) an isolated human monoclonal antibody or
antigen-binding fragment thereof that specifically binds to an
epitope in the extracellular domain of the matrix 2 ectodomain
(M2e) polypeptide of an influenza virus.
2. The composition of claim 1, wherein said isolated human
monoclonal antibody or antigen-binding fragment thereof that
specifically binds an epitope of the M2e polypeptide is selected
from the group consisting of: (a) an antibody or antigen-binding
fragment thereof comprising a heavy chain variable (VH) domain and
a light chain variable (VL) domain, wherein the VH domain and the
VL domain each comprise three complementarity determining regions 1
to 3 (CDR1-3), and wherein each CDR comprises the following amino
acid sequences: VH CDR1: SEQ ID NOs: 72, 103, 179, 187, 203, 211,
228, 252, 260, 268, 284, 293, or 301; VH CDR2: SEQ ID NOs: 74, 105,
180, 188, 204, 212, 229, 237, 253, 261, 269, 285, or 294; VH CDR3
SEQ ID NOs: 76, 107, 181, 189, 197, 205, 213, 230, 238, 254, 262,
270, 286, or 295; VL CDR1: SEQ ID NOs: 59, 92, 184, 192, 208, 192,
233, 241, 265, or 273; VL CDR2: SEQ ID NOs: 61, 94, 185, 193, 209,
217, 226, 234, 258, 274, or 282; and VL CDR3: SEQ ID NOs: 63, 96,
186, 194, 210, 218, 243, 259, 267, 275, 291, or 300; and (b) an
antibody or antigen-binding fragment thereof comprising a heavy
chain variable (VH) domain and a light chain variable (VL) domain,
wherein the VH domain and the VL domain each comprise three
complementarity determining regions 1 to 3 (CDR1-3), and wherein
each CDR comprises the following amino acid sequences: VH CDR1: SEQ
ID NOs: 109, 112, 182, 190, 206, 214, 239, 255, 263, 271, 287, 296,
or 304; VH CDR2: SEQ ID NOs: 110, 113, 183, 191, 207, 215, 232,
240, 256, 264, 272, 288, or 297; VH CDR3 SEQ ID NOs: 76, 107, 181,
189, 197, 205, 213, 230, 238, 254, 262, 270, 286, or 295; VL CDR1:
SEQ ID NOs: 59, 92, 184, 192, 208, 192, 223, 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.
3. The composition of claim 1, wherein said isolated human antibody
that specifically binds an epitope of the HA glycoprotein comprises
a heavy chain variable region (VH) domain and a light chain
variable (VL) domain, wherein the VH domain and the VL domain each
comprise three complementarity determining regions 1 to 3 (CDR1-3),
and wherein each CDR comprises the following amino acid sequences:
VH CDR1: SEQ ID NOs: 247, 571, 586, 597, 603, 609, 615, 627, 633,
637, 643, 649, 658, 664, 670, 303, 251, 242, or 222; VH CDR2: SEQ
ID NOs: 248, 572, 587, 592, 598, 604, 610, 616, 628, 634, 638, 644,
650, 655, 659, 665, 671, 306, 249, 307, or 221; VH CDR3: SEQ ID
NOs: 568, 573, 588, 593, 599, 605, 611, 617, 629, 635, 639, 645,
651, 656, 660, 666, 672, 725, 246, 290, or 220; VL CDR1: SEQ ID
NOs: 569, 574, 577, 580, 583, 589, 594, 612, 618, 621, 624, 640,
646, 652, 661, 667, 285, 289, 245, 224, or 219; VL CDR2: SEQ ID
NOs: 570, 575, 578, 581, 584, 590, 595, 601, 607, 613, 619, 622,
625, 631, 653, 662, 668, 305, 223, or 231; VL CDR3: SEQ ID NOs:
289, 576, 579, 582, 585, 591, 596, 602, 608, 614, 620, 623, 626,
632, 636, 642, 648, 654, 657, 663, 669, 308, 250, 227, or 280.
4. The composition of claim 1, wherein said epitope of the HA
glycoprotein is 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).
5. The composition of claim 1, wherein said epitope of the M2e
polypeptide is a discontinuous epitope.
6. The composition of claim 1, wherein said epitope of the M2e
polypeptide comprises, a) the amino acid at positions 2, 5, and 6
of MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1); or b) the amino acid at
positions 2, 5, and 6 of SLLTEV (SEQ ID NO: 42).
7. The composition of claim 1 comprising: (a) an isolated human
anti-HA antibody, or an antigen-binding fragment thereof,
comprising a heavy chain variable region (VH) domain, wherein the
VH domain comprises the following amino acid sequences: SEQ ID NOs
309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357,
361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409,
199, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 483,
489, 495, 501, 507, 513, 519, 525, 531, 537, 543, 550, 556, or 562,
and a light chain variable (VL) domain, wherein the VL domain
comprises the following amino acid sequences: SEQ ID NOs 310, 314,
318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366,
370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 420,
426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492, 498,
504, 510, 516, 522, 528, 534, 540, 547, 553, 559, or 565; and (b)
an isolated anti-matrix 2 ectodomain (M2e) antibody, or
antigen-binding fragment thereof, comprising a heavy chain variable
(VH) domain, wherein the VH domain comprises the following amino
acid sequences: SEQ ID NOs 44, 277, 276, 50, 236, 235, 116, 120,
124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, or
176, and a light chain variable (VL) domain, wherein the VL domain
comprises 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.
8. A multivalent vaccine composition comprising the composition of
claim 1.
9. A pharmaceutical composition comprising the composition of claim
1 and a pharmaceutical carrier.
10. A method for stimulating an immune response in a subject,
comprising administering to the subject the composition of claim
9.
11. A method for the treatment of an influenza virus infection in a
subject in need thereof, comprising administering to said subject
the composition of claim 9.
12. The method of claim 11, wherein the subject has been exposed to
an influenza virus.
13. The method of claim 12, wherein the subject has not be
diagnosed with an influenza infection.
14. A method for the prevention of an influenza virus infection in
a subject in need thereof, comprising administering to said subject
the vaccine of claim 8, prior to exposure of said subject to an
influenza virus.
15. The method of claim 11, wherein the method further comprises
administering an anti-viral drug, a viral entry inhibitor or a
viral attachment inhibitor.
16. The method of claim 15, wherein said anti-viral drug is a
neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or
an M2 ion channel.
17. The method of claim 16, wherein said M2 ion channel inhibitor
is amantadine or rimantadine.
18. The method of claim 16, wherein said neuraminidase inhibitor is
zanamivir or oseltamivir phosphate.
19. The method of claim 11, further comprising administering a
second anti-Influenza A antibody.
20. The method of claim 19, wherein said antibody is administered
prior to or after exposure to Influenza virus.
21. The method of claim 12, wherein the subject is at risk of
contracting an influenza infection.
22. The method of claim 11, wherein said composition is
administered at a dose sufficient to promote viral clearance or
eliminate influenza infected cells.
23. A method for determining the presence of an Influenza virus
infection in a subject, comprising the steps of: (a) contacting a
biological sample obtained from the subject with the antibody of
claim 1; (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.
24. The method of claim 23, wherein the control value is determined
by contacting a control sample obtained from the subject with the
antibody according to claim 1 and detecting an amount of the
antibody that binds to the control sample.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
13/372,870, filed Feb. 14, 2012, which claims the benefit of
provisional application U.S. Ser. No. 61/442,733, filed Feb. 14,
2011, the contents of which are herein incorporated by reference in
their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file named
"THER018C01US_SeqList.txt," which was created on Dec. 22, 2014 and
is 909 KB in size, are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to prevention,
diagnosis, therapy and monitoring of influenza infection. The
invention is more specifically related to compositions containing a
combination of human antibodies raised against either the influenza
hemagglutinin or matrix 2 protein. Such compositions are useful in
pharmaceutical compositions for the prevention and treatment of
influenza, and for the diagnosis and monitoring of influenza
infection.
BACKGROUND OF THE INVENTION
[0004] Influenza virus infects 5-20% of the population and results
in 30,000-50,000 deaths each year in the U.S. Disease caused by
influenza A viral infections is typified by its cyclical nature.
Antigenic drift and shift allow for different A strains to emerge
every year. Added to that, the threat of highly pathogenic strains
entering into the general population has stressed the need for
novel therapies for flu infections.
SUMMARY OF THE INVENTION
[0005] The invention provides diagnostic, prophylactic, and
therapeutic compositions including a human antibody raised against
the Influenza hemagglutinin protein and a human monoclonal antibody
raised against the Influenza M2 protein. Moreover, the invention
provides diagnostic, prophylactic, and therapeutic compositions
including an isolated human antibody raised against an epitope of
the Influenza hemagglutinin protein and an isolated human
monoclonal antibody raised against an epitope of the Influenza M2
protein. Furthermore, these compositions are pharmaceutical
compositions that include a pharmaceutical carrier. These
compositions address a long-felt need in the art for pharmaceutical
compositions that both strongly neutralizes Influenza virus
infection and recognizes constant regions within proteins common to
all Influenza strains.
[0006] Specifically, the invention provides a composition
including: (a) an isolated human antibody that specifically binds
to an epitope of the hemagglutinin (HA) glycoprotein of an
influenza virus; and (b) an isolated human monoclonal antibody that
specifically binds to an epitope in the extracellular domain of the
matrix 2 ectodomain (M2e) polypeptide of an influenza virus. In
certain embodiments of this composition, the isolated human
monoclonal antibody that specifically binds an epitope of the M2e
polypeptide is TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23,
3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05,
3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10,
3260_D19, 3362_B11, or 3242_P05. Moreover, the isolated human
antibody that specifically binds an epitope of the HA glycoprotein
is optionally TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523
(5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527
(5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530
(5248_H10), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533
(5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536
(5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539
(5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542
(5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545
(5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548
(5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551
(5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554
(5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557
(5081_A04), TCN 558 (5248_H10b), TCN-559 (5097_G08), TCN-560
(5084_P10), TCN-504 (3251_K17), SC06-141, SC06-255, SC06-257,
SC06-260, SC06-261, SC06-262, SC06-268, SC06-272, SC06-296,
SC06-301, SC06-307, SC06-310, SC06-314, SC06-323, SC06-325,
SC06-327, SC06-328, SC06-329, SC06-331, SC06-332, SC06-334,
SC06-336, SC06-339, SC06-342, SC06-343, SC06-344, CR6141, CR6255,
CR6257, CR6260, CR6261, CR6262, CR6268, CR6272, CR6296, CR6301,
CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329,
CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, CR6344, 2A,
D7, D8, F10, G17, H40, A66, D80, E88, E90, or H98.
[0007] The epitope of the HA glycoprotein is optionally
GVTNKVNSIIDK (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO: 283),
GVTNKENSIIDK (SEQ ID NO: 202), GVTNKVNRIIDK (SEQ ID NO: 201),
GITNKVNSVIEK (SEQ ID NO: 281), GITNKENSVIEK (SEQ ID NO: 257),
GITNKVNSIIDK (SEQ ID NO: 225), and KITSKVNNIVDK (SEQ ID NO: 216).
The influenza hemaglutinin (HA) glycoprotein includes an HA1 and
HA2 subunit. Exemplary epitopes of the HA glycoprotein include the
HA1 subunit, HA2 subunit, or both the HA1 and HA2 subunits.
Alternatively, or in addition, the epitope of the M2e polypeptide
is a discontinuous epitope. For example, the epitope of the M2e
polypeptide includes the amino acid at positions 2, 5, and 6 of
MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1) or the amino acid at
positions 2, 5, and 6 of SLLTEV (SEQ ID NO: 42).
[0008] The invention further provides a composition including: (a)
an isolated human anti-HA antibody, or an antigen-binding fragment
thereof, including a heavy chain variable region (VH) domain and a
light chain variable (VL) domain, wherein the VH domain and the VL
domain each contain three complementarity determining regions 1 to
3 (CDR1-3), and wherein each CDR includes the following amino acid
sequences: VH CDR1: SEQ ID NOs: 247, 571, 586, 597, 603, 609, 615,
627, 633, 637, 643, 649, 658, 664, 670, 303, 251, 242, or 222; VH
CDR2: SEQ ID NOs: 248, 572, 587, 592, 598, 604, 610, 616, 628, 634,
638, 644, 650, 655, 659, 665, 671, 306, 249, 307, or 221; VH CDR3:
SEQ ID NOs: 568, 573, 588, 593, 599, 605, 611, 617, 629, 635, 639,
645, 651, 656, 660, 666, 672, 725, 246, 290, or 220; VL CDR1: SEQ
ID NOs: 569, 574, 577, 580, 583, 589, 594, 612, 618, 621, 624, 640,
646, 652, 661, 667, 285, 289, 245, 224, or 219; VL CDR2: SEQ ID
NOs: 570, 575, 578, 581, 584, 590, 595, 601, 607, 613, 619, 622,
625, 631, 653, 662, 668, 305, 223, or 231; VL CDR3: SEQ ID NOs:
289, 576, 579, 582, 585, 591, 596, 602, 608, 614, 620, 623, 626,
632, 636, 642, 648, 654, 657, 663, 669, 308, 250, 227, or 280; and
(b) an isolated anti-matrix 2 ectodomain (M2e) antibody, or
antigen-binding fragment thereof, including a heavy chain variable
(VH) domain and a light chain variable (VL) domain, wherein the VH
domain and the VL domain each contain three complementarity
determining regions 1 to 3 (CDR1-3), and wherein each CDR includes
the following amino acid sequences: VH CDR1: SEQ ID NOs: 72, 103,
179, 187, 203, 211, 228, 252, 260, 268, 284, 293, or 301; VH CDR2:
SEQ ID NOs: 74, 105, 180, 188, 204, 212, 229, 237, 253, 261, 269,
285, or 294; VH CDR3 SEQ ID NOs: 76, 107, 181, 189, 197, 205, 213,
230, 238, 254, 262, 270, 286, or 295; VL CDR1: SEQ ID NOs: 59, 92,
184, 192, 208, 192, 233, 241, 265, or 273; VL CDR2: SEQ ID NOs: 61,
94, 185, 193, 209, 217, 226, 234, 258, 274, or 282; and VL CDR3:
SEQ ID NOs: 63, 96, 186, 194, 210, 218, 243, 259, 267, 275, 291, or
300.
[0009] Alternatively, or in addition, the invention provides a
composition including: (a) an isolated human anti-HA antibody, or
an antigen-binding fragment thereof, including a heavy chain
variable region (VH) domain and a light chain variable (VL) domain,
wherein the VH domain and the VL domain each contain three
complementarity determining regions 1 to 3 (CDR1-3), and wherein
each CDR includes the following amino acid sequences: VH CDR1: SEQ
ID NOs: 247, 571, 586, 597, 603, 609, 615, 627, 633, 637, 643, 649,
658, 664, 670, 303, 251, 242, or 222; VH CDR2: SEQ ID NOs: 248,
572, 587, 592, 598, 604, 610, 616, 628, 634, 638, 644, 650, 655,
659, 665, 671, 306, 249, 307, or 221; VH CDR3: SEQ ID NOs: 568,
573, 588, 593, 599, 605, 611, 617, 629, 635, 639, 645, 651, 656,
660, 666, 672, 725, 246, 290, or 220; VL CDR1: SEQ ID NOs: 569,
574, 577, 580, 583, 589, 594, 612, 618, 621, 624, 640, 646, 652,
661, 667, 285, 289, 245, 224, or 219; VL CDR2: SEQ ID NOs: 570,
575, 578, 581, 584, 590, 595, 601, 607, 613, 619, 622, 625, 631,
653, 662, 668, 305, 223, or 231; VL CDR3: SEQ ID NOs: 289, 576,
579, 582, 585, 591, 596, 602, 608, 614, 620, 623, 626, 632, 636,
642, 648, 654, 657, 663, 669, 308, 250, 227, or 280; and (b) an
isolated anti-matrix 2 ectodomain (M2e) antibody, or
antigen-binding fragment thereof, including a heavy chain variable
(VH) domain and a light chain variable (VL) domain, wherein the VH
domain and the VL domain each contain three complementarity
determining regions 1 to 3 (CDR1-3), and wherein each CDR includes
the following amino acid sequences: VH CDR1: SEQ ID NOs: 109, 112,
182, 190, 206, 214, 239, 255, 263, 271, 287, 296, or 304; VH CDR2:
SEQ ID NOs: 110, 113, 183, 191, 207, 215, 232, 240, 256, 264, 272,
288, or 297; VH CDR3 SEQ ID NOs: 76, 107, 181, 189, 197, 205, 213,
230, 238, 254, 262, 270, 286, or 295; VL CDR1: SEQ ID NOs: 59, 92,
184, 192, 208, 192, 233, 241, 265, or 273; VL CDR2: SEQ ID NOs: 61,
94, 185, 193, 209, 217, 226, 234, 258, 274, or 282; and VL CDR3:
SEQ ID NOs: 63, 96, 186, 194, 210, 218, 243, 259, 267, 275, 291, or
300.
[0010] The invention provides a composition including: (a) an
isolated human anti-HA antibody, or an antigen-binding fragment
thereof, including a heavy chain variable region (VH) domain,
wherein the VH domain includes the following amino acid sequences:
SEQ ID NOs 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349,
353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401,
405, 409, 199, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471,
477, 483, 489, 495, 501, 507, 513, 519, 525, 531, 537, 543, 550,
556, or 562, and a light chain variable (VL) domain, wherein the VL
domain includes the following amino acid sequences: SEQ ID NOs 310,
314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362,
366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414,
420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 486, 492,
498, 504, 510, 516, 522, 528, 534, 540, 547, 553, 559, or 565; and
(b) an isolated anti-matrix 2 ectodomain (M2e) antibody, or
antigen-binding fragment thereof, including a heavy chain variable
(VH) domain, wherein the VH domain includes the following amino
acid sequences: SEQ ID NOs 44, 277, 276, 50, 236, 235, 116, 120,
124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, or
176, and a light chain variable (VL) domain, wherein the VL domain
includes the following amino acid sequences: SEQ ID NOs 46, 292,
52, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162,
166, 170, or 178.
[0011] Furthermore, the invention provides a multivalent vaccine
composition including any of the compositions described herein
containing an isolated human anti-HA antibody, or an
antigen-binding fragment thereof and an isolated anti-matrix 2
ectodomain (M2e) antibody, or antigen-binding fragment thereof.
Alternatively, the multivalent vaccine includes antibodies that
bind to the epitopes to which the antibodies of the invention bind.
Exemplary antibodies of the invention include, but are not limited
to, TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10,
3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13,
3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19,
3362_B11, 3242_P05, TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523
(5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527
(5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530
(5248_H10), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533
(5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536
(5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539
(5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542
(5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545
(5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548
(5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551
(5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554
(5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557
(5081_A04), TCN 558 (5248_H10b), TCN-559 (5097_G08), TCN-560
(5084_P10), TCN-504 (3251_K17), SC06-141, SC06-255, SC06-257,
SC06-260, SC06-261, SC06-262, SC06-268, SC06-272, SC06-296,
SC06-301, SC06-307, SC06-310, SC06-314, SC06-323, SC06-325,
SC06-327, SC06-328, SC06-329, SC06-331, SC06-332, SC06-334,
SC06-336, SC06-339, SC06-342, SC06-343, SC06-344, CR6141, CR6255,
CR6257, CR6260, CR6261, CR6262, CR6268, CR6272, CR6296, CR6301,
CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329,
CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, CR6344, D7,
D8, F10, G17, H40, A66, D80, E88, E90, and H98. For example, the
multivalent vaccine may include one or more of the following
epitopes: GVTNKVNSIIDK (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO:
283), GVTNKENSIIDK (SEQ ID NO: 202), GVTNKVNRIIDK (SEQ ID NO: 201),
GITNKVNSVIEK (SEQ ID NO: 281), GITNKENSVIEK (SEQ ID NO: 257),
GITNKVNSIIDK (SEQ ID NO: 225), KITSKVNNIVDK (SEQ ID NO: 216),
MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1), and
MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1) provided in its native
conformation.
[0012] The multivalent vaccine also includes a composition
including: (a) a human antibody that specifically binds to an
epitope of the hemagglutinin (HA) glycoprotein of an influenza
virus; and (b) a human monoclonal antibody that specifically binds
to an epitope in the extracellular domain of the matrix 2
ectodomain (M2e) polypeptide of an influenza virus.
[0013] The invention provides a pharmaceutical composition
including any one of the compositions described herein. Moreover,
the pharmaceutical composition includes a pharmaceutical
carrier.
[0014] The invention provides a method for stimulating an immune
response in a subject, including administering to the subject the
pharmaceutical composition described herein. The pharmaceutical
composition may administered prior to or after exposure of the
subject to an Influenza virus.
[0015] The invention also provides a method for the treatment of an
influenza virus infection in a subject in need thereof, including
administering to the subject the pharmaceutical composition
described herein. The subjection may have been exposed to an
influenza virus. Alternatively, or in addition, the subject has not
been diagnosed with an influenza infection. The pharmaceutical
composition may administered prior to or after exposure of the
subject to an Influenza virus. Preferably, the pharmaceutical
composition is administered at a dose sufficient to promote viral
clearance or eliminate influenza infected cells.
[0016] The invention further provides a method for the prevention
of an influenza virus infection in a subject in need thereof,
including administering to the subject a vaccine composition
described herein, prior to exposure of the subject to an influenza
virus. In certain embodiments of this method, the subject is at
risk of contracting an influenza infection. The pharmaceutical
composition may administered prior to or after exposure of the
subject to an Influenza virus. Preferably, the pharmaceutical
composition is administered at a dose sufficient to promote viral
clearance or eliminate influenza infected cells.
[0017] The treatment and prevention methods provided by the
invention further include administering an anti-viral drug, a viral
entry inhibitor or a viral attachment inhibitor. Exemplary
anti-viral drugs include, but are not limited to, a neuraminidase
inhibitor, a HA inhibitor, a sialic acid inhibitor, or an M2 ion
channel inhibitor. In certain aspects of these methods, the M2 ion
channel inhibitor is amantadine or rimantadine. In other aspects of
these methods, the neuraminidase inhibitor is zanamivir or
oseltamivir phosphate. The antiviral drug may administered prior to
or after exposure of the subject to an Influenza virus.
[0018] The treatment and prevention methods provided by the
invention further include administering a second anti-Influenza A
antibody. The second antibody is optionally an antibody described
herein. The second antibody may administered prior to or after
exposure of the subject to an Influenza virus.
[0019] The invention provides a method for determining the presence
of an Influenza virus infection in a subject, including the steps
of: (a) contacting a biological sample obtained from the subject
with any one of the antibodies or pharmaceutical compositions
described herein; (b) detecting an amount of the antibody that
binds to the biological sample; and (c) comparing the amount of
antibody that binds to the biological sample to a control value,
and therefrom determining the presence of the Influenza virus in
the subject. Optionally, the control value is determined by
contacting a control sample obtained from the subject with any one
of the antibodies or pharmaceutical compositions described herein
and detecting an amount of the antibody that binds to the control
sample.
[0020] The invention also provides a diagnostic kit including any
one of the antibodies, compositions, or pharmaceutical compositions
described herein.
[0021] The invention further provides a prophylactic kit including
a vaccine composition described herein. Preferably, the vaccine is
a multivalent vaccine. The term "multivalent vaccine" describes a
single vaccine that elicits an immune response either to more than
one infectious agent, e.g. the influenza HA glycoprotein and the
influenza M2e polypeptide, or to several different epitopes of a
molecule, e.g. HA epitopes shown in SEQ ID NOs 198, 283, 202, 201,
281, 257, 225, and 216. Alternatively, or in addition, the term
multivalent vaccine is meant to describe the administration of a
combination of human antibodies raised against more than one
infectious agent, e.g. the influenza HA glycoprotein and the
influenza M2e polypeptide.
[0022] Other features and advantages of the invention will be
apparent from and are encompassed by the following detailed
description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows the binding of three antibodies of the present
invention and control hu14C2 antibody to 293-HEK cells transfected
with an M2 expression construct or control vector, in the presence
or absence of free M2 peptide.
[0024] FIGS. 2A and B are graphs showing human monoclonal antibody
binding to influenza A/Puerto Rico/8/32.
[0025] FIG. 3A is a chart showing amino acid sequences of
extracellular domains of M2 variants (SEQ ID NOS 1-3, 679 &
5-40, respectively, in order of appearance).
[0026] FIGS. 3B and C are bar charts showing binding of human
monoclonal anti-influenza antibody binding to M2 variants shown in
FIG. 3A.
[0027] FIGS. 4A and B are bar charts showing binding of human
monoclonal anti-influenza antibody binding to M2 peptides subjected
to alanine scanning mutagenesis.
[0028] FIG. 5 is a series of bar charts showing binding of MAbs
8i10 and 23K12 to M2 protein representing influenza strain
A/HK/483/1997 sequence that was stably expressed in the CHO cell
line DG44.
[0029] FIG. 6A is a chart showing cross reactivity binding of
anti-M2 antibodies to variant M2 peptides (SEQ ID NOS 680-704,
respectively, in order of appearance).
[0030] FIG. 6B is a chart showing binding activity of M2 antibodies
to truncated M2 peptides (SEQ ID NOS 680, 705-724 &19,
respectively, in order of appearance).
[0031] FIG. 7 is a graph showing survival of influenza infected
mice treated with human anti-influenza monoclonal antibodies.
[0032] FIG. 8 is an illustration showing the anti-M2 antibodies
bind a highly conserved region in the N-Terminus of M2e (SEQ ID NO:
19).
[0033] FIG. 9 is a graph showing anti-M2 rHMAb clones from crude
supernatant bound to influenza on ELISA, whereas the control
anti-M2e mAb 14C2 did not readily bind virus.
[0034] FIG. 10 is a series of photographs showing anti-M2 rHMAbs
bound to cells infected with influenza. MDCK cells were or were not
infected with influenza A/PR/8/32 and Ab binding from crude
supernatant was tested 24 hours later. Data were gathered from the
FMAT plate scanner.
[0035] FIG. 11 is a graph showing anti-M2 rHMAb clones from crude
supernatant bound to cells transfected with the influenza subtypes
H3N2, HK483, and VN1203 M2 proteins. Plasmids encoding full length
M2 cDNAs corresponding to influenza strains H3N2, HK483, and
VN1203, as well as a mock plasmid control, were transiently
transfected into 293 cells. The 14C2, 8i10, 23K12, and 21B15 mABs
were tested for binding to the transfectants, and were detected
with an AF647-conjugated anti-human IgG secondary antibody. Shown
are the mean fluorescence intensities of the specific mAB bound
after FACS analysis.
[0036] FIGS. 12A-B are amino acid sequences of the variable regions
of anti-M2e mAbs. Framework regions 1-4 (FR 1-4) and
complementarity determining regions 1-3 (CDR 1-3) for VH and Vk are
shown. FR, CDR, and gene names are defined using the nomenclature
in the IMGT database (IMGT.RTM., the International ImMunoGeneTics
Information System.RTM.). Grey boxes denote identity with the
germline sequence which is shown in light blue boxes, hyphens
denote gaps, and white boxes are amino acid replacement mutations
from the germline.
[0037] FIG. 13 is a graph depicting the results of a competition
binding analysis of a panel of anti-M2e mAbs with TCN-032 Fab. The
indicated anti-M2e mAbs were used to bind to the stable CHO
transfectant expressing M2 of A/Hong Kong/483/97 that had
previously been treated with or without 10 .mu.g/mL TCN-032 Fab
fragment. The anti-M2e mAb bound to the cell surface was detected
with goat anti-huIgG FcAlexafluor488 FACS and analyzed by flow
cytometry. The results are derived from one experiment.
[0038] FIG. 14A is a graph depicting the ability of anti-M2e mAbs
TCN-032 and TCN-031 to bind virus particles and virus-infected
cells but not M2e-derived synthetic peptide. Purified influenza
virus (A/Puerto Rico/8/34) was coated at 10 .mu.g/ml on ELISA wells
and binding of anti-M2e mAbs TCN-031, TCN-032, ch14C2, and the HCMV
mAbs 2N9 was evaluated using HRP-labeled goat anti-human Fc.
Results shown are representative of 3 experiments.
[0039] FIG. 14B is a graph depicting the ability of anti-M2e mAbs
TCN-032 and TCN-031 to bind virus particles and virus-infected
cells but not M2e-derived synthetic peptide. 23mer synthetic
peptide of M2 derived from A/Fort Worth/1/50 was coated at 1
.mu.g/ml on ELISA wells and binding of mAbs TCN-031, TCN-032,
ch14C2, and 2N9 were evaluated as in panel a. Results shown are
representative of 3 experiments.
[0040] FIG. 14C is a graph depicting the ability of anti-M2e mAbs
TCN-032 and TCN-031 to bind virus particles and virus-infected
cells but not M2e-derived synthetic peptide. MDCK cells were
infected with A/Puerto Rico/8/34 (PR8) and subsequently stained
with mAbs TCN-031, TCN-032, ch14C2 and the HCMV mAb 5J12. Binding
of antibodies was detected using Alexafluor 647-conjugated goat
anti-Human IgG H&L antibody and quantified by flow cytometry.
Results shown are representative of 3 experiments.
[0041] FIG. 14D is a series of photographs depicting HEK 293 cells
stably transfected with the M2 ectodomain of A/Fort Worth/1/50
(D20) were stained with transient transfection supernatant
containing mAbs TCN-031, TCN-032, or the control ch14C2 and
analyzed by FMAT for binding to M2 in the presence or absence of 5
ug/ml M2e peptide. Mock transfected cells are 293 cells stably
transfected with vector alone. Results shown are representative of
one experiment.
[0042] FIGS. 15A-D are graphs depicting the Therapeutic efficacy of
anti-M2 mAbs TCN-031 and TCN-032 in mice. Mice (n=10) were infected
by intranasal inoculation with 5.times..sub.LD50 A/Vietnam/1203/04
(H5N1) (panels A-B) or (n=5) with 5.times..sub.LD50 A/Puerto Rico
8/34 (H1N1) (panels C-D), followed by 3 intraperitoneal (ip)
injections with mAbs at 24, 72, and 120 hours post-infection (a
total of 3 mAb injections per mouse) and weighed daily for 14 days.
Percentage survival is shown in a and c, whereas percent weight
change of mice is shown in B and D. The results shown for the
treatment study of mice infected with A/Vietnam/1203/04 (H5N1) are
representative of 2 experiments.
[0043] FIG. 16 is a series of graphs depicting the viral titers in
lung, liver, and brain of mice treated with anti-M2e mAbs TCN-031
and TCN-032 after challenge with H5N1 A/Vietnam/1203/04. BALB/C
mice (n=19) were treated i.p. injection of a 400 .mu.g/200 .mu.L
dose of TCN-031, TCN-032, control human mAb 2N9, control chimeric
mAb ch14C2, PBS, or left untreated. Tissue viral titers were
determined from 3 mice per group at 3 and 6 days post-infection in
the lungs (as an indicator of local replication) and in liver and
brain (as an indicator of the systemic spread which is
characteristic of H5N1 infection).
[0044] FIG. 17 is a graph depicting the ability of TCN-031 and
TCN-032 can potentiate cytolysis by NK cells. MDCK cells were
infected with A/Solomon Island/3/2006 (H1N1) virus, and were
treated with mAbs TCN-031, TCN-032, or the subclass-matched
negative control mAb 2N9. The cells were then challenged with
purified human NK cells, and the lactate dehydrogenase released as
a result of cell lysis was measured through light absorbance. The
results are representative of two separate experiments with two
different normal human donors.
[0045] FIG. 18 is a graph depicting complement-dependent cytolysis
(CDC) of M2-expressing cells bound with anti-M2 mAb. The stable
transfectant expressing M2 of A/Hong Kong/483/97 and a mock control
were treated with the indicated mAbs and subsequently challenged
with human complement. Lysed cells were visualized by Propidium
Iodide staining followed by FACS analysis. The data are
representative of two experiments.
[0046] FIGS. 19A-C are graphs depicting binding of anti-M2e mAbs
TCN-031 and TCN-032 to M2 mutants indicates the epitope is located
in the highly conserved N-terminal of M2e. Mutants with alanine
substituted at each position of the M2 ectodomain of A/Fort
Worth/1/50 (D20) (A) or forty wild-type M2 mutants including
A/Vietnam/1203/04 (VN) and A/Hong Kong/483/97 (HK) (B) were
transiently transfected into 293 cells. The identity of each
wild-type M2 mutant is listed in Table 6. Transfected cells were
stained with mAbs TCN-031, TCN-032, or the control ch14C2 and
analyzed by FACS for binding to M2 at 24 hours post-transfection.
mAbs TCN-031 and TCN-032 do not bind variants with amino acid
substitutions at positions 1, 4, or 5 of M2e. (C) The deduced
epitope for TCN-031 and TCN-032 occurs in a highly conserved region
of M2e and is distinct from that found for ch14C2. Results shown
for (A) and (B) are representative of 3 experiments.
[0047] FIG. 20 is a graph depicting mAbs TCN-031 and TCN-032
recognize the same region on M2e. The CHO transfectant stably
expressing M2 for A/Hong Kong/483/97 as stained with 10 .mu.g/mL
TCN-031, TCN-032, or 2N9, followed by detection with
Alexafluor647-labeled TCN-031 (TCN-031AF647) or TCN-032
(TCN-032AF647) and analysis by flow cytometry. The results are
representative of three experiments.
[0048] FIG. 21 is a graph depicting anti-M2e mAbs TCN-031 and
TCN-032 bind cells that have been infected with H1N1
A/California/4/09. MDCK cells were infected with Influenza A strain
H1N1 A/Memphis/14/96, H1N1 A/California/4/09, or mock infected.
Twenty four hours post-infection cells were stained with mAbs
TCN-031, TCN-032, or the control ch14C2 and analyzed by FACS for
binding to M2. Results shown are for one experiment.
DETAILED DESCRIPTION
[0049] Influenza viruses consist of three types, A, B and C.
Influenza A viruses infect a wide variety of birds and mammals,
including humans, horses, marine mammals, pigs, ferrets, and
chickens. In animals most influenza A viruses cause mild localized
infections of the respiratory and intestinal tract. However, highly
pathogenic influenza A strains such as H5N1 exist that cause
systemic infections in poultry in which mortality may reach 100%.
Animals infected with influenza A often act as a reservoir for the
influenza viruses and certain subtypes have been shown to cross the
species barrier to humans.
[0050] Influenza A viruses can be classified into subtypes based on
allelic variations in antigenic regions of two genes that encode
surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase
(NA) which are required for viral attachment and cellular release.
Other major viral proteins include the nucleoprotein, the
nucleocapsid structural protein, membrane proteins (M1 and M2),
polymerases (PA, PB and PB2) and non-structural proteins (NS1 and
NS2). Currently, sixteen subtypes of HA (H1-H16) and nine NA
(N1-N9) antigenic variants are known in influenza A virus.
Previously, only three subtypes have been known to circulate in
humans (H1N1, H1N2, and H3N2).
[0051] However, in recent years, the pathogenic H5N1 subtype of
avian influenza A has been reported to cross the species barrier
and infect humans as documented in Hong Kong in 1997 and 2003,
leading to the death of several patients. In humans, the avian
influenza virus infects cells of the respiratory tract as well as
the intestinal tract, liver, spleen, kidneys and other organs.
Symptoms of avian influenza infection include fever, respiratory
difficulties including shortness of breath and cough, lymphopenia,
diarrhea and difficulties regulating blood sugar levels. In
contrast to seasonal influenza, the group most at risk is healthy
adults, which make up the bulk of the population. Due to the high
pathogenicity of certain avian influenza A subtypes, particularly
H5N1, and their demonstrated ability to cross over to infect
humans, there is a significant economic and public health risk
associated with these viral strains, including a real epidemic and
pandemic threat. The scale of the threat is illustrated by the 1918
influenza pandemic which killed over 50 million people.
[0052] Currently, no effective vaccines for H5N1 infection are
available, so passive immunotherapy with immunoglobulins may be an
alternative strategy. Use of passive immunization during the 1918
pandemic reportedly halved the death rate. In view of their
therapeutic benefit in humans, there is thus a need for antibodies,
preferably human antibodies, capable of neutralizing influenza
infection, including H5N1.
[0053] The invention provides compositions including human
antibodies raised against two influenza proteins, hemagglutinin
(HA) and matrix 2 ectodomain (M2e), and shows that these
compositions can be used in medicine, in particular for diagnosis,
prevention and treatment of influenza infections, including
H5N1.
HuM2e Antibodies
[0054] The present invention provides fully human monoclonal
antibodies specifically directed against M2e. Optionally, the
antibody is isolated form a B-cell from a human donor. Exemplary
monoclonal antibodies include TCN-032 (8I10), 21B15, TCN-031
(23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19,
3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23,
3248_P18, 3253_P10, 3260_D19, 3362_B11, and 3242_P05. described
herein. Alternatively, the monoclonal antibody is an antibody that
binds to the same epitope as TCN-032 (8I10), 21B15, TCN-031
(23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19,
3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23,
3248_P18, 3253_P10, 3260_D19, 3362_B11, and 3242_P05. The
antibodies respectively referred to herein are huM2e antibodies.
The huM2e antibody has one or more of the following
characteristics: a) binds to an epitope in the extracellular domain
of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus;
b) binds to influenza A infected cells; or c) binds to influenza A
virus.
[0055] The epitope that huM2e antibody binds to is a non-linear
epitope of a M2 polypeptide. Preferably, the epitope includes the
amino terminal region of the M2e polypeptide. More preferably the
epitope wholly or partially includes the amino acid sequence SLLTEV
(SEQ ID NO: 42). Most preferably, the epitope includes the amino
acid at position 2, 5 and 6 of the M2e polypeptide when numbered in
accordance with SEQ ID NO: 1. The amino acid at position 2 is a
serine; at position 5 is a threonine; and at position 6 is a
glutamic acid.
[0056] A huM2e antibody contains a heavy chain variable having the
amino acid sequence of SEQ ID NOs: 44, 277, 276, 50, 236, 235, 116,
120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168,
172, or 176 and a light chain variable having the amino acid
sequence of SEQ ID NOs: 46, 52, 118, 122, 126, 130, 134, 138, 142,
146, 150, 154, 158, 162, 166, 170, 174, or 178. Preferably, the
three heavy chain CDRs include an amino acid sequence at least 90%,
92%, 95%, 97% 98%, 99% or more identical to the amino acid sequence
of SEQ ID NOs: 72, 74, 76, 103, 105, 107, 179, 180, 181, 187, 188,
189, 197, 203, 204, 205, 21, 212, 213, 228, 229, 230, 237, 238,
252, 253, 254, 260, 261, 262, 268, 269, 270, 284, 285, 286, 293,
294, 295, and 301 (as determined by the Kabat method) or SEQ ID
NOs: 109, 110, 76, 112, 113, 107, 182, 183, 181, 190, 191, 189,
197, 206, 207, 205, 214, 215, 213, 232, 230, 239, 240, 238, 255,
256, 254, 263, 264, 262, 271, 272, 270, 287, 288, 286, 296, 297,
295, and 304 (as determined by the Chothia method) and a light
chain with three CDRs that include an amino acid sequence at least
90%, 92%, 95%, 97% 98%, 99% or more identical to the amino acid
sequence of SEQ ID NOs: 59, 60, 61, 92, 94, 96, 184, 185, 186, 192,
193, 194, 208, 209, 210, 217, 218, 226, 223, 234, 241, 243, 258,
259, 265, 267, 273, 274, 275, 282, 291, and 300 (as determined by
the Kabat method) or SEQ ID NOs: 59, 60, 61, 92, 94, 96, 184, 185,
186, 192, 193, 194, 208, 209, 210, 217, 218, 226, 223, 234, 241,
243, 258, 259, 265, 267, 273, 274, 275, 282, 291, and 300 (as
determined by the Chothia method). The antibody binds M2e.
[0057] 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.
[0058] The M2e antibodies of the invention include a variable heavy
chain (V.sub.H) region encoded by a human IgHV4 or the IgHV3
germline gene sequence. A IgHV4 germline gene sequence are shown,
e.g., in Accession numbers L10088, M29812, M95114, X56360 and
M95117. IgHV3 germline gene sequence are shown, e.g., in Accession
numbers X92218, X70208, Z27504, M99679 and AB019437. The M2e
antibodies of the invention include a V.sub.H region that is
encoded by a nucleic acid sequence that is at least 80% homologous
to the IgHV4 or the IgHV3 germline gene sequence. Preferably, the
nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to
the IgHV4 or the IgHV3 germline gene sequence, and more preferably,
at least 98%, 99% homologous to the IgHV4 or the IgHV3 germline
gene sequence. The V.sub.H region of the M2e antibody is at least
80% homologous to the amino acid sequence of the V.sub.H region
encoded by the IgHV4 or the IgHV3 V.sub.H germline gene sequence.
Preferably, the amino acid sequence of V.sub.H region of the M2e
antibody is at least 90%, 95%, 96%, 97% homologous to the amino
acid sequence encoded by the IgHV4 or the IgHV3 germline gene
sequence, and more preferably, at least 98%, 99% homologous to the
sequence encoded by the IgHV4 or the IgHV3 germline gene
sequence.
[0059] The M2e antibodies of the invention also include a variable
light chain (V.sub.L) region encoded by a human IgKV1 germline gene
sequence. A human IgKV1 V.sub.L germline gene sequence is shown,
e.g., Accession numbers X59315, X59312, X59318, J00248, and Y14865.
Alternatively, the M2e antibodies include a V.sub.L region that is
encoded by a nucleic acid sequence that is at least 80% homologous
to the IgKV1 germline gene sequence. Preferably, the nucleic acid
sequence is at least 90%, 95%, 96%, 97% homologous to the IgKV1
germline gene sequence, and more preferably, at least 98%, 99%
homologous to the IgKV1 germline gene sequence. The V.sub.L region
of the M2e antibody is at least 80% homologous to the amino acid
sequence of the V.sub.L region encoded the IgKV1 germline gene
sequence. Preferably, the amino acid sequence of V.sub.L region of
the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the
amino acid sequence encoded by the IgKV1 germline gene sequence,
and more preferably, at least 98%, 99% homologous to the sequence
encoded by e the IgKV1 germline gene sequence.
[0060] In another aspect the invention provides a composition
including an huM2e antibody according to the invention. In various
aspects the composition further includes an anti-viral drug, a
viral entry inhibitor or a viral attachment inhibitor. The
anti-viral drug is for example a neuraminidase inhibitor, a HA
inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor.
The M2 ion channel inhibitor is for example amantadine or
rimantadine. The neuraminidase inhibitor for example zanamivir, or
oseltamivir phosphate. In a further aspect the composition further
includes a second anti-influenza A antibody.
[0061] In a further aspect the huM2e antibodies according to the
invention are operably-linked to a therapeutic agent or a
detectable label.
[0062] Additionally, the invention provides methods for stimulating
an immune response, treating, preventing or alleviating a symptom
of an influenza viral infection by administering an huM2e antibody
to a subject
[0063] Optionally, the subject is further administered with a
second agent such as, but not limited to, an influenza virus
antibody, an anti-viral drug such as a neuraminidase inhibitor, a
HA inhibitor, a sialic acid inhibitor or an M2 ion channel
inhibitor, a viral entry inhibitor or a viral attachment inhibitor.
The M2 ion channel inhibitor is, for example, amantadine or
rimantadine. The neuraminidase inhibitor is, for example, zanamivir
or oseltamivir phosphate. The subject is suffering from or is
predisposed to developing an influenza virus infection, such as,
for example, an autoimmune disease or an inflammatory disorder.
[0064] In another aspect, the invention provides methods of
administering the huM2e antibody of the invention to a subject
prior to, and/or after exposure to an influenza virus. For example,
the huM2e antibody of the invention is used to treat or prevent
rejection influenza infection. The huM2e antibody is administered
at a dose sufficient to promote viral clearance or eliminate
influenza A infected cells.
[0065] Also included in the invention is a method for determining
the presence of an influenza virus infection in a patient, by
contacting a biological sample obtained from the patient with a
humM2e antibody; detecting an amount of the antibody that binds to
the biological sample; and comparing the amount of antibody that
binds to the biological sample to a control value.
[0066] The invention further provides a diagnostic kit comprising a
huM2e antibody.
[0067] Other features and advantages of the invention will be
apparent from and are encompassed by the following detailed
description and claims.
[0068] The present invention provides fully human monoclonal
antibodies specific against the extracellular domain of the matrix
2 (M2) polypeptide. The antibodies are respectively referred to
herein as huM2e antibodies.
[0069] M2 is a 96 amino acid transmembrane protein present as a
homotetramer on the surface of influenza virus and virally infected
cells. M2 contains a 23 amino acid ectodomain (M2e) that is highly
conserved across influenza A strains. Few amino acid changes have
occurred since the 1918 pandemic strain thus M2e is an attractive
target for influenza therapies. In prior studies, monoclonal
antibodies specific to the M2 ectodomain (M2e) were derived upon
immunizations with a peptide corresponding to the linear sequence
of M2e. In contrast, the present invention provides a novel process
whereby full-length M2 is expressed in cell lines, which allows for
the identification of human antibodies that bound this
cell-expressed M2e. The huM2e antibodies have been shown to bind
conformational determinants on the M2-transfected cells, as well as
native M2, either on influenza infected cells, or on the virus
itself. The huM2e antibodies did not bind the linear M2e peptide,
but they do bind several natural M2 variants, also expressed upon
cDNA transfection into cell lines. Thus, this invention has allowed
for the identification and production of human monoclonal
antibodies that exhibit novel specificity for a very broad range of
influenza A virus strains. These antibodies may be used
diagnostically to identify influenza A infection and
therapeutically to treat influenza A infection.
[0070] The huM2e antibodies of the invention have one or more of
the following characteristics: the huM2e antibody binds a) to an
epitope in the extracellular domain of the matrix 2 (M2)
polypeptide of an influenza virus; b) binds to influenza A infected
cells; and/or c) binds to influenza A virus (i.e., virons). The
huM2e antibodies of the invention eliminate influenza infected
cells through immune effector mechanisms, such as ADCC, and promote
direct viral clearance by binding to influenza virons. The huM2e
antibodies of the invention bind to the amino-terminal region of
the M2e polypeptide. Preferably, the huM2e antibodies of the
invention bind to the amino-terminal region of the M2e polypeptide
wherein the N-terminal methionine residue is absent. Exemplary M2e
sequences include those sequences listed on Table 1 below
TABLE-US-00001 TABLE 1 Type Name Subtype M2E Sequence SEQ ID NO A
BREVIG H1N1 MSLLTEVETPTRNEWGCRCNDSSD SEQ ID NO: 1 MISSION.1.1918 A
FORT H1N1 MSLLTEVETPTKNEWECRCNDSSD SEQ ID NO: 2 MONMOUTH.1.1947 A
.SINGAPORE.02.2005 H3N2 MSLLTEVETPIRNEWECRCNDSSD SEQ ID NO: 3 A
WISCONSIN.10.98 H1N1 MSLLTEVETPIRNGWECKCNDSSD SEQ ID NO: 4 A
WISCONSIN.301.1976 H1N1 MSLLTEVETPIRSEWGCRCNDSSD SEQ ID NO: 5 A
PANAMA.1.66 H2N2 MSFLPEVETPIRNEWGCRCNDSSD SEQ ID NO: 6 A NEW H3N2
MSLLTEVETPIRNEWGCRCNDSSN SEQ ID NO: 7 YORK.321.1999 A CARACAS.1.71
H3N2 MSLLTEVETPIRKEWGCRCNDSSD SEQ ID NO: 8 A TAIWAN.3.71 H3N2
MSFLTEVETPIRNEWGCRCNDSSD SEQ ID NO: 9 A WUHAN.359.95 H3N2
MSLPTEVETPIRSEWGCRCNDSSD SEQ ID NO: 10 A HONG H3N2
MSLLPEVETPIRNEWGCRCNDSSD SEQ ID NO: 11 KONG.1144.99 A HONG H3N2
MSLLPEVETPIRNGWGCRCNDSSD SEQ ID NO: 12 KONG.1180.99 A HONG H3N2
MSLLTEVETPTRNGWECRCSGSSD SEQ ID NO: 13 KONG.1774.99 A NEW
YORK.217.02 H1N2 MSLLTEVETPIRNEWEYRCNDSSD SEQ ID NO: 14 A NEW H1N2
MSLLTEVETPIRNEWEYRCSDSSD SEQ ID NO: 15 YORK.300.2003 A
SWINE.SPAIN.54008. H3N2 MSLLTEVETPTRNGWECRYSDSSD SEQ ID NO: 16 2004
A GUANGZHOU.333.99 H9N2 MSFLTEVETLTRNGWECRCSDSSD SEQ ID NO: 17 A
HONG H9N2 MSLLTEVETLTRNGWECKCRDSSD SEQ ID NO: 18 KONG.1073.99 A
HONG KONG.1.68 H3N2 MSLLTEVETPIRNEWGCRCNDSSD SEQ ID NO: 19 A
SWINE.HONG H3N2 MSLLTEVETPIRSEWGCRCNDSGD SEQ ID NO: 20
KONG.126.1982 A NEW H3N2 MSLLTEVETPIRNEWECRCNGSSD SEQ ID NO: 21
YORK.703.1995 A SWINE.QUEBEC.192. H1N1 MSLPTEVETPIRNEWGCRCNDSSD SEQ
ID NO: 22 81 A PUERTO RICO.8.34 H1N1 MSLLTEVETPIRNEWGCRCNGSSD SEQ
ID NO: 23 A HONG KONG.485.97 H5N1 MSLLTEVDTLTRNGWGCRCSDSSD SEQ ID
NO: 24 A HONG KONG.542.97 H5N1 MSLLTEVETLTKNGWGCRCSDSSD SEQ ID NO:
25 A SILKY H9N2 MSLLTEVETPTRNGWECKCSDSSD SEQ ID NO: 26
CHICKEN.SHANTOU. 1826.2004 A CHICKEN.TAIWAN.0305. H6N1
MSLLTEVETHTRNGWECKCSDSSD SEQ ID NO: 27 4 A QUAIL.ARKANSAS.16309-
H7N3NSA MSLLTEVKTPTRNGWECKCSDSSD SEQ ID NO: 28 7.94 A HONG
KONG.486.97 H5N1 MSLLTEVETLTRNGWGCRCSDSSD SEQ ID NO: 29 A
CHICKEN.PENNSYLVANIA. H7N2NSB MSLLTEVETPTRDGWECKCSDSSD SEQ ID NO:
30 13552-1.98 A CHICKEN.HEILONGJIANG. H9N2 MSLLTEVETPTRNGWGCRCSDSSD
SEQ ID NO: 31 48.01 A SWINE.KOREA.S5.2005 H1N2
MSLLTEVETPTRNGWECKCNDSSD SEQ ID NO: 32 A HONG H9N2
MSLLTEVETLTRNGWECKCSDSSD SEQ ID NO: 33 KONG.1073.99 A
WISCONSIN.3523.88 H1N1 MSLLTEVETPIRNEWGCKCNDSSD SEQ ID NO: 34 A
X-31 VACCINE H3N2 MSFLTEVETPIRNEWGCRCNGSSD SEQ ID NO: 35 STRAIN A
CHICKEN.ROSTOCK. H7N1 MSLLTEVETPTRNGWECRCNDSSD SEQ ID NO: 36 8.1934
A ENVIRONMENT.NEW H7N2 MSLLTEVETPIRKGWECNCSDSSD SEQ ID NO: 37
YORK.16326- 1.2005 A INDONESIA.560H.2006 H5N1
MSLLTEVETPTRNEWECRCSDSSD SEQ ID NO: 38 A CHICKEN.HONG H9N2
MSLLTGVETHTRNGWGCKCSDSSD SEQ ID NO: 39 KONG.SF1.03 A
CHICKEN.HONGKONG. H9N2 MSLLPEVETHTRNGWGCRCSDSSD SEQ ID NO: 40
YU427.03
[0071] In one embodiment, the huM2e antibodies of the invention
bind to a M2e that wholly or partially includes the amino acid
residues from position 2 to position 7 of M2e when numbered in
accordance with SEQ ID NO: 1. For example, the huM2e antibodies of
the invention bind wholly or partially to the amino acid sequence
SLLTEVET (SEQ ID NO: 41) Most preferably, the huM2e antibodies of
the invention bind wholly or partially to the amino acid sequence
SLLTEV (SEQ ID NO: 42). Preferably, the huM2e antibodies of the
invention bind to non-linear epitope of the M2e protein. For
example, the huM2e antibodies bind to an epitope comprising
position 2, 5, and 6 of the M2e polypeptide when numbered in
accordance to SEQ ID NO: 1 where the amino acid at a) position 2 is
a serine; b) position 5 is a threonine; and c) position 6 is a
glutamic acid. Exemplary huM2e monoclonal antibodies that bind to
this epitope are the TCN-032 (8I10), 21B15, TCN-031 (23K12),
3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19, 3244_H04,
3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23, 3248_P18,
3253_P10, 3260_D19, 3362_B11, and 3242_P05 antibodies described
herein.
[0072] The TCN-032 (8I10) antibody includes a heavy chain variable
region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown
below in SEQ ID NO: 43, a short heavy chain variable region (SEQ ID
NO: 277) encoded by the nucleic acid sequence shown below in SEQ ID
NO: 278, a long heavy chain variable region (SEQ ID NO: 276)
encoded by the nucleic acid sequence shown below in SEQ ID NO: 196,
and a light chain variable region (SEQ ID NO: 46) encoded by the
nucleic acid sequence shown in SEQ ID NO: 45.
[0073] The amino acids encompassing the CDRs as defined by Chothia,
C. et al. (1989, Nature, 342: 877-883) are underlined and those
defined by Kabat E. A. et al. (1991, Sequences of Proteins of
Immunological Interest, 5.sup.th edit., NIH Publication no. 91-3242
U.S. Department of Heath and Human Services.) are highlighted in
bold in the sequences below.
[0074] The heavy chain CDRs of the TCN-032 (8I10) antibody have the
following sequences per Kabat definition: NYYWS (SEQ ID NO: 72),
FIYYGGNTKYNPSLKS (SEQ ID NO: 74) and ASCSGGYCILD (SEQ ID NO: 76).
The light chain CDRs of the TCN-032 (8I10) antibody have the
following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO:
59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).
[0075] The heavy chain CDRs of the TCN-032 (8I10) antibody have the
following sequences per Chothia definition: GSSISN (SEQ ID NO:
109), FIYYGGNTK (SEQ ID NO: 110) and ASCSGGYCILD (SEQ ID NO: 76).
The light chain CDRs of the TCN-032 (8I10) antibody have the
following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO:
59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).
[0076] TCN-032 (8I10) VH Nucleotide Sequence: (SEQ ID NO: 43)
TABLE-US-00002 CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT
GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT
ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT
CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT
CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT
AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCG
[0077] TCN-032 (8I10) VH Amino Acid Sequence: (SEQ ID NO: 44)
Kabat Bold, Chothia Underlined
TABLE-US-00003 [0078] 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
[0079] TCN-032 (8I10) VH Short Nucleotide Sequence: (SEQ ID NO:
278)
TABLE-US-00004 CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT
GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT
ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT
CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT
CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT
AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGT
[0080] TCN-032 (8I10) VH Short Amino Acid Sequence: (SEQ ID NO:
277)
TABLE-US-00005 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
[0081] TCN-032 (8I10) VH Long Nucleotide Sequence: (SEQ ID NO:
196)
TABLE-US-00006 CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT
GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT
ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT
CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT
CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT
AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCGAGC
[0082] TCN-032 (8I10) VH Long Amino Acid Sequence: (SEQ ID NO:
276)
Kabat Bold, Chothia Underlined
TABLE-US-00007 [0083] 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
[0084] TCN-032 (8I10) VL nucleotide sequence: (SEQ ID NO: 45)
TABLE-US-00008 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAA
ATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCT
GCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGA
GGGACCAGGGTGGAGATCAAAC
[0085] TCN-032 (8I10) VL Amino Acid Sequence: (SEQ ID NO: 46)
Kabat Bold, Chothia underlined
TABLE-US-00009 D I Q M T Q S P S S L S A S V G D R V T I T C R A S
Q N I Y K Y L N W Y Q Q R P G K A P K G L I S A A S G L Q S G V P S
R F S G S G S G T D F T L T I T S L Q P E D F A T Y Y C Q Q S Y S P
P L T F G G G T R V E I K
[0086] The 21B15 antibody includes a heavy chain variable region
(SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in
SEQ ID NO: 47, a short heavy chain variable region (SEQ ID NO: 277)
encoded by the nucleic acid sequence shown below in SEQ ID NO: 278,
a long heavy chain variable region (SEQ ID NO: 276) encoded by the
nucleic acid sequence shown below in SEQ ID NO: 196, and a light
chain variable region (SEQ ID NO: 46) encoded by the nucleic acid
sequence shown in SEQ ID NO: 48.
[0087] 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.
[0088] The heavy chain CDRs of the 21B15 antibody have the
following sequences per Kabat definition: NYYWS (SEQ ID NO: 72),
FIYYGGNTKYNPSLKS (SEQ ID NO: 74) and ASCSGGYCILD (SEQ ID NO: 76).
The light chain CDRs of the 21B15 antibody have the following
sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 59),
AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).
[0089] The heavy chain CDRs of the 21B15 antibody have the
following sequences per Chothia definition: GSSISN (SEQ ID NO:
109), FIYYGGNTK (SEQ ID NO: 110) and ASCSGGYCILD (SEQ ID NO: 76).
The light chain CDRs of the 21B15 antibody have the following
sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 59),
AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQ ID NO: 63).
[0090] 21B15 VH Nucleotide Sequence: (SEQ ID NO: 47)
TABLE-US-00010 CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT
GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT
ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT
CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT
CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT
AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCG
[0091] 21B15 VH Amino Acid Sequence: (SEQ ID NO: 44)
Kabat Bold, Chothia Underlined
TABLE-US-00011 [0092] 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
[0093] B15 VH Short Nucleotide Sequence: (SEQ ID NO: 278)
TABLE-US-00012 CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT
GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT
ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT
CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT
CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT
AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGT
[0094] 21B15 VH Short Amino Acid Sequence: (SEQ ID NO: 277)
Kabat Bold, Chothia Underlined
TABLE-US-00013 [0095] 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
[0096] 21B15 VH Long Nucleotide Sequence: (SEQ ID NO: 196)
TABLE-US-00014 CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACT
GGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTT
ATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGT
CACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCT
CTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGT
AGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCGAGC
[0097] 21B15 VH Long Amino Acid Sequence: (SEQ ID NO: 276)
Kabat Bold, Chothia Underlined
TABLE-US-00015 [0098] 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
[0099] 21B15 VL Nucleotide Sequence: (SEQ ID NO: 48)
TABLE-US-00016 GACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAA
ATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCT
GCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGA
GGGACCAGGGTGGATATCAAAC
[0100] 21B15 VL Amino Acid Sequence: (SEQ ID NO: 292)
Kabat Bold, Chothia Underlined
TABLE-US-00017 [0101] D I Q V T Q S P S S L S A S V G D R V T I T C
R A S Q N I Y K Y L N W Y Q Q R P G K A P K G L I S A A S G L Q S G
V P S R F S G S G S G T D F T L T I T S L Q P E D F A T Y Y C Q Q S
Y S P P L T F G G G T R V D I K
[0102] The TCN-031 (23K12) antibody includes a heavy chain variable
region (SEQ ID NO: 50) encoded by the nucleic acid sequence shown
below in SEQ ID NO: 49, a short heavy chain variable region (SEQ ID
NO: 236) encoded by the nucleic acid sequence shown below in SEQ ID
NO: 244, a long heavy chain variable region (SEQ ID NO: 195)
encoded by the nucleic acid sequence shown below in SEQ ID NO: 235,
and a light chain variable region (SEQ ID NO: 52) encoded by the
nucleic acid sequence shown in SEQ ID NO: 51.
[0103] 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.
[0104] The heavy chain CDRs of the TCN-031 (23K12) antibody have
the following sequences per Kabat definition: SNYMS (SEQ ID NO:
103), VIYSGGSTYYADSVK (SEQ ID NO: 105) and CLSRMRGYGLDV (SEQ ID NO:
107). The light chain CDRs of the TCN-031 (23K12) antibody have the
following sequences per Kabat definition: RTSQSISSYLN (SEQ ID NO:
92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA (SEQ ID NO:
96).
[0105] The heavy chain CDRs of the TCN-031 (23K12) antibody have
the following sequences per Chothia definition: GFTVSSN (SEQ ID NO:
112), VIYSGGSTY (SEQ ID NO: 113) and CLSRMRGYGLDV (SEQ ID NO: 107).
The light chain CDRs of the TCN-031 (23K12) antibody have the
following sequences per Chothia definition: RTSQSISSYLN (SEQ ID NO:
92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA (SEQ ID NO:
96).
[0106] TCN-031 (23K12) VH Nucleotide Sequence: (SEQ ID NO: 49)
TABLE-US-00018 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACA
TGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT
ATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATT
CTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACA
GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGC
AGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCG
[0107] TCN-031 (23K12) VH Amino Acid Sequence: (SEQ ID NO: 50)
Kabat Bold, Chothia Underlined
TABLE-US-00019 [0108] E V Q L V E S G G G L V Q P G G S L R I S C A
A S G F T V S S N Y M S W V R Q A P G K G L E W V S V I Y S G G S T
Y Y A D S V K G R F S F S R D N S K N T V F L Q M N S L R A E D T A
V Y Y C A R C L S R M R G Y G L D V W G Q G T T V T V S
[0109] TCN-031 (23K12) VH Short Nucleotide Sequence: (SEQ ID NO:
244)
TABLE-US-00020 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACA
TGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT
ATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATT
CTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACA
GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGC
AGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCAC CGT
[0110] TCN-031 (23K12) VH Short Amino Acid Sequence: (SEQ ID NO:
236)
Kabat Bold, Chothia Underlined
TABLE-US-00021 [0111] E V Q L V E S G G G L V Q P G G S L R I S C A
A S G F T V S S N Y M S W V R Q A P G K G L E W V S V I Y S G G S T
Y Y A D S V K G R F S F S R D N S K N T V F L Q M N S L R A E D T A
V Y Y C A R C L S R M R G Y G L D V W G Q G T T V T V S
[0112] TCN-031 (23K12) VH Long Nucleotide Sequence: (SEQ ID NO:
195)
TABLE-US-00022 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACA
TGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT
ATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATT
CTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACA
GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGC
AGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCGAGC
[0113] TCN-031 (23K12) VH Long Amino Acid Sequence: (SEQ ID NO:
235)
Kabat Bold, Chothia Underlined
TABLE-US-00023 [0114] E V Q L V E S G G G L V Q P G G S L R I S C A
A S G F T V S S N Y M S W V R Q A P G K G L E W V S V I Y S G G S T
Y Y A D S V K G R F S F S R D N S K N T V F L Q M N S L R A E D T A
V Y Y C A R C L S R M R G Y G L D V W G Q G T T V T V S S
[0115] TCN-031 (23K12) VL Nucleotide Sequence: (SEQ ID NO: 51)
TABLE-US-00024 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTG
CAACCTACTACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGG
ACCAAGCTGGAGATCAAA
[0116] TCN-031 (23K12) VL Amino Acid Sequence: (SEQ ID NO: 52)
Kabat Bold, Chothia Underlined
TABLE-US-00025 [0117] 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
[0118] The 3241_G23 antibody (also referred to herein as G23)
includes a heavy chain variable region (SEQ ID NO: 116) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 115, and a
light chain variable region (SEQ ID NO: 118) encoded by the nucleic
acid sequence shown in SEQ ID NO: 117.
[0119] 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.
[0120] The heavy chain CDRs of the G23 antibody have the following
sequences per Kabat definition: GGGYSWN (SEQ ID NO: 179),
FMFHSGSPRYNPTLKS (SEQ ID NO: 180) and VGQMDKYYAMDV (SEQ ID NO:
181). The light chain CDRs of the G23 antibody have the following
sequences per Kabat definition: RASQSIGAYVN (SEQ ID NO: 184),
GASNLQS (SEQ ID NO: 185) and QQTYSTPIT (SEQ ID NO: 186).
[0121] The heavy chain CDRs of the G23 antibody have the following
sequences per Chothia definition: GGPVSGGG (SEQ ID NO: 182),
FMFHSGSPR (SEQ ID NO: 183) and VGQMDKYYAMDV (SEQ ID NO: 181). The
light chain CDRs of the G23 antibody have the following sequences
per Chothia definition: RASQSIGAYVN (SEQ ID NO: 184), GASNLQS (SEQ
ID NO: 185) and QQTYSTPIT (SEQ ID NO: 186).
[0122] 3241_G23 VH Nucleotide Sequence (SEQ ID NO: 115)
TABLE-US-00026 CAGGTGCAGCTGCAGCAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACTTGCACTGTCTCTGGTGGCCCCGTCAGCGGTGGTGGTT
ACTCCTGGAACTGGATCCGCCAACGCCCAGGACAGGGCCTGGAGTGGGTT
GGGTTCATGTTTCACAGTGGGAGTCCCCGCTACAATCCGACCCTCAAGAG
TCGAATTACCATCTCAGTCGACACGTCTAAGAACCTGGTCTCCCTGAAGC
TGAGCTCTGTGACGGCCGCGGACACGGCCGTGTATTTTTGTGCGCGAGTG
GGGCAGATGGACAAGTACTATGCCATGGACGTCTGGGGCCAAGGGACCAC
GGTCACCGTCTCGAGC
[0123] 3241_G23 VH Amino Acid Sequence (SEQ ID NO: 116)
Kabat Bold, Chothia Underlined
TABLE-US-00027 [0124]
QVQLQQSGPGLVKPSQTLSLTCTVSGGPVSGGGYSWNWIRQRPGQGLEWV
GFMFHSGSPRYNPTLKSRITISVDTSKNLVSLKLSSVTAADTAVYFCARV
GQMDKYYAMDVWGQGTTVTVSS
[0125] 3241_G23 VL Nucleotide Sequence (SEQ ID NO: 117)
TABLE-US-00028 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTTCCTCTGTCGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGGCGCCTATGTAA
ATTGGTATCAACAGAAAGCAGGGAAAGCCCCCCAGGTCCTGATCTTTGGT
GCTTCCAATTTACAAAGCGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGACTTTG
CAACTTACTTCTGTCAACAGACTTACAGTACCCCGATCACCTTCGGCCAA
GGGACACGACTGGAGATTAAACG
[0126] 3241_G23 VL Amino Acid Sequence (SEQ ID NO: 118)
Kabat Bold, Chothia Underlined
TABLE-US-00029 [0127]
DIQMTQSPSSLSSSVGDRVTITCRASQSIGAYVNWYQQKAGKAPQVLIFG
ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTYSTPITFGQ GTRLEIK
[0128] The 3244_I10 antibody (also referred 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.
[0129] 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.
[0130] The heavy chain CDRs of the 110 antibody have the following
sequences per Kabat definition: SDYWS (SEQ ID NO: 187),
FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDAKFSGSYYVAS (SEQ ID NO:
189). The light chain CDRs of the 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).
[0131] 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) and QQSYNTPLI (SEQ ID NO: 194).
[0132] 3244_I10 VH Nucleotide Sequence (SEQ ID NO: 119)
TABLE-US-00030 CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACAC
CCTGGCCCTCACTTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACT
GGAGCTGGATCCGGCAACCCCCAGGGAGGGGACTGGACTGGATCGGATTC
TTCTATAACGGCGGAAGCACCAAGTACAATCCCTCCCTCAAGAGTCGAGT
CACCATTTCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGACCT
CTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGCC
AAATTTAGTGGGAGCTACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGT
CACCGTCTCGAGC
[0133] 3244_I10 VH Amino Acid Sequence (SEQ ID NO: 120)
TABLE-US-00031 QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGF
FYNGGSTKYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDA
KFSGSYYVASWGQGTRVTVSS
[0134] 3244_I10 VL Nucleotide Sequence (SEQ ID NO: 121)
TABLE-US-00032 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA
ATTGGTATCAGCAGCAACCTGGGAAAGCCCCTAAGGTCCTCATTTTTGGT
GCAACCAACTTGCAAAGTGGGGTCCCATCTCGCTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAATACCCCCCTCATTTTTGGCCAG
GGGACCAAGCTGGAGATCAAACG
[0135] 3244_I10 VL Amino Acid Sequence (SEQ ID NO: 122)
TABLE-US-00033 DIQMTQSPSSLSASVGDRVTISCRASQSISTYLNWYQQQPGKAPKVLIFG
ATNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLIFGQ GTKLEIK
[0136] The 3243_J07 antibody (also referred to herein as J07)
includes a heavy chain variable region (SEQ ID NO: 124) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 123, and a
light chain variable region (SEQ ID NO: 126) encoded by the nucleic
acid sequence shown in SEQ ID NO: 125.
[0137] 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.
[0138] The heavy chain CDRs of the J07 antibody have the following
sequences per Kabat definition: SDYWS (SEQ ID NO: 187),
FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDVKFSGSYYVAS (SEQ ID NO:
197). The light chain CDRs of the J07 antibody have the following
sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192),
GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
[0139] The heavy chain CDRs of the J07 antibody have the following
sequences per Chothia definition: GGSITS (SEQ ID NO: 190),
FFYNGGSTK (SEQ ID NO: 191) and HDVKFSGSYYVAS (SEQ ID NO: 197). The
light chain CDRs of the J07 antibody have the following sequences
per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ
ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
[0140] 3243_J07 VH Nucleotide Sequence (SEQ ID NO: 123)
TABLE-US-00034 CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACAC
CCTGGCCCTCACTTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACT
GGAGCTGGATCCGGCAACCCCCAGGGAGGGGACTGGACTGGATCGGATTC
TTCTATAACGGCGGGAGCACCAAGTACAATCCCTCCCTCAAGAGTCGAGT
CACCATATCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGACCT
CTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGTC
AAATTTAGTGGGAGCTACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGT
CACCGTCTCGAGC
[0141] 3243_J07 VH Amino Acid Sequence (SEQ ID NO: 124)
TABLE-US-00035 QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGF
FYNGGSTKYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDV
KFSGSYYVASWGQGTRVTVSS
[0142] 3243_J07 VL Nucleotide Sequence (SEQ ID NO: 125)
TABLE-US-00036 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA
ATTGGTATCAGCAGCAACCTGGGAAAGCCCCTAAGGTCCTGATCTCTGGT
GCAACCAACTTGCAAAGTGGGGTCCCATCTCGCTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAATACCCCCCTCATTTTTGGCCAG
GGGACCAAGCTGGAGATCAAACG
[0143] 3243_J07 VL Amino Acid Sequence (SEQ ID NO: 126)
TABLE-US-00037 DIQMTQSPSSLSASVGDRVTISCRASQSISTYLNWYQQQPGKAPKVLISG
ATNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLIFGQ GTKLEIK
[0144] The 3259_J21 antibody (also referred to herein as J21)
includes a heavy chain variable region (SEQ ID NO: 128) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 127, and a
light chain variable region (SEQ ID NO: 130) encoded by the nucleic
acid sequence shown in SEQ ID NO: 129.
[0145] 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.
[0146] The heavy chain CDRs of the J21 antibody have the following
sequences per Kabat definition: SYNWI (SEQ ID NO: 203),
HIYDYGRTFYNSSLQS (SEQ ID NO: 204) and PLGILHYYAMDL (SEQ ID NO:
205). The light chain CDRs of the J21 antibody have the following
sequences per Kabat definition: RASQSIDKFLN (SEQ ID NO: 208),
GASNLHS (SEQ ID NO: 209) and QQSFSVPA (SEQ ID NO: 210).
[0147] The heavy chain CDRs of the J21 antibody have the following
sequences per Chothia definition: GGSISS (SEQ ID NO: 206),
HIYDYGRTF (SEQ ID NO: 207) and PLGILHYYAMDL (SEQ ID NO: 205). The
light chain CDRs of the J21 antibody have the following sequences
per Chothia definition: RASQSIDKFLN (SEQ ID NO: 208), GASNLHS (SEQ
ID NO: 209) and QQSFSVPA (SEQ ID NO: 210).
[0148] 3259_J21 VH Nucleotide Sequence (SEQ ID NO: 127)
TABLE-US-00038 CAGGTGCAGCTGCAGGAGTCGGGCCCACGAGTGGTGAGGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCGGGGGGCTCCATCAGTTCTTACAACT
GGATTTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGATTGGGCAC
ATATATGACTATGGGAGGACCTTCTACAACTCCTCCCTCCAGAGTCGACC
TACCATATCTGTAGACGCGTCCAAGAATCAGCTCTCCCTGCGATTGACCT
CTGTGACCGCCTCAGACACGGCCGTCTATTACTGTGCGAGACCTCTCGGT
ATACTCCACTACTACGCGATGGACCTCTGGGGCCAAGGGACCACGGTCAC CGTCTCGAGC
[0149] 3259_J21 VH Amino Acid Sequence (SEQ ID NO: 128)
TABLE-US-00039 QVQLQESGPRVVRPSETLSLTCTVSGGSISSYNWIWIRQPPGKGLEWIGH
IYDYGRTFYNSSLQSRPTISVDASKNQLSLRLTSVTASDTAVYYCARPLG
ILHYYAMDLWGQGTTVTVSS
[0150] 3259_J21 VL Nucleotide Sequence (SEQ ID NO: 129)
TABLE-US-00040 GACATCCAGATGACCCAGTCTCCATTATCCGTGTCTGTATCTGTCGGGGA
CAGGGTCACCATCGCTTGCCGGGCAAGTCAGAGTATTGACAAGTTTTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGGT
GCCTCCAATTTGCACAGTGGGGCCCCATCAAGGTTCAGTGCCAGTGGGTC
TGGGACAGACTTCACTCTAACAATCACCAATATACAGACTGAAGATTTCG
CAACTTACCTCTGTCAACAGAGTTTCAGTGTCCCCGCTTTCGGCGGAGGG
ACCAAGGTTGAGATCAAACG
[0151] 3259_J21 VL Amino Acid Sequence (SEQ ID NO: 130)
TABLE-US-00041 DIQMTQSPLSVSVSVGDRVTIACRASQSIDKFLNWYQQKPGKAPKLLIYG
ASNLHSGAPSRFSASGSGTDFTLTITNIQTEDFATYLCQQSFSVPAFGGG TKVEIK
[0152] The 3245_O19 antibody (also referred to herein as 019)
includes a heavy chain variable region (SEQ ID NO: 132) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 131, and a
light chain variable region (SEQ ID NO: 134) encoded by the nucleic
acid sequence shown in SEQ ID NO: 133.
[0153] 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.
[0154] 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 following
sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192),
GASTLQS (SEQ ID NO: 217) and QQTYSIPL (SEQ ID NO: 218).
[0155] The heavy chain CDRs of the 019 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 019 antibody have the following sequences
per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GASTLQS (SEQ
ID NO: 217) and QQTYSIPL (SEQ ID NO: 218).
[0156] 3245_O19 VH Nucleotide Sequence (SEQ ID NO:131)
TABLE-US-00042 GAGGTGCAACTGGTGGAGTCTGGAGGGGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTACGGCCTCTGGGTTAAGTGTCAGTTCCACCTACA
TGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTCTCAGTT
TTTTATAGTGAGACCAGGACGTACTACGCAGACTCCGTGAAGGGCCGATT
CACCGTCTCCAGACACAATTCCAACAACACGCTCTATCTTCAGATGAACA
GCCTGAGAGTTGAAGACACGGCCGTGTATTATTGTGCGAGAGTCCAGAGA
TTGTCGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC GAGC
[0157] 3245_O19 VH Amino Acid Sequence (SEQ ID NO: 132)
TABLE-US-00043 EVQLVESGGGLVQPGGSLRLSCTASGLSVSSTYMNWVRQAPGKGLEWVSV
FYSETRTYYADSVKGRFTVSRHNSNNTLYLQMNSLRVEDTAVYYCARVQR
LSYGMDVWGQGTTVTVSS
[0158] 3245_O19 VL Nucleotide Sequence (SEQ ID NO: 133)
TABLE-US-00044 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA
ATTGGTATCAGAAGAGACCAGGGAAAGCCCCTAAACTCCTGGTCTATGGT
GCATCCACTTTGCAGAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCGCCAGTCTGCAACCTGAAGATTCTG
CAACTTACTACTGTCAACAGACTTACAGTATCCCCCTCTTCGGCCAGGGG
ACACGGCTGGAGATTAAACG
[0159] 3245_O19 VL Amino Acid Sequence (SEQ ID NO: 134)
TABLE-US-00045 DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQKRPGKAPKLLVYG
ASTLQSGVPSRFSGSGSGTDFTLTIASLQPEDSATYYCQQTYSIPLFGQG TRLEIK
[0160] The 3244_H04 antibody (also referred to herein as H04)
includes a heavy chain variable region (SEQ ID NO: 136) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 135, and a
light chain variable region (SEQ ID NO: 138) encoded by the nucleic
acid sequence shown in SEQ ID NO: 137.
[0161] 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.
[0162] The heavy chain CDRs of the H04 antibody have the following
sequences per Kabat definition: STYMN (SEQ ID NO: 211),
VFYSETRTYYADSVKG (SEQ ID NO: 212) and VQRLSYGMDV (SEQ ID NO: 213).
The light chain CDRs of the H04 antibody have the following
sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192),
GASSLQS (SEQ ID NO: 226) and QQTYSIPL (SEQ ID NO: 218).
[0163] 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).
[0164] 3244_H04 VH Nucleotide Sequence (SEQ ID NO: 135)
TABLE-US-00046 GAGGTGCAGCTGGTGGAATCTGGAGGGGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTACAGCCTCTGGGTTAAGCGTCAGTTCCACCTACA
TGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTCTCAGTT
TTTTATAGTGAAACCAGGACGTATTACGCAGACTCCGTGAAGGGCCGATT
CACCGTCTCCAGACACAATTCCAACAACACGCTGTATCTTCAAATGAACA
GCCTGAGAGCTGAAGACACGGCCGTGTATTATTGTGCGAGAGTCCAGAGA
CTGTCATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC GAGC
[0165] 3244_H04 VH Amino Acid Sequence (SEQ ID NO: 136)
TABLE-US-00047 EVQLVESGGGLVQPGGSLRLSCTASGLSVSSTYMNWVRQAPGKGLEWVSV
FYSETRTYYADSVKGRFTVSRHNSNNTLYLQMNSLRAEDTAVYYCARVQR
LSYGMDVWGQGTTVTVSS
[0166] 3244_H1104 VL Nucleotide Sequence (SEQ ID NO: 137)
TABLE-US-00048 GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA
ATTGGTATCAGAAGAGACCAGGGAAAGCCCCTAAACTCCTGGTCTATGGT
GCATCCAGTTTGCAGAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCGCCAGTCTGCAACCTGAAGATTCTG
CAGTTTATTACTGTCAACAGACTTACAGTATCCCCCTCTTCGGCCAGGGG
ACACGACTGGAGATTAAACG
[0167] 3244_H04 VL Amino Acid Sequence (SEQ ID NO: 138)
TABLE-US-00049 DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQKRPGKAPKLLVYG
ASSLQSGVPSRFSGSGSGTDFTLTIASLQPEDSAVYYCQQTYSIPLFGQG TRLEIK
[0168] The 3136_G05 antibody (also referred to herein as G05)
includes a heavy chain variable region (SEQ ID NO: 140) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 139, and a
light chain variable region (SEQ ID NO: 142) encoded by the nucleic
acid sequence shown in SEQ ID NO: 141.
[0169] 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.
[0170] The heavy chain CDRs of the G05 antibody have the following
sequences per Kabat definition: SDFWS (SEQ ID NO: 228),
YVYNRGSTKYSPSLKS (SEQ ID NO: 229) and NGRSSTSWGIDV (SEQ ID NO:
230). The light chain CDRs of the 3136_G05 antibody have the
following sequences per Kabat definition: RASQSISTYLH (SEQ ID NO:
233), AASSLQS (SEQ ID NO: 234) and QQSYSPPLT (SEQ ID NO: 63).
[0171] The heavy chain CDRs of the 3136_G05 antibody have the
following sequences per Chothia definition: GGSISS (SEQ ID NO:
206), YVYNRGSTK (SEQ ID NO: 232) and NGRSSTSWGIDV (SEQ ID NO: 230).
The light chain CDRs of the 3136_G05 antibody have the following
sequences per Chothia definition: RASQSISTYLH (SEQ ID NO: 233),
AASSLQS (SEQ ID NO: 234) and QQSYSPPLT (SEQ ID NO: 63).
[0172] 3136_G05 VH Nucleotide Sequence (SEQ ID NO: 139)
TABLE-US-00050 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCGGAGAC
CCTGTCCCTCACCTGCAGTGTCTCTGGTGGCTCCATTAGTAGTGATTTCT
GGAGTTGGATCCGACAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT
GTCTATAACAGAGGGAGCACTAAGTACAGTCCCTCCCTCAAGAGTCGAGT
CACCATATCAGCAGACATGTCCAAGAACCAGTTTTCCCTGAATATGAGTT
CTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAAAAATGGTCGA
AGTAGCACCAGTTGGGGCATCGACGTCTGGGGCAAAGGGACCACGGTCAC CGTCTCGAGC
[0173] 3136_G05 VH Amino Acid Sequence (SEQ ID NO: 140)
TABLE-US-00051 QVQLQESGPGLVKPSETLSLTCSVSGGSISSDFWSWIRQPPGKGLEWIGY
VYNRGSTKYSPSLKSRVTISADMSKNQFSLNMSSVTAADTAVYYCAKNGR
SSTSWGIDVWGKGTTVTVSS
[0174] 3136_G05 VL Nucleotide Sequence (SEQ ID NO: 141)
TABLE-US-00052 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGA
CAGACTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAC
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTAGATC
AGGAACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGATGACTTTG
CAACTTACTACTGTCAACAGAGTTACAGTCCCCCCCTCACTTTCGGCCCT
GGGACCAAAGTGGATATGAAACG
[0175] 3136_G05 VL Amino Acid Sequence (SEQ ID NO: 142)
TABLE-US-00053 DIQMTQSPSSLSASVGDRLTITCRASQSISTYLHWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSRSGTDFTLTISSLQPDDFATYYCQQSYSPPLTFGP GTKVDMK
[0176] The 3252_C13 antibody (also referred to herein as C13)
includes a heavy chain variable region (SEQ ID NO: 144) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 143, and a
light chain variable region (SEQ ID NO: 146) encoded by the nucleic
acid sequence shown in SEQ ID NO: 145.
[0177] 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.
[0178] The heavy chain CDRs of the C13 antibody have the following
sequences per Kabat definition: SDYWS (SEQ ID NO: 187),
YIYNRGSTKYTPSLKS (SEQ ID NO: 237) and HVGGHTYGIDY (SEQ ID NO: 238).
The light chain CDRs of the C13 antibody have the following
sequences per Kabat definition: RASQSISNYLN (SEQ ID NO: 241),
AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
[0179] The heavy chain CDRs of the C13 antibody have the following
sequences per Chothia definition: GASISS (SEQ ID NO: 239),
YIYNRGSTK (SEQ ID NO: 240) and HVGGHTYGIDY (SEQ ID NO: 238). The
light chain CDRs of the C13 antibody have the following sequences
per Chothia definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ
ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
[0180] 3252_C13 VH Nucleotide Sequence (SEQ ID NO: 143)
TABLE-US-00054 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGCCTCCATCAGTAGTGACTACT
GGAGCTGGATCCGGCTGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT
ATCTATAATAGAGGGAGTACCAAGTACACCCCCTCCCTGAAGAGTCGAGT
CACCATATCACTAGACACGGCCGAGAACCAGTTCTCCCTGAGGCTGAGGT
CGGTGACCGCCGCAGACACGGCCATCTATTACTGTGCGAGACATGTAGGT
GGCCACACCTATGGAATTGATTACTGGGGCCAGGGAACCCTGGTCACCGT CTCGAGC
[0181] 3252_C13 VH Amino Acid Sequence (SEQ ID NO: 144)
TABLE-US-00055 QVQLQESGPGLVKPSETLSLTCTVSGASISSDYWSWIRLPPGKGLEWIGY
IYNRGSTKYTPSLKSRVTISLDTAENQFSLRLRSVTAADTAIYYCARHVG
GHTYGIDYWGQGTLVTVSS
[0182] 3252_C13 VL Nucleotide Sequence (SEQ ID NO: 145)
TABLE-US-00056 GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCCTCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAACTATTTAA
ATTGGTATCAACACAAACCTGGGGAAGCCCCCAAGCTCCTGAACTATGCT
GCGTCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTTCAACCTGAAGATTTTG
CCACTTACTACTGTCAACAGAGTTACAATACTCCGATCACCTTCGGCCAA
GGGACACGACTGGAAATTAAACG
[0183] 3252_C13 VL Amino Acid Sequence (SEQ ID NO: 146)
TABLE-US-00057 DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQHKPGEAPKLLNYA
ASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSYNTPITFGQ GTRLEIK
[0184] The 3259_J06 antibody (also referred to herein as J06)
includes a heavy chain variable region (SEQ ID NO: 148) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 147, and a
light chain variable region (SEQ ID NO: 150) encoded by the nucleic
acid sequence shown in SEQ ID NO: 149.
[0185] 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.
[0186] The heavy chain CDRs of the J06 antibody have the following
sequences per Kabat definition: SDYWS (SEQ ID NO: 187),
YIYNRGSTKYTPSLKS (SEQ ID NO: 237) and HVGGHTYGIDY (SEQ ID NO: 238).
The light chain CDRs of the J06 antibody have the following
sequences per Kabat definition: RASQSISNYLN (SEQ ID NO: 241),
AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
[0187] The heavy chain CDRs of the J06 antibody have the following
sequences per Chothia definition: GASISS (SEQ ID NO: 239),
YIYNRGSTK (SEQ ID NO: 240) and HVGGHTYGIDY (SEQ ID NO: 238). The
light chain CDRs of the J06 antibody have the following sequences
per Chothia definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ
ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
[0188] 3255_J06 VH Nucleotide Sequence (SEQ ID NO: 147)
TABLE-US-00058 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGCCTCCATCAGTAGTGACTACT
GGAGCTGGATCCGGCTGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT
ATCTATAATAGAGGGAGTACCAAGTACACCCCCTCCCTGAAGAGTCGAGT
CACCATATCACTAGACACGGCCGAGAACCAGTTCTCCCTGAGGCTGAGGT
CGGTGACCGCCGCAGACACGGCCGTCTATTACTGTGCGAGACATGTGGGT
GGCCACACCTATGGAATTGATTACTGGGGCCAGGGAACCCTGGTCACCGT CTCGAGC
[0189] 3255_J06 VH Amino Acid Sequence (SEQ ID NO: 148)
TABLE-US-00059 QVQLQESGPGLVKPSETLSLTCTVSGASISSDYWSWIRLPPGKGLEWIGY
IYNRGSTKYTPSLKSRVTISLDTAENQFSLRLRSVTAADTAVYYCARHVG
GHTYGIDYWGQGTLVTVSS
[0190] 3255_J06 VL Nucleotide Sequence (SEQ ID NO: 149)
TABLE-US-00060 GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCCTCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAACTATTTAA
ATTGGTATCAACACAAACCTGGGGAAGCCCCCAAGCTCCTGAACTATGCT
GCGTCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGCCAGTGGATC
TGGGACAGATTTCACTCTCAGCATCAGCGGTCTTCAACCTGAAGATTTTG
CCACTTACTACTGTCAACAGAGCTACAATACTCCGATCACCTTCGGCCCA
GGGACACGACTGGAAATTAAACG
[0191] 3255_J06 VL Amino Acid Sequence (SEQ ID NO: 150)
TABLE-US-00061 DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQHKPGEAPKLLNYA
ASSLQSGVPSRFSASGSGTDFTLSISGLQPEDFATYYCQQSYNTPITFGP GTRLEIK
[0192] The 3410.sub.--1123 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.
[0193] 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.
[0194] The heavy chain CDRs of the 3410_I23 antibody have the
following sequences per Kabat definition: SYSWS (SEQ ID NO: 252),
YLYYSGSTKYNPSLKS (SEQ ID NO: 253) and TGSESTTGYGMDV (SEQ ID NO:
254). The light chain CDRs of the 3410_I23 antibody have the
following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO:
192), AASSLHS (SEQ ID NO: 258) and QQSYSPPIT (SEQ ID NO: 259).
[0195] The heavy chain CDRs of the 3410_I23 antibody have the
following sequences per Chothia definition: GDSISS (SEQ ID NO:
255), YLYYSGSTK (SEQ ID NO: 256) and TGSESTTGYGMDV (SEQ ID NO:
254). The light chain CDRs of the 3410_I23 antibody have the
following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO:
192), AASSLHS (SEQ ID NO: 258) and QQSYSPPIT (SEQ ID NO: 259).
[0196] 3420_I23 VH Nucleotide Sequence (SEQ ID NO: 151)
TABLE-US-00062 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCGTCACCTGCAAAGTCTCTGGTGACTCCATCAGTAGTTATTCCT
GGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGGTTGGCTAT
TTGTATTATAGTGGGAGCACCAAGTACAACCCCTCCCTCAAGAGTCGAAC
CACCATATCAGTAGACACGTCCACGAACCAGTTGTCCCTGAAGTTGAGTT
TTGTGACCGCCGCGGACACGGCCGTGTATTTCTGTGCGAGAACCGGCTCG
GAATCTACTACCGGCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGT
CACCGTCTCGAGC
[0197] 3420_I23 VH Amino Acid Sequence (SEQ ID NO: 152)
TABLE-US-00063 QVQLQESGPGLVKPSETLSVTCKVSGDSISSYSWSWIRQPPGKGLEWVGY
LYYSGSTKYNPSLKSRTTISVDTSTNQLSLKLSFVTAADTAVYFCARTGS
ESTTGYGMDVWGQGTTVTVSS
[0198] 3420_I23 VL Nucleotide Sequence (SEQ ID NO: 153)
TABLE-US-00064 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCGCTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAGTCCCCCGATCACCTTCGGCCAA
GGGACACGACTGGAGATTAAACG
[0199] 3420_I23 VL Amino Acid Sequence (SEQ ID NO: 154)
TABLE-US-00065 DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYA
ASSLHSGVPSRFSGSGSGTDFALTISSLQPEDFATYYCQQSYSPPITFGQ GTRLEIK
[0200] The 3139_P23 antibody (also referred to herein as P23)
includes a heavy chain variable region (SEQ ID NO: 156) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 155, and a
light chain variable region (SEQ ID NO:158) encoded by the nucleic
acid sequence shown in SEQ ID NO:157.
[0201] 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.
[0202] The heavy chain CDRs of the P23 antibody have the following
sequences per Kabat definition: NSFWG (SEQ ID NO: 260),
YVYNSGNTKYNPSLKS (SEQ ID NO: 261) and HDDASHGYSIS (SEQ ID NO: 262).
The light chain CDRs of the 3139_P23 antibody have the following
sequences per Kabat definition: RASQTISTYLN (SEQ ID NO: 265),
AASGLQS (SEQ ID NO: 61) and QQSYNTPLT (SEQ ID NO: 267).
[0203] The heavy chain CDRs of the 3139_P23 antibody have the
following sequences per Chothia definition: GGSISN (SEQ ID NO:
263), YVYNSGNTK (SEQ ID NO: 264) and HDDASHGYSIS (SEQ ID NO: 262).
The light chain CDRs of the 3139_P23 antibody have the following
sequences per Chothia definition: RASQTISTYLN (SEQ ID NO: 265),
AASGLQS (SEQ ID NO: 61) and QQSYNTPLT (SEQ ID NO: 267).
TABLE-US-00066 CAGGTGCAGCTGCAGGAGTCGGGCCCAAGACTGGTGAAGCCTTCGGAGAG
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATTAGTAATTCCTTCT
GGGGCTGGATCCGGCAGCCCCCAGGGGAGGGACTGGAGTGGATTGGTTAT
GTCTATAACAGTGGCAACACCAAGTACAATCCCTCCCTCAAGAGTCGAGT
CACCATTTCGCGCGACACGTCCAAGAGTCAACTCTACATGAAGCTGAGGT
CTGTGACCGCCGCTGACACGGCCGTGTACTACTGTGCGAGGCATGACGAC
GCAAGTCATGGCTACAGCATCTCCTGGGGCCACGGAACCCTGGTCACCGT CTCGAGC
[0204] 3139_P23 VH Amino Acid Sequence (SEQ ID NO: 156)
TABLE-US-00067 QVQLQESGPRLVKPSESLSLTCTVSGGSISNSFWGWIRQPPGEGLEWIGY
VYNSGNTKYNPSLKSRVTISRDTSKSQLYMKLRSVTAADTAVYYCARHDD
ASHGYSISWGHGTLVTVSS
[0205] 3139_P23 VL Nucleotide Sequence (SEQ ID NO: 157)
TABLE-US-00068 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAGTACTTATTTAA
ATTGGTATCAACAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCGGTTTGCAAAGTGGAGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTTCAACCTGAAGATTTTG
CAACTTACTTCTGTCAACAGAGTTACAATACTCCCCTGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAA
[0206] 3139_P23 VL Amino Acid Sequence (SEQ ID NO: 158)
TABLE-US-00069 DIQMTQSPSSLSASVGDRVTITCRASQTISTYLNWYQQKSGKAPKLLIYA
ASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSYNTPLTFGQ GTKVEIK
[0207] The 3248_P18 antibody (also referred to herein as P18)
includes a heavy chain variable region (SEQ ID NO: 160) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 159, and a
light chain variable region (SEQ ID NO: 162) encoded by the nucleic
acid sequence shown in SEQ ID NO: 161.
[0208] 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.
[0209] The heavy chain CDRs of the 3248_P18 antibody have the
following sequences per Kabat definition: AYHWS (SEQ ID NO: 268),
HIFDSGSTYYNPSLKS (SEQ ID NO: 269) and PLGSRYYYGMDV (SEQ ID NO:
270). The light chain CDRs of the 3248_P18 antibody have the
following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO:
273), GASTLQN (SEQ ID NO: 274) and QQSYSVPA (SEQ ID NO: 275).
[0210] The heavy chain CDRs of the 3248_P18 antibody have the
following sequences per Chothia definition: GGSISA (SEQ ID NO:
271), HIFDSGSTY (SEQ ID NO: 272) and PLGSRYYYGMDV (SEQ ID NO: 270).
The light chain CDRs of the 3248_P18 antibody have the following
sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273),
GASTLQN (SEQ ID NO: 274) and QQSYSVPA (SEQ ID NO: 275).
[0211] 3248_P18 VH Nucleotide Sequence (SEQ ID NO: 159)
TABLE-US-00070 CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCGGGTGGCTCCATCAGTGCTTACCACT
GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGCAC
ATCTTTGACAGTGGGAGCACTTACTACAACCCCTCCCTTAAGAGTCGAGT
CACCATATCACTAGACGCGTCCAAGAACCAGCTCTCCCTGAGATTGACCT
CTGTGACCGCCTCAGACACGGCCATATATTACTGTGCGAGACCTCTCGGG
AGTCGGTACTATTACGGAATGGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCGAGC
[0212] 3248_P18 VH Amino Acid Sequence (SEQ ID NO: 160)
TABLE-US-00071 QVQLQESGPGLVKPSETLSLTCTVSGGSISAYHWSWIRQPPGKGLEWIGH
IFDSGSTYYNPSLKSRVTISLDASKNQLSLRLTSVTASDTAIYYCARPLG
SRYYYGMDVWGQGTTVTVSS
[0213] 3248_P18 VL Nucleotide Sequence (SEQ ID NO: 161)
TABLE-US-00072 GACATCCAGATGACCCAGTCTCCGTCCTCCCTGTCTGCATCTGTCGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCAGGTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GCCTCCACTTTGCAAAATGGGGCCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTCCG
CAACTTACCTCTGTCAACAGAGTTACAGTGTCCCTGCTTTCGGCGGAGGA
ACCAAGGTGGAGGTCAAA
[0214] 3248_P18 VL Amino Acid Sequence (SEQ ID NO: 162)
TABLE-US-00073 DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYG
ASTLQNGAPSRFSGSGSGTDFTLTISSLQPEDSATYLCQQSYSVPAFGGG TKVEVK
[0215] The 3253_P10 antibody (also referred to herein as P10)
includes a heavy chain variable region (SEQ ID NO: 164) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 163, and a
light chain variable region (SEQ ID NO: 166) encoded by the nucleic
acid sequence shown in SEQ ID NO: 165.
[0216] 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.
[0217] The heavy chain CDRs of the 3253_P10 antibody have the
following sequences per Kabat definition: SDYWS (SEQ ID NO: 187),
FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDAKFSGSYYVAS (SEQ ID NO:
189). The light chain CDRs of the 3253_P10 antibody have the
following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO:
192), GATDLQS (SEQ ID NO: 282) and QQSYNTPLI (SEQ ID NO: 194).
[0218] The heavy chain CDRs of the 3253_P10 antibody have the
following sequences per Chothia definition: GGSITS (SEQ ID NO:
190), FFYNGGSTK (SEQ ID NO: 191) and HDAKFSGSYYVAS (SEQ ID NO:
189). The light chain CDRs of the 3253_P10 antibody have the
following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO:
192), GATDLQS (SEQ ID NO: 282) and QQSYNTPLI (SEQ ID NO: 194).
[0219] 3253_P10 VH Nucleotide Sequence (SEQ ID NO: 163)
TABLE-US-00074 CAGGTCCAGCTGCAGGAGTCGGGCCCAGGACTGCTGAAGCCTTCGGACAC
CCTGGCCCTCACTTGCACTGTCTCTGGTGGCTCCATCACCAGTGACTACT
GGAGCTGGATCCGGCAACCCCCAGGGAGGGGACTGGACTGGATCGGATTC
TTCTATAACGGCGGGAGCACCAAGTACAATCCCTCCCTCAAGAGTCGAGT
CACCATATCAGCGGACACGTCCAAGAACCAGTTGTCCCTGAAATTGACCT
CTGTGACCGCCGCAGACACGGGCGTGTATTATTGTGCGAGACATGATGCC
AAATTTAGTGGGAGCTACTACGTTGCCTCCTGGGGCCAGGGAACCCGAGT
CACCGTCTCGAGC
[0220] 3253_P10 VH Amino Acid Sequence (SEQ ID NO: 164)
TABLE-US-00075 QVQLQESGPGLLKPSDTLALTCTVSGGSITSDYWSWIRQPPGRGLDWIGF
FYNGGSTKYNPSLKSRVTISADTSKNQLSLKLTSVTAADTGVYYCARHDA
KFSGSYYVASWGQGTRVTVSS
[0221] 3253_P10 VL Nucleotide Sequence (SEQ ID NO: 165)
TABLE-US-00076 GACATCCAGATGACCCAGTCTCCCTCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCTCTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAA
ATTGGTATCAGCAGCAACCTGGGAAAGCCCCTAAGGTCCTGATCTCTGGT
GCAACCGACTTGCAAAGTGGGGTCCCATCTCGCTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAATACCCCCCTCATTTTTGGCCAG
GGGACCAAGCTGGAGATCAAA
[0222] 3253_P10 VL Amino Acid Sequence (SEQ ID NO: 166)
TABLE-US-00077 DIQMTQSPSSLSASVGDRVTISCRASQSISTYLNWYQQQPGKAPKVLISG
ATDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLIFGQ GTKLEIK
[0223] The 3260_D19 antibody (also referred to herein as D19)
includes a heavy chain variable region (SEQ ID NO: 168) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 167, and a
light chain variable region (SEQ ID NO: 170) encoded by the nucleic
acid sequence shown in SEQ ID NO: 169.
[0224] 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.
[0225] The heavy chain CDRs of the 3260_D19 antibody have the
following sequences per Kabat definition: DNYIN (SEQ ID NO: 284),
VFYSADRTSYADSVKG (SEQ ID NO: 285) and VQKSYYGMDV (SEQ ID NO: 286).
The light chain CDRs of the 3260_D19 antibody have the following
sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273),
GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).
[0226] The heavy chain CDRs of the 3260_D19 antibody have the
following sequences per Chothia definition: GFSVSD (SEQ ID NO:
287), VFYSADRTS (SEQ ID NO: 288) and VQKSYYGMDV (SEQ ID NO: 286).
The light chain CDRs of the 3260_D19 antibody have the following
sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273),
GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).
[0227] 3260_D19 VH Nucleotide Sequence (SEQ ID NO: 167)
TABLE-US-00078 GACATGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCCGCCGGGGGGGTC
CCTGAGACTCTCCTGCGCAGCCTCTGGGTTTTCCGTCAGTGACAACTACA
TAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGACTGGGTCTCAGTC
TTTTATAGTGCTGATAGAACATCCTACGCAGACTCCGTGAAGGGCCGATT
CACCGTCTCCAGCCACGATTCCAAGAACACAGTGTACCTTCAAATGAACA
GTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTGCGAGAGTTCAGAAG
TCCTATTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC GAGC
[0228] 3260_D19 VH Amino Acid Sequence (SEQ ID NO: 168)
TABLE-US-00079 DMQLVESGGGLVPPGGSLRLSCAASGFSVSDNYINWVRQAPGKGLDWVSV
FYSADRTSYADSVKGRFTVSSHDSKNTVYLQMNSLRAEDTAVYYCARVQK
SYYGMDVWGQGTTVTVSS
[0229] 3260_D19 VL Nucleotide Sequence (SEQ ID NO: 169)
TABLE-US-00080 GGCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTTAA
ATTGGTATCTGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCTGGT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCACTGGGTC
TGGGACAGAATTCACTCTCACCATCAGCAGTTTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGACTTTCAGTATCCCTCTTTTTGGCCAGGGG
ACCAAGGTGGAGATCAAA
[0230] 3260_D19 VL Amino Acid Sequence (SEQ ID NO: 170)
TABLE-US-00081 GIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYLQKPGKAPKLLISG
ASSLQSGVPSRFSGTGSGTEFTLTISSLQPEDFATYYCQQTFSIPLFGQG TKVEIK
[0231] The 3362_B11 antibody (also referred to herein as B11)
includes a heavy chain variable region (SEQ ID NO: 172) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 171, and a
light chain variable region (SEQ ID NO: 174) encoded by the nucleic
acid sequence shown in SEQ ID NO: 173.
[0232] 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.
[0233] The heavy chain CDRs of the B11 antibody have the following
sequences per Kabat definition: SGAYYWT (SEQ ID NO: 293),
YIYYSGNTYYNPSLKS (SEQ ID NO: 294) and AASTSVLGYGMDV (SEQ ID NO:
295). The light chain CDRs of the B11 antibody have the following
sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273),
AASSLQS (SEQ ID NO: 234) and QQSYSTPLT (SEQ ID NO: 300).
[0234] The heavy chain CDRs of the B11 antibody have the following
sequences per Chothia definition: GDSITSGA (SEQ ID NO: 296),
YIYYSGNTY (SEQ ID NO: 297) and AASTSVLGYGMDV (SEQ ID NO: 295). The
light chain CDRs of the B11 antibody have the following sequences
per Chothia definition: RASQSISRYLN (SEQ ID NO: 273), AASSLQS (SEQ
ID NO: 234) and QQSYSTPLT (SEQ ID NO: 300).
[0235] 3362_B11 VH Nucleotide Sequence (SEQ ID NO: 171)
TABLE-US-00082 CAGGTGCAGCTGCAGGCGTCGGGCCCAGGACTGGTGAAGCCTTCAGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCACCAGTGGTGCTT
ACTACTGGACCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT
GGGTACATCTATTACAGTGGGAACACCTACTACAACCCGTCCCTCAAGAG
TCGAGTTACCATATCACTAGACACGTCTAAGAACCAGTTCTCCCTGAAGG
TGAACTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGCGAGCT
GCTTCGACTTCAGTGCTAGGATACGGTATGGACGTCTGGGGCCAAGGGAC
CACGGTCACCGTCTCGAGC
[0236] 3362_B11 VH Amino Acid Sequence (SEQ ID NO: 172)
TABLE-US-00083 QVQLQASGPGLVKPSETLSLTCTVSGDSITSGAYYWTWIRQHPGKGLEWI
GYIYYSGNTYYNPSLKSRVTISLDTSKNQFSLKVNSVTAADTAVYYCARA
ASTSVLGYGMDVWGQGTTVTVSS
[0237] 3362_B11 VL Nucleotide Sequence (SEQ ID NO: 173)
TABLE-US-00084 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTTAA
ATTGGTATCAGCAGGAACCAGGGAAGGCCCCTAAGCTCCTGGTCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATAAGCAGTCTTCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTATAGTACCCCCCTCACCTTCGGCCAA
GGGACACGACTGGAGATTAAA
[0238] 3362_B11 VH Amino Acid Sequence (SEQ ID NO: 174)
TABLE-US-00085 DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQEPGKAPKLLVYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQ GTRLEIK
[0239] The 3242_P05 antibody (also referred to herein as P05)
includes a heavy chain variable region (SEQ ID NO: 176) encoded by
the nucleic acid sequence shown below in SEQ ID NO: 175, and a
light chain variable region (SEQ ID NO: 178) encoded by the nucleic
acid sequence shown in SEQ ID NO: 177.
[0240] 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.
[0241] The heavy chain CDRs of the 3242_P05 antibody have the
following sequences per Kabat definition: VSDNYIN (SEQ ID NO: 301),
VFYSADRTSYADSVKG (SEQ ID NO: 285) and VQKSYYGMDV (SEQ ID NO: 286).
The light chain CDRs of the 3242_P05 antibody have the following
sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273),
GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).
[0242] The heavy chain CDRs of the 3242_P05 antibody have the
following sequences per Chothia definition: SGFSV (SEQ ID NO: 304),
VFYSADRTS (SEQ ID NO: 288) and VQKSYYGMDV (SEQ ID NO: 286). The
light chain CDRs of the 3242_P05 antibody have the following
sequences per Chothia definition: The light chain CDRs of the
3242_P05 antibody have the following sequences per Kabat
definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226)
and QQTFSIPL (SEQ ID NO: 291).
[0243] 3242_P05 VH Nucleotide Sequence (SEQ ID NO: 175)
TABLE-US-00086 GACATGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCCGCCGGGGGGGTC
CCTGAGACTCTCCTGCGCAGCCTCTGGGTTTTCCGTCAGTGACAACTACA
TAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGACTGGGTCTCAGTC
TTTTATAGTGCTGATAGAACATCCTACGCAGACTCCGTGAAGGGCCGATT
CACCGTCTCCAGCCACGATTCCAAGAACACAGTGTACCTTCAAATGAACA
GTCTGAGAGCTGAGGACACGGCCGTTTATTACTGTGCGAGAGTTCAGAAG
TCCTATTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC GAGC
[0244] 3242_P05 VH Amino Acid Sequence (SEQ ID NO: 176)
TABLE-US-00087 DMQLVESGGGLVPPGGSLRLSCAASGFSVSDNYINWVRQAPGKGLDWVSV
FYSADRTSYADSVKGRFTVSSHDSKNTVYLQMNSLRAEDTAVYYCARVQK
SYYGMDVWGQGTTVTVSS
[0245] 3242_P05 VL Nucleotide Sequence (SEQ ID NO: 177)
TABLE-US-00088 GGCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGATATTTAA
ATTGGTATCTGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCTGGT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCACTGGGTC
TGGGACAGAATTCACTCTCACCATCAGCAGTTTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGACTTTCAGTATCCCTCTTTTTGGCCAGGGG
ACCAAGGTGGAGATCAAA
[0246] 3242_P05 VL Amino Acid Sequence (SEQ ID NO: 178)
TABLE-US-00089 GIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYLQKPGKAPKLLISG
ASSLQSGVPSRFSGTGSGTEFTLTISSLQPEDFATYYCQQTFSIPLFGQG TKVEIK
[0247] HuM2e antibodies of the invention also include antibodies
that include a heavy chain variable amino acid sequence that is at
least 90%, 92%, 95%, 97% 98%, 99% or more identical the amino acid
sequence of SEQ ID NO: 44, 277, 276, 50, 236, 235, 116, 120, 124,
128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, or 176.
and/or a light chain variable amino acid that is at least 90%, 92%,
95%, 97% 98%, 99% or more identical the amino acid sequence of SEQ
ID NO: 46, 52, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154,
158, 162, 166, 170, 174, 178.
[0248] Alternatively, the monoclonal antibody is an antibody that
binds to the same epitope as TCN-032 (8I10), 21B15, TCN-031
(23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19,
3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23,
3248_P18, 3253_P10, 3260_D19, 3362_B11, or 3242_P05.
[0249] 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.
[0250] The M2e antibodies of the invention include a variable heavy
chain (V.sub.H) region encoded by a human IgHV4 or the IgHV3
germline gene sequence. An IgHV4 germline gene sequence is shown,
e.g., in Accession numbers L10088, M29812, M95114, X56360 and
M95117. An IgHV3 germline gene sequence is shown, e.g., in
Accession numbers X92218, X70208, Z27504, M99679 and AB019437. The
M2e antibodies of the invention include a V.sub.H region that is
encoded by a nucleic acid sequence that is at least 80% homologous
to the IgHV4 or the IgHV3 germline gene sequence. Preferably, the
nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to
the IgHV4 or the IgHV3 germline gene sequence, and more preferably,
at least 98%, 99% homologous to the IgHV4 or the IgHV3 germline
gene sequence. The V.sub.H region of the M2e antibody is at least
80% homologous to the amino acid sequence of the V.sub.H region
encoded by the IgHV4 or the IgHV3 V.sub.H germline gene sequence.
Preferably, the amino acid sequence of V.sub.H region of the M2e
antibody is at least 90%, 95%, 96%, 97% homologous to the amino
acid sequence encoded by the IgHV4 or the IgHV3 germline gene
sequence, and more preferably, at least 98%, 99% homologous to the
sequence encoded by the IgHV4 or the IgHV3 germline gene
sequence.
[0251] The M2e antibodies of the invention also include a variable
light chain (V.sub.L) region encoded by a human IgKV1 germline gene
sequence. A human IgKV1 V.sub.L germline gene sequence is shown,
e.g., Accession numbers X59315, X59312, X59318, J00248, and Y14865.
Alternatively, the M2e antibodies include a V.sub.L region that is
encoded by a nucleic acid sequence that is at least 80% homologous
to the IgKV1 germline gene sequence. Preferably, the nucleic acid
sequence is at least 90%, 95%, 96%, 97% homologous to the IgKV1
germline gene sequence, and more preferably, at least 98%, 99%
homologous to the IgKV1 germline gene sequence. The V.sub.L region
of the M2e antibody is at least 80% homologous to the amino acid
sequence of the V.sub.L region encoded the IgKV1 germline gene
sequence. Preferably, the amino acid sequence of V.sub.L region of
the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the
amino acid sequence encoded by the IgKV1 germline gene sequence,
and more preferably, at least 98%, 99% homologous to the sequence
encoded by e the IgKV1 germline gene sequence.
HA Antibodies I
[0252] The HA antibodies of the invention may also be capable of
specifically binding to one or more fragments of influenza virus
H5N1, such as the surface glycoproteins, hemagglutinin (HA) and
neuraminidase (NA), which are required for viral attachment and
cellular release, or membrane proteins (M1 and M2). In a specific
embodiment, the HA antibodies of the invention are capable of
specifically binding to the HA molecule of H5N1 strains. They may
be capable of specifically binding to the HA1 and/or HA2 subunit of
the HA molecule. They may be capable of specifically binding to
linear or structural and/or conformational epitopes on the HA1
and/or HA2 subunit of the HA molecule. The HA molecule may be
purified from viruses or recombinantly produced and optionally
isolated before use. Alternatively, HA may be expressed on the
surface of cells.
[0253] For diagnostic purposes, the HA antibodies may also be
capable of specifically binding to proteins not present on the
surface of H5N1 including the nucleoprotein, the nucleocapsid
structural protein, polymerases (PA, PB and PB2), and
non-structural proteins (NS1 and NS2). The nucleotide and/or amino
acid sequence of proteins of various H5N1 strains can be found in
the GenBank-database, NCBI Influenza Virus Sequence Database,
Influenza Sequence Database (ISD), EMBL-database and/or other
databases. It is well within the reach of the skilled person to
find such sequences in the respective databases.
[0254] In another embodiment the HA antibodies of the invention are
capable of specifically binding to a fragment of the
above-mentioned proteins and/or polypeptides, wherein the fragment
at least includes an antigenic determinant recognized by the HA
antibodies of the invention. An "antigenic determinant" as used
herein is a moiety that is capable of binding to an HA antibody of
the invention with sufficiently high affinity to form a detectable
antigen-antibody complex. As used herein, the terms "antigenic
determinant" and "epitope" are equivalents. The HA antibodies of
the invention may or may not be capable of specifically binding to
the extracellular part of HA (also called herein soluble HA
(sHA)).
[0255] The HA antibodies of the invention can be intact
immunoglobulin molecules such as polyclonal or monoclonal
antibodies or the HA antibodies can be antigen-binding fragments
including, but not limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd,
complementarity determining region (CDR) fragments, single-chain
antibodies (scFv), bivalent single-chain antibodies, single-chain
phage antibodies, diabodies, triabodies, tetrabodies, and
(poly)peptides that contain at least a fragment of an
immunoglobulin that is sufficient to confer specific antigen
binding to influenza virus H5N1 strains or a fragment thereof. In a
preferred embodiment the HA antibodies are human monoclonal
antibodies.
[0256] HA antibodies can be used in non-isolated or isolated form.
Furthermore, the HA antibodies can be used alone or in a mixture
including at least one HA antibody (or variant or fragment
thereof). Thus, HA antibodies can be used in combination, e.g., as
a pharmaceutical composition comprising two or more antibodies of
the invention, variants or fragments thereof. For example,
antibodies having different, but complementary activities can be
combined in a single therapy to achieve a desired prophylactic,
therapeutic or diagnostic effect, but alternatively, antibodies
having identical activities can also be combined in a single
therapy to achieve a desired prophylactic, therapeutic or
diagnostic effect. Optionally, the mixture further includes at
least one other therapeutic agent. Preferably, the therapeutic
agent such as, e.g., M2 inhibitors (e.g., amantidine, rimantadine)
and/or neuraminidase inhibitors (e.g., zanamivir, oseltamivir) is
useful in the prophylaxis and/or treatment of an influenza virus
H5N1 infection.
[0257] Typically, HA antibodies can bind to their binding partners,
i.e. influenza virus H5N1 or fragments thereof, with an affinity
constant (Kd-value) that is lower than 0.2.times.10.sup.-4 M,
1.0.times.10.sup.-5 M, 1.0.times.10.sup.-6 M, 1.0.times.10.sup.-7
M, preferably lower than 1.0.times.10.sup.-5 M, more preferably
lower than 1.0.times.10.sup.-9 M, more preferably lower than
1.0.times.10.sup.-10 M, even more preferably lower than
1.0.times.10.sup.-11 M, and in particular lower than
1.0.times.10.sup.-12 M. The affinity constants can vary for
antibody isotypes. For example, affinity binding for an IgM isotype
refers to a binding affinity of at least about 1.0.times.10.sup.-7
M. Affinity constants can for instance be measured using surface
plasmon resonance, for example using the BIACORE system (Pharmacia
Biosensor AB, Uppsala, Sweden).
[0258] HA antibodies may bind to influenza virus H5N1 or a fragment
thereof in soluble form such as for instance in a sample or in
suspension or may bind to influenza virus H5N1 or a fragment
thereof bound or attached to a carrier or substrate, e.g.,
microtiter plates, membranes and beads, etc. Carriers or substrates
may be made of glass, plastic (e.g., polystyrene), polysaccharides,
nylon, nitrocellulose, or Teflon, etc. The surface of such supports
may be solid or porous and of any convenient shape. Furthermore,
the HA antibodies may bind to influenza virus H5N1 in
purified/isolated or non purified/non-isolated form.
[0259] HA antibodies exhibit neutralizing activity. Neutralizing
activity can for instance be measured as described in International
Patent Application PCT/EP2007/059356 (Publication No. WO
2008/028946, the contents of which are incorporated herein in their
entirety). Alternative assays measuring neutralizing activity are
described in for instance WHO Manual on Animal Influenza Diagnosis
and Surveillance, Geneva: World Health Organization, 2005, version
2002.5.
[0260] The invention relates to an isolated human HA antibody that
recognizes and binds to an epitope in the HA2 subunit of the
influenza haemagglutinin protein (HA), characterized in that said
HA antibody has neutralizing activity against an influenza virus,
for instance, including HA of the H5 subtype. Examples of influenza
strains that contain such a HA of the H5 subtype and that are
important strains in view of pandemic threats are H5N1, H5N2, H5N8,
and H5N9. Particularly preferred are HA antibodies that at least
neutralize the H5N1 influenza strain. Preferably, HA antibodies do
not depend on an epitope in the HA1 subunit of the HA protein for
binding to said HA protein.
DEFINITIONS
[0261] The term "human HA antibody" describes an intact
immunoglobulin including monoclonal antibodies, such as chimeric,
humanized or human monoclonal antibodies, or to an antigen-binding
and/or variable domain comprising fragment of an immunoglobulin
that competes with the intact immunoglobulin for specific binding
to the binding partner of the immunoglobulin, e.g. H5N1. Regardless
of structure, the antigen binding fragment binds with the same
antigen that is recognized by the intact immunoglobulin. An
antigen-binding fragment can comprise a peptide or polypeptide
comprising an amino acid sequence of at least 2, 5, 10, 15, 20, 25,
30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250
contiguous amino acid residues of the amino acid sequence of the HA
antibody.
[0262] The term "HA antibody", includes all immunoglobulin classes
and subclasses known in the art. Depending on the amino acid
sequence of the constant domain of their heavy chains, HA
antibodies can be divided into the five major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into subclasses (isotypes), e.g., IgA1, IgA2,
IgG1, IgG2, IgG3 and IgG4.
[0263] Antigen-binding fragments include, inter alia, Fab, F(ab'),
F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR)
fragments, single-chain antibodies (scFv), bivalent single-chain
antibodies, single-chain phage antibodies, diabodies, triabodies,
tetrabodies, (poly)peptides that contain at least a fragment of an
immunoglobulin that is sufficient to confer specific antigen
binding to the (poly)peptide, etc. The above fragments may be
produced synthetically or by enzymatic or chemical cleavage of
intact immunoglobulins or they may be genetically engineered by
recombinant DNA techniques. The methods of production are well
known in the art and are described, for example, in Antibodies: A
Laboratory Manual, Edited by: E. Harlow and D, Lane (1988), Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which is
incorporated herein by reference. An HA antibody or antigen-binding
fragment thereof may have one or more binding sites. If there is
more than one binding site, the binding sites may be identical to
one another or they may be different.
[0264] With respect to HA antibodies, the term "complementarity
determining regions" (CDR) as used herein means sequences within
the variable regions of HA antibodies, such as immunoglobulins,
that usually contribute to a large extent to the antigen binding
site which is complementary in shape and charge distribution to the
epitope recognized on the antigen. The CDR regions of HA antibodies
can be specific for linear epitopes, discontinuous epitopes, or
conformational epitopes of proteins or protein fragments, either as
present on the protein in its native conformation or, in some
cases, as present on the proteins as denatured, e.g., by
solubilization in SDS. Epitopes of HA antibodies may also consist
of posttranslational modifications of proteins.
[0265] The term "functional variant", as used herein, refers to an
HA antibody that includes a nucleotide and/or amino acid sequence
that is altered by one or more nucleotides and/or amino acids
compared to the nucleotide and/or amino acid sequences of the
parental HA antibody and that is still capable of competing for
binding to the binding partner, e.g. H5N1, with the parental HA
antibody. In other words, the modifications in the amino acid
and/or nucleotide sequence of the parental HA antibody do not
significantly affect or alter the binding characteristics of the HA
antibody encoded by the nucleotide sequence or containing the amino
acid sequence, i.e. the antibody is still able to recognize and
bind its target. The functional variant may have conservative
sequence modifications including nucleotide and amino acid
substitutions, additions and deletions. These modifications can be
introduced by standard techniques known in the art, such as
site-directed mutagenesis and random PCR-mediated mutagenesis, and
may include natural as well as non-natural nucleotides and amino
acids.
[0266] Conservative amino acid substitutions include the ones in
which the amino acid residue is replaced with an amino acid residue
having similar structural or chemical properties. Families of amino
acid residues having similar side chains have been defined in the
art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
asparagine, glutamine, serine, threonine, tyrosine, cysteine,
tryptophan), non-polar side chains (e.g., glycine, alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
It will be clear to the skilled artisan that other classifications
of amino acid residue families than the one used above can also be
employed. Furthermore, a HA antibody functional variant may have
non-conservative amino acid substitutions, e.g., replacement of an
amino acid with an amino acid residue having different structural
or chemical properties. Similar minor variations may also include
amino acid deletions or insertions, or both. Guidance in
determining which amino acid residues may be substituted, inserted,
or deleted without abolishing immunological activity may be found
using computer programs well known in the art.
[0267] A mutation in a nucleotide sequence can be a single
alteration made at a locus (a point mutation), such as transition
or transversion mutations, or alternatively, multiple nucleotides
may be inserted, deleted or changed at a single locus. In addition,
one or more alterations may be made at any number of loci within a
nucleotide sequence. The mutations may be performed by any suitable
method known in the art.
[0268] The term "human", when applied to HA antibodies, refers to
molecules that are either directly derived from a human or based
upon a human sequence. When an HA antibody is derived from or based
on a human sequence and subsequently modified, it is still to be
considered human as used throughout the specification. In other
words, the term human, when applied to HA antibodies is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences or based on variable
or constant regions occurring in a human or human lymphocyte and
modified in some form. Thus, the human HA antibodies may include
amino acid residues not encoded by human germline immunoglobulin
sequences, contain substitutions and/or deletions (e.g., mutations
introduced by for instance random or site-specific mutagenesis in
vitro or by somatic mutation in vivo). "Based on" as used herein
refers to the situation that a nucleic acid sequence may be exactly
copied from a template, or with minor mutations, such as by
error-prone PCR methods, or synthetically made matching the
template exactly or with minor modifications. Semi-synthetic
molecules based on human sequences are also considered to be human
as used herein.
Single Chain HA Antibodies
[0269] The heavy chain of an HA antibody is derived from a germ
line V (variable) gene such as, for example, the VH1 or VH3
germline gene (see, Tomlinson I M, Williams S C, Ignatovitch O,
Corbett S J, Winter G. V-BASE Sequence Directory. Cambridge, United
Kingdom: MRC Centre for Protein Engineering (1997)). The HA
antibodies of the invention include a V.sub.H region that is
encoded by a nucleic acid sequence that is at least 80% homologous
to the VH1 or VH3 germline gene sequence. Preferably, the nucleic
acid sequence is at least 90%, 95%, 96%, 97% homologous to the VH1
or VH3 germline gene sequence, and more preferably, at least 98%,
99% homologous to the VH1 or VH3 germline gene sequence. The
V.sub.H region of the HA antibody is at least 80% homologous to the
amino acid sequence of the V.sub.H region encoded by the VH1 or VH3
V.sub.H germline gene sequence. Preferably, the amino acid sequence
of V.sub.H region of the HA antibody is at least 90%, 95%, 96%, 97%
homologous to the amino acid sequence encoded by the VH1 or VH3
germline gene sequence, and more preferably, at least 98%, 99%
homologous to the sequence encoded by the VH1 or VH3 germline gene
sequence.
[0270] In certain aspects of the invention the VH1 germline gene is
VH1 (1-2), VH1 (1-18), VH1 (3-23), or VH1 (1-69). In other aspects
of the invention the VH3 germline gene is VH3 (3-21)
[0271] The HA antibodies of the invention also include a variable
light chain (V.sub.L) region encoded by a human germline gene
sequence selected from the group consisting of VKI, VKII, VKIII,
VKIV, VL1, VL2, and VL3 (see, Tomlinson I M, Williams S C,
Ignatovitch O, Corbett S J, Winter G. V-BASE Sequence Directory.
Cambridge, United Kingdom: MRC Centre for Protein Engineering
(1997)). Alternatively, the HA antibodies include a V.sub.L region
that is encoded by a nucleic acid sequence that is at least 80%
homologous to the germline gene sequence of VKI, VKII, VKIII, VKIV,
VL1, VL2, or VL3. Preferably, the nucleic acid sequence is at least
90%, 95%, 96%, 97% homologous to the germline gene sequence of VKI,
VKII, VKIII, VKIV, VL1, VL2, or VL3, and more preferably, at least
98%, 99% homologous to the germline gene sequence of VKI, VKII,
VKIII, VKIV, VL1, VL2, or VL3. The V.sub.L region of the HA
antibody is at least 80% homologous to the amino acid sequence of
the V.sub.L region encoded the germline gene sequence of VKI, VKII,
VKIII, VKIV, VL1, VL2, or VL3. Preferably, the amino acid sequence
of V.sub.L region of the HA antibody is at least 90%, 95%, 96%, 97%
homologous to the amino acid sequence encoded by the germline gene
sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or VL3, and more
preferably, at least 98%, 99% homologous to the sequence encoded by
the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1, VL2, or
VL3.
[0272] In certain aspects of the invention the VKI germline gene is
VKI (A20), the VKII germline gene is VKII (A3), the VKIII germline
gene is VKIII (A27), and the VKIV germline gene is VKIV (B3). In
other aspects of the invention, the VL1 germline gene is VL1
(V1-13), VL1 (V1-16), VL1 (V1-17), or. VL1 (V1-19). Alternatively,
the VL2 germline gene is VL2 (V1-3) or VL2 (V1-4). Furthermore, the
VL3 germline gene is VL3 (V2-14).
[0273] Specific combinations of a VH and HL-locus are provided for
each HA antibody described below.
[0274] The CDR regions of the HA antibodies of the invention were
determined according to Kabat et al. (1991) as described in
Sequences of Proteins of Immunological Interest. In certain
embodiments of the invention, HA antibodies contain two, three,
four, five or all six CDR regions as disclosed herein. Preferably,
HA antibodies contain at least two of the CDRs disclosed
herein.
[0275] 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).
[0276] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-141
antibody have the following CDR sequences: GYYVY (HCDR1, SEQ ID NO:
247), WISAYNGNTNYAQKFQG (HCDR2, SEQ ID NO: 248) and SRSLDV (HCDR3,
SEQ ID NO: 568). The light chain CDRs of the SC06-141 antibody have
the following CDR sequences: KSSQSVLYSSNNKNYLA (LCDR1, SEQ ID NO:
569), WASTRES (LCDR2, SEQ ID NO: 570) and QQYYSTPLT (LCDR3, SEQ ID
NO: 289).
[0277] SC06-141 Nucleotide Sequence (SEQ ID NO: 311)
TABLE-US-00090 gaggtccagc tggtgcagtc tggggctgag gtgaagaagc
ctggggcctc agtgaaggtc 60 tcctgcaagg cttctgggta caccttcacc
ggctactatg tgtactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 gcacagaagt
tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagaagtaga
300 tccctggacg tctggggcca agggaccacg gtcaccgtct cgagcggtac
gggcggttca 360 ggcggaaccg gcagcggcac tggcgggtcg acggatgttg
tgatgactca gtctccagac 420 tccctggctg tgtctctggg cgagagggcc
accatcaact gcaagtccag ccagagtgtt 480 ttatacagct ccaacaataa
gaactactta gcttggtacc agcagaaacc aggacagcct 540 cctaagctgc
tcatttactg ggcatctacc cgggaatccg gggtccctga ccgattcagt 600
ggcagcgggt ctgggacaga tttcactctc accatcagca gcctgcaggc tgaagatgtg
660 gcagtttatt actgtcagca atattatagt actcctctca ctttcggcgg
agggaccaaa 720 gtggatatca aacgt 735
[0278] SC06-141 Amino Acid Sequence (SEQ ID NO: 312)
TABLE-US-00091 EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGW
ISAYNGNTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSR
SLDVWGQGTTVTVSSGTGGSGGTGSGTGGSTDVVMTQSPDSLAVSLGERA
TINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFS
GSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVDIKR
[0279] SC06-141 VH Amino Acid Sequence (SEQ ID NO: 309)
TABLE-US-00092 EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGW
ISAYNGNTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSR
SLDVWGQGTTVTVSS
[0280] SC06-141 VL Amino Acid Sequence (SEQ ID NO: 310)
TABLE-US-00093 DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP
KWYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPL TFGGGTKVDIKR
[0281] The SC06-255 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 313) and a light chain
variable region (SEQ ID NO: 314) encoded by the nucleic acid
sequence shown in SEQ ID NO: 315 and the amino acid sequence shown
in SEQ ID NO: 316. The VH-locus is VH1 (1-69) and the VL locus is
VL1 (V1-16).
[0282] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-255
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV
(HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-255
antibody have the following CDR sequences: SGSTFNIGSNAVD (LCDR1,
SEQ ID NO: 574), SNNQRPS (LCDR2, SEQ ID NO: 575) and AAWDDILNVPV
(LCDR3, SEQ ID NO: 576).
[0283] SC06-255 Nucleotide Sequence (SEQ ID NO: 315)
TABLE-US-00094 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gtcctatgtg 420 ctgactcagc caccctcagc gtctgggacc
cccgggcaga gggtcaccat ctcttgttct 480 ggaagcacgt tcaacatcgg
aagtaatgct gtagactggt accggcagct cccaggaacg 540 gcccccaaac
tcctcatcta tagtaataat cagcggccct caggggtccc tgaccgattc 600
tctggctcca ggtctggcac ctcagcctcc ctggccatca gtgggctcca gtctgaggat
660 gaggctgatt attactgtgc agcatgggat gacatcctga atgttccggt
attcggcgga 720 gggaccaagc tgaccgtcct aggt 744
[0284] SC06-255 Amino Acid Sequence (SEQ ID NO: 316)
TABLE-US-00095 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSASGT
PGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIYSNNQRPSGVPDRF
SGSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPVFGGGTKLTVLG
[0285] SC06-255 VH Amino Acid Sequence (SEQ ID NO: 313)
TABLE-US-00096 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM GYQVRETMDVWGKG
TTVTVSS
[0286] SC06-255 VL Amino Acid Sequence (SEQ ID NO: 314)
TABLE-US-00097 SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIY
SNNQRPSGVPDRFSGSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPV FGGGTKLTVLG
[0287] The SC06-257 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 317) and a light chain
variable region (SEQ ID NO: 318) encoded by the nucleic acid
sequence shown in SEQ ID NO: 319 and the amino acid sequence shown
in SEQ ID NO: 320. The VH-locus is VH1 (1-69) and the VL locus is
VL2 (V1-4).
[0288] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-257
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV
(HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-257
antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDR1,
SEQ ID NO: 577), EVSNRPS (LCDR2, SEQ ID NO: 578) and SSYTSSSTY
(LCDR3, SEQ ID NO: 579).
[0289] SC06-257 Nucleotide Sequence (SEQ ID NO: 319)
TABLE-US-00098 caggtccagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gcagtctgcc 420 ctgactcagc ctgccgccgt gtctgggtct
cctggacagt cgatcaccat ctcctgcact 480 ggaaccagca gtgacgttgg
tggttataac tatgtctcct ggtaccaaca gcacccaggc 540 aaagccccca
aactcatgat ttatgaggtc agtaatcggc cctcaggggt ttctaatcgc 600
ttctctggct ccaagtctgg caacacggcc tccctgacca tctctgggct ccaggctgag
660 gacgaggctg attattactg cagctcatat acaagcagca gcacttatgt
cttcggaact 720 gggaccaagg tcaccgtcct aggt 744
[0290] SC06-257 Amino Acid Sequence (SEQ ID NO: 320)
TABLE-US-00099 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTQSALTQPAAVSGS
PGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNR
[0291] SC06-257 VH Amino Acid Sequence (SEQ ID NO: 317)
TABLE-US-00100 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0292] SC06-257 VL Amino Acid Sequence (SEQ ID NO: 318)
TABLE-US-00101 QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYV FGTGTKVTVLG
[0293] The SC6-260 HA-specific single-cham Fv antibody includes a
heavy chain variable region (SEQ ID NO: 321) and a light chain
variable region (SEQ ID NO: 322) encoded by the nucleic acid
sequence shown in SEQ ID NO: 323 and the amino acid sequence shown
in SEQ ID NO: 324. The VH-locus is VH1 (1-69) and the VL locus is
VL1 (V1-17).
[0294] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-260
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV
(HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-260
antibody have the following CDR sequences: SGSRSNVGDNSVY (LCDR1,
SEQ ID NO: 580), KNTQRPS (LCDR2, SEQ ID NO: 581) and VAWDDSVDGYV
(LCDR3, SEQ ID NO: 582).
[0295] SC06-260 Nucleotide Sequence (SEQ ID NO: 323)
TABLE-US-00102 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gtcctatgtg 420 ctgactcagc caccctcagt ctctgggacc
cccgggcaga gggtcaccat ctcttgctct 480 ggaagccgct ccaacgtcgg
agataattct gtatattggt atcaacacgt cccagaaatg 540 gcccccaaac
tcctcgtcta taagaatact caacggccct caggagtccc tgcccggttt 600
tccggctcca agtctggcac ttcagcctcc ctggccatca ttggcctcca gtccggcgat
660 gaggctgatt attattgtgt ggcatgggat gacagcgtag atggctatgt
cttcggatct 720 gggaccaagg tcaccgtcct aggt 744
[0296] SC06-260 Amino Acid Sequence (SEQ ID NO: 324)
TABLE-US-00103 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSVSGT
PGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVYKNTQRPSGVPARF
SGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYVFGSGTKVTVLG
[0297] SC06-260 VH Amino Acid Sequence (SEQ ID NO: 321)
TABLE-US-00104 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0298] SC06-260 VL Amino Acid Sequence (SEQ ID NO: 322)
TABLE-US-00105 SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVY
KNTQRPSGVPARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYV FGSGTKVTVLG
[0299] The SC06-261 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 325) and a light chain
variable region (SEQ ID NO: 326) encoded by the nucleic acid
sequence shown in SEQ ID NO: 327 and the amino acid sequence shown
in SEQ ID NO: 328. The VH-locus is VH1 (1-69) and the VL locus is
VL1 (V1-19).
[0300] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-261
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV
(HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-261
antibody have the following CDR sequences: SGSSSNIGNDYVS (LCDR1,
SEQ ID NO: 583), DNNKRPS (LCDR2, SEQ ID NO: 584) and ATWDRRPTAYVV
(LCDR3, SEQ ID NO: 585).
[0301] SC06-261 Nucleotide Sequence (SEQ ID NO: 327)
TABLE-US-00106 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gcagtctgtg 420 ttgacgcagc cgccctcagt gtctgcggcc
ccaggacaga aggtcaccat ctcctgctct 480 ggaagcagct ccaacattgg
gaatgattat gtatcctggt accagcagct cccaggaaca 540 gcccccaaac
tcctcattta tgacaataat aagcgaccct cagggattcc tgaccgattc 600
tctggctcca agtctggcac gtcagccacc ctgggcatca ccggactcca gactggggac
660 gaggccaact attactgcgc aacatgggat cgccgcccga ctgcttatgt
tgtcttcggc 720 ggagggacca agctgaccgt cctaggt 747
[0302] SC06-261 Amino Acid Sequence (SEQ ID NO: 328)
TABLE-US-00107 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTQSVLTQPPSVSAA
PGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRF
SGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVLG
[0303] SC06-261 VH Amino Acid Sequence (SEQ ID NO: 325)
TABLE-US-00108 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0304] SC06-261 VL Amino Acid Sequence (SEQ ID NO: 326)
TABLE-US-00109 SVLTQFFSVSAAFGQKVTISCSGSSSNIGNDYVSWYQQLFGTAFKLLIYD
NNKRPSGIFDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVV FGGGTKLTVLG
[0305] 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).
[0306] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-262
antibody have the following CDR sequences: GSAIS (HCDR1, SEQ ID NO:
586), GISPLFGTTNYAQKFQG (HCDR2, SEQ ID NO: 587) and GPKYYSEYMDV
(HCDR3, SEQ ID NO: 588). The light chain CDRs of the SC06-262
antibody have the following CDR sequences: RASQGISSYLA (LCDR1, SEQ
ID NO: 589), DASTLRS (LCDR2, SEQ ID NO: 590) and QRYNSAPPI (LCDR3,
SEQ ID NO: 591).
[0307] SC06-262 Nucleotide Sequence (SEQ ID NO: 331)
TABLE-US-00110 caggtacagc tgcagcagtc aggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg tttccggagt cattttcagc
ggcagtgcga tcagctgggt gcgacaggcc 120 cctggacaag gccttgagtg
gatgggaggg atcagccctc tctttggcac aacaaattac 180 gcacaaaagt
tccagggcag agtcacgatt accgcggacc aatccacgaa cacaacctac 240
atggaggtga acagcctgag atatgaggac acggccgtgt atttctgtgc gcgaggtcca
300 aaatattaca gtgagtacat ggacgtctgg ggcaaaggga ccacggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgga catccagatg 420 acccagtctc catcctccct gtctgcatct
gtaggagaca gagtcaccat cacttgccgg 480 gcgagtcagg gcattagcag
ttatttagcc tggtatcagc agaagccagg gaaagttcct 540 acactcctga
tctatgatgc atccactttg cgatcagggg tcccatctcg cttcagtggc 600
agtggatctg cgacagattt cactctcacc atcagcagcc tgcagcctga agatgttgca
660 acttattact gtcaaaggta taacagtgcc cccccgatca ccttcggcca
agggacacga 720 ctggagatta aacgt 735
[0308] SC06-262 Amino Acid Sequence (SEQ ID NO: 332)
TABLE-US-00111 QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGG
ISPLFGTTNYAQKFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGP
KYYSEYMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTDIQMTQSPSSLSAS
VGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYDASTLRSGVPSRFSG
SGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFGQGTRLEIKR
[0309] SC06-262 VH Amino Acid Sequence (SEQ ID NO: 329)
TABLE-US-00112 QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGG
ISPLFGTTNYAQKFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGP
KYYSEYMDVWGKGTTVTVSS
[0310] SC06-262 VL Amino Acid Sequence (SEQ ID NO: 330)
TABLE-US-00113 DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYD
ASTLRSGVPSRFSGSGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFG QGTRLEIKR
[0311] The SC06-268 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 333) and a light chain
variable region (SEQ ID NO: 334) encoded by the nucleic acid
sequence shown in SEQ ID NO: 335 and the amino acid sequence shown
in SEQ ID NO: 336. The VH-locus is VH1 (1-69) and the VL locus is
VL3 (V2-14).
[0312] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-268
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
571), GIMGMFGTTNYAQKFQG (HCDR2, SEQ ID NO: 592) and SSGYYPEYFQD
(HCDR3, SEQ ID NO: 593). The light chain CDRs of the SC06-268
antibody have the following CDR sequences: SGHKLGDKYVS (LCDR1, SEQ
ID NO: 594), QDNRRPS (LCDR2, SEQ ID NO: 595) and QAWDSSTA (LCDR3,
SEQ ID NO: 596).
[0313] SC06-268 Nucleotide Sequence (SEQ ID NO: 335)
TABLE-US-00114 caggtccagc tggtacagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagt
agttatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggagga atcatgggta tgtttggcac aactaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aattcacgag cgcagcctac 240
atggagctga ggagcctgag atctgaggac acggccgtct actactgtgc gaggtctagt
300 ggttattacc ccgaatactt ccaggactgg ggccagggca ccctggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgca gtctgtgctg 420 actcagccac cctcagagtc cgtgtcccca
ggacagacag ccagcgtcac ctgctctgga 480 cataaattgg gggataaata
tgtttcgtgg tatcagcaga agccaggcca gtcccctgta 540 ttactcatct
atcaagataa caggcggccc tcagggatcc ctgagcgatt cataggctcc 600
aactctggga acacagccac tctgaccatc agcgggaccc aggctctgga tgaggctgac
660 tattactgtc aggcgtggga cagcagcact gcggttttcg gcggagggac
caagctgacc 720 gtcctaggt 729
[0314] SC06-268 Amino Acid Sequence (SEQ ID NO: 336)
TABLE-US-00115 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IMGMFGTTNYAQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCARSS
GYYPEYFQDWGQGTLVTVSSGTGGSGGTGSGTGGSTQSVLTQPPSESVSP
GQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQDNRRPSGIPERFIGS
NSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGGTKLTVLG
[0315] SC06-268 VH Amino Acid Sequence (SEQ ID NO: 333)
TABLE-US-00116 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IMGMFGTTNYAQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCARSS
GYYPEYFQDWGQGTLVTVSS
[0316] SC06-268 VL Amino Acid Sequence (SEQ ID NO: 334)
TABLE-US-00117 QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQD
NRRPSGIPERFIGSNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGG TKLTVLG
[0317] The SC06-272 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 337) and a light chain
variable region (SEQ ID NO: 338) encoded by the nucleic acid
sequence shown in SEQ ID NO: 339 and the amino acid sequence shown
in SEQ ID NO: 340. The VH-locus is VH1 (1-69) and the VL locus is
VL2 (V1-3).
[0318] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-272
antibody have the following CDR sequences: SYAIT (HCDR1, SEQ ID NO:
597), GIIGMFGSTNYAQNFQG (HCDR2, SEQ ID NO: 598) and STGYYPAYLHH
(HCDR3, SEQ ID NO: 599). The light chain CDRs of the SC06-272
antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDR1,
SEQ ID NO: 577), DVSKRPS (LCDR2, SEQ ID NO: 601) and SSYTSSSTHV
(LCDR3, SEQ ID NO: 602).
[0319] SC06-272 Nucleotide Sequence (SEQ ID NO: 339)
TABLE-US-00118 cagatgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttctcc
agttatgcta tcacctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcggta tgtttggttc aacaaactac 180 gcacagaact
tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240
atggagctga gcagcctcag atctgaggac acggccgtgt attactgtgc gagaagtact
300 ggttattacc ctgcatacct ccaccactgg ggccagggca ccctggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgca gtctgccctg 420 actcagcctc gctcagtgtc cgggtctcct
ggacagtcag tcaccatctc ctgcactgga 480 accagcagtg atgttggtgg
ttataactat gtctcctggt accaacagca cccaggcaaa 540 gcccccaaac
tcatgattta tgatgtcagt aagcggccct caggggtccc tgatcgcttc 600
tctggctcca agtctggcaa cacggcctcc ctgaccatct ctgggctcca ggctgaggat
660 gaggctgatt attactgcag ctcatataca agcagcagca ctcatgtctt
cggaactggg 720 accaaggtca ccgtcctagg t 741
[0320] SC06-272 Amino Acid Sequence (SEQ ID NO: 340)
TABLE-US-00119 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGG
IIGMFGSTYAQNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTG
YYPAYLHHWGQGTLVTVSSGTGGSGGTGSGTGGSTQSALTQPRSVSGSPG
QSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFS
GSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVLG
[0321] SC06-272 VH Amino Acid Sequence (SEQ ID NO: 337)
TABLE-US-00120 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGG
IIGMFGSTNYAQNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARST
GYYPAYLHHWGQGTLVTVSS
[0322] SC06-272 VL Amino Acid Sequence (SEQ ID NO: 338)
TABLE-US-00121 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHV FGTGTKVTVLG
[0323] The SC06-296 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 341) and a light chain
variable region (SEQ ID NO: 342) encoded by the nucleic acid
sequence shown in SEQ ID NO: 343 and the amino acid sequence shown
in SEQ ID NO: 344. The VH-locus is VH1 (1-2) and the VL locus is
VKIII (A27).
[0324] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-296
antibody have the following CDR sequences: SYYMH (HCDR1, SEQ ID NO:
603), WINPNSGGTNYAQKFQG (HCDR2, SEQ ID NO: 604) and EGKWGPQAAFDI
(HCDR3, SEQ ID NO: 605). The light chain CDRs of the SC06-296
antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ
ID NO: 646), DASSRAT (LCDR2, SEQ ID NO: 607) and QQYGSSLW (LCDR3,
SEQ ID NO: 608).
[0325] SC06-296 Nucleotide Sequence (SEQ ID NO: 343)
TABLE-US-00122 gaggtgcagc tggtggagac cggggctgag gtgaagaagc
ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc
agctactata tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggatgg atcaacccta acagtggtgg cacaaactat 180 gcacagaagt
ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagagggg
300 aaatggggac ctcaagcggc ttttgatatc tggggccaag ggacaatggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac ggaaattgtg 420 atgacgcagt ctccaggcac cctgtctttg
tctccagggg aaagagccac cctctcctgc 480 agggccagtc agagtgttag
cagcagctac ttagcctggt accagcagaa acctggccag 540 gctcccaggc
tcctcatcta tgatgcatcc agcagggcca ctgacatccc agacaggttc 600
agtggcagtg ggtctgggac agacttcact ctcaccatca gcagactgga gcctgaagat
660 tttgcagtgt attactgtca gcagtatggt agctcacttt ggacgttcgg
ccaagggacc 720 aaggtggaga tcaaacgt 738
[0326] SC06-296 Amino Acid Sequence (SEQ ID NO: 344)
TABLE-US-00123 EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREG
KWGPQAAFDIWGQGTMVTVSSGTGGSGGTGSGTGGSTEIVMTQSPGTLSL
SPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATDIPDRF
SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFGQGTKVEIKR
[0327] SC06-296 VH amino acid sequence (SEQ ID NO: 341)
TABLE-US-00124 EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREG
KWGPQAAFDIWGQGTMVTVSS
[0328] SC06-296 VL Amino Acid Sequence (SEQ ID NO: 342)
TABLE-US-00125 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY
DASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFG QGTKVEIKR
[0329] The SC06-301 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 345) and a light chain
variable region (SEQ ID NO: 346) encoded by the nucleic acid
sequence shown in SEQ ID NO: 347 and the amino acid sequence shown
in SEQ ID NO: 348. The VH-locus is VH1 (3-23) and the VL locus is
VKII (A3).
[0330] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-301
antibody have the following CDR sequences: IYAMS (HCDR1, SEQ ID NO:
609), AISSSGDSTYYADSVKG (HCDR2, SEQ ID NO: 610) and AYGYTFDP
(HCDR3, SEQ ID NO: 611). The light chain CDRs of the SC06-301
antibody have the following CDR sequences: RSSQSLLHSNGYNYLD (LCDR1,
SEQ ID NO: 612), LGSNRAS (LCDR2, SEQ ID NO: 613) and MQALQTPL
(LCDR3, SEQ ID NO: 614).
[0331] SC06-301 Nucleotide Sequence (SEQ ID NO: 347)
TABLE-US-00126 gaggtgcagc tggtagagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc
atctatgcca tgagctgggt ccgccaggca 120 ccagggaagg ggctggagtg
ggtctcagct attagtagta gtggtgatag cacatactac 180 gcagactccg
tgaagggccg gttcaccatc tccagagaca acgccaggaa cacgctgtat 240
ctgcaaatga acagtctgag agccgaggac acggctgtgt attactgtgc gagagcgtat
300 ggctacacgt tcgacccctg gggccaggga accctggtca ccgtctcgag
cggtacgggc 360 ggttcaggcg gaaccggcag cggcactggc gggtcgacgg
aaattgtgct gactcagtct 420 ccactctccc tgcccgtcac ccctggagag
ccggcctcca tctcctgcag gtctagtcag 480 agcctcctgc atagtaatgg
atacaactat ttggattggt acctgcagaa gccagggcag 540 tctccacagc
tcctgatcta tttgggttct aatcgggcct ccggggtccc tgacaggttc 600
agtggcagtg gatcaggcac agattttaca ctgaaaatca gcagagtgga ggctgaggat
660 gttggggttt attactgcat gcaagctcta caaactcccc tcactttcgg
cggagggacc 720 aaggtggaga tcaaacgt 738
[0332] SC06-301 Amino Acid Sequence (SEQ ID NO: 348)
TABLE-US-00127 EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSA
ISSSGDSTYYADSVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAY
GYTFDPWGQGTLVTVSSGTGGSGGTGSGTGGSTEIVLTQSPLSLPVTPGE
PASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRF
SGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKR
[0333] SC06-301 VH Amino Acid Sequence (SEQ ID NO: 345)
TABLE-US-00128 EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSA
ISSSGDSTYYADSVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAY
GYTFDPWGQGTLVTVSS
[0334] SC06-301 VL Amino Acid Sequence (SEQ ID NO: 346)
TABLE-US-00129 EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP
LTFGGGTKVEIKR
[0335] The SC06-307 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 349) and a light chain
variable region (SEQ ID NO: 350) encoded by the nucleic acid
sequence shown in SEQ ID NO: 351 and the amino acid sequence shown
in SEQ ID NO: 352. The VH-locus is VH3 (3-21) and the VL locus is
VKIII (A27).
[0336] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-307
antibody have the following CDR sequences: SYSMN (HCDR1, SEQ ID NO:
615), SISSSSSYIYYVDSVKG (HCDR2, SEQ ID NO: 616) and GGGSYGAYEGFDY
(HCDR3, SEQ ID NO: 617). The light chain CDRs of the SC06-307
antibody have the following CDR sequences: RASQRVSSYLA (LCDR1, SEQ
ID NO: 618), GASTRAA (LCDR2, SEQ ID NO: 619) and QQYGRTPLT (LCDR3,
SEQ ID NO: 620).
[0337] SC06-307 Nucleotide Sequence (SEQ ID NO: 351)
TABLE-US-00130 caggtccagc tggtgcagtc tgggggaggc ctggtcaagc
ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt
agctatagca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg
ggtctcatcc attagtagta gtagtagtta catatactac 180 gtagactcag
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagaggtggt
300 gggagctacg gggcctacga aggctttgac tactggggcc agggcaccct
ggtcaccgtc 360 tcgagcggta cgggcggttc aggcggaacc ggcagcggca
ctggcgggtc gacggaaatt 420 gtgctgactc agtctccagg caccctgtct
ttgtctccag gggaaagagc caccctctcc 480 tgcagggcca gtcagcgtgt
tagcagctac ttagcctggt accaacagaa acctggccag 540 gctcccaggc
tcctcatcta tggtgcatcc accagggccg ctggcatccc agacaggttc 600
agtggcagtg ggtctgggac agacttcact ctcaccatca gcagactgga gcctgaagat
660 tctgcagtgt attactgtca gcagtatggt aggacaccgc tcactttcgg
cggagggacc 720 aaggtggaga tcaaacgt 738
[0338] SC06-307 Amino Acid Sequence (SEQ ID NO: 352)
TABLE-US-00131 QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSS
ISSSSSYIYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGG
GSYGAYEGFDYWGQGTLVTVSSGTGGSGGTGSGTGGSTEIVLTQSPGTLS
LSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYGASTRAAGIPDRF
SGSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGGGTKVEIKR
[0339] SC06-307 VH Amino Acid Sequence (SEQ ID NO: 349)
TABLE-US-00132 QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSS
ISSSSSYIYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGG
GSYGAYEGFDYWGQGTLVTVSS
[0340] SC06-307 VL Amino Acid Sequence (SEQ ID NO: 350)
TABLE-US-00133 EIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYG
ASTRAAGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGG GTKVEIKR
[0341] The SC06-310 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 353) and a light chain
variable region (SEQ ID NO: 354) encoded by the nucleic acid
sequence shown in SEQ ID NO: 355 and the amino acid sequence shown
in SEQ ID NO: 356. The VH-locus is VH1 (1-69) and the VL locus is
VL3 (V2-14).
[0342] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-310
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV
(HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-310
antibody have the following CDR sequences: GGNNIGSKSVH (LCDR1, SEQ
ID NO: 621), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDHAV
(LCDR3, SEQ ID NO: 623).
[0343] SC06-310 Nucleotide Sequence (SEQ ID NO: 355)
TABLE-US-00134 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gtcctatgtg 420 ctgactcagc caccctcggt gtcagtggcc
ccaggacaga cggccaggat tacctgtggg 480 ggaaacaaca ttggaagtaa
aagtgtgcac tggtaccagc 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 tcctcggt 738
[0344] SC6-310 Amino Acid Sequence (SEQ ID NO: 356)
TABLE-US-00135 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSVSVA
PGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG
SNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFGGGTQLTVLG
[0345] SC06-310 VH Amino Acid Sequence (SEQ ID NO: 353)
TABLE-US-00136 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0346] SC06-310 VL Amino Acid Sequence (SEQ ID NO: 354)
TABLE-US-00137 SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFG GGTQLTVLG
[0347] The SC06-314 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 357) and a light chain
variable region (SEQ ID NO: 358) encoded by the nucleic acid
sequence shown in SEQ ID NO: 359 and the amino acid sequence shown
in SEQ ID NO: 360. The VH-locus is VH1 (1-69) and the VL locus is
VL1 (V1-17).
[0348] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-314
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
571), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV
(HCDR3, SEQ ID NO: 573). The light chain CDRs of the SC06-314
antibody have the following CDR sequences: SGSSSNIGSNYVY (LCDR1,
SEQ ID NO: 624), RDGQRPS (LCDR2, SEQ ID NO: 625) and ATWDDNLSGPV
(LCDR3, SEQ ID NO: 626).
[0349] SC06-314 Nucleotide Sequence (SEQ ID NO: 359)
TABLE-US-00138 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gtcctatgtg 420 ctgactcagc caccctcagc gtctgggacc
cccgggcaga gggtcaccat ctcttgttct 480 ggaagcagct ccaacatcgg
aagtaattat gtatactggt accagcagct cccaggcacg 540 gcccccaaac
tcctcatcta tagggatggt cagcggccct caggggtccc tgaccgattc 600
tctggctcca agtctggcac ctcagcctcc ctggccatca gtggactccg gtccgatgat
660 gaggctgatt attactgtgc aacatgggat gacaacctga gtggtccagt
attcggcgga 720 gggaccaagc tgaccgtcct aggt 744
[0350] SC06-314 Amino Acid Sequence (SEQ ID NO: 360)
TABLE-US-00139 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSASGT
PGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRDGQRPSGVPDRF
SGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPVFGGGTKLTVLG
[0351] SC06-314 VH Amino Acid Sequence (SEQ ID NO: 357)
TABLE-US-00140 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMG
YQVRETMDVWGKGTTVTVSS
[0352] SC06-314 VL Amino Acid Sequence (SEQ ID NO: 358)
TABLE-US-00141 SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIY
RDGQRPSGVPDRFSGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPV FGGGTKLTVLG
[0353] The SC06-323 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 361) and a light chain
variable region (SEQ ID NO: 362) encoded by the nucleic acid
sequence shown in SEQ ID NO: 363 and the amino acid sequence shown
in SEQ ID NO: 364. The VH-locus is VH1 (1-69) and the VL locus is
VKIII (A27).
[0354] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-323
antibody have the following CDR sequences: SYGIS (HCDR1, SEQ ID NO:
627), DIIGMFGSTNYAQNFQG (HCDR2, SEQ ID NO: 628) and SSGYYPAYLPH
(HCDR3, SEQ ID NO: 629). The light chain CDRs of the SC06-323
antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ
ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSPRT (LCDR3,
SEQ ID NO: 632).
[0355] SC06-323 Nucleotide Sequence (SEQ ID NO: 363)
TABLE-US-00142 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
cagggtcctc ggtgaaggtc 60 tcctgtaagg cctctggagg caccttctcc
agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggagac atcatcggta tgtttggttc aacaaactac 180 gcacagaact
tccagggcag actcacgatt accgcggacg aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaagtagt
300 ggttattacc ctgcatacct cccccactgg ggccagggca ccttggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgga aattgtgttg 420 acccagtctc caggcaccct gtctttgtct
ccaggggaaa gagccaccct ctcctgcagg 480 gccagtcaga gtgttagcag
cagctactta gcctggtacc agcagaaacc tggccaggct 540 cccaggctcc
tcatctatgg tgcatccagc agggccactg gcatcccaga caggttcagt 600
ggcagtgggt ctgggacaga cttcactctc accatcagca gactggagcc tgaagatttt
660 gcagtgtatt actgtcagca gtatggtagc tcacccagaa ctttcggcgg
agggaccaag 720 gtggagatca aacgt 735
[0356] SC06-323 Amino Acid Sequence (SEQ ID NO: 364)
TABLE-US-00143 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGD
IIGMFGSTNYAQNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSS
GYYPAYLPHWGQGTLVTVSSGTGGSGGTGSGTGGSTEIVLTQSPGTLSLS
PGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR
[0357] SC06-323 VH Amino Acid Sequence (SEQ ID NO: 361)
TABLE-US-00144 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGD
IIGMFGSTNYAQNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSS
GYYPAYLPHWGQGTLVTVSS
[0358] SC06-323 VL Amino Acid Sequence (SEQ ID NO: 362)
TABLE-US-00145 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFG GGTKVEIKR
[0359] The SC06-325 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 365) and a light chain
variable region (SEQ ID NO: 366) encoded by the nucleic acid
sequence shown in SEQ ID NO: 367 and the amino acid sequence shown
in SEQ ID NO: 368. The VH-locus is VH1 (1-69) and the VL locus is
VL2 (V1-4).
[0360] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-325
antibody have the following CDR sequences: FYSMS (HCDR1, SEQ ID NO:
633), GIIPMFGTTNYAQKFQG (HCDR2, SEQ ID NO: 634) and GDKGIYYYYMDV
(HCDR3, SEQ ID NO: 635). The light chain CDRs of the SC06-325
antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDR1,
SEQ ID NO: 577), EVSNRPS (LCDR2, SEQ ID NO: 578) and SSYTSSSTLV
(LCDR3, SEQ ID NO: 636).
[0361] SC06-325 Nucleotide Sequence (SEQ ID NO: 367)
TABLE-US-00146 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
cggggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc
ttctattcta tgagctgggt gcgacaggcc 120 cctggacaag gacttgagtg
gatgggaggg atcatcccta tgtttggtac aacaaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggtcg aatccacgag cacagcctac 240
atggaggtga gcagcctgag atctgaggac acggccgttt attactgtgc gagaggtgat
300 aagggtatct actactacta catggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gcagtctgcc 420 ctgactcagc ctgcctccgt gtctgggtct
cctggacagt cgatcaccat ctcctgcact 480 ggaaccagca gtgacgttgg
tggttataac tatgtctcct ggtaccaaca gcacccaggc 540 aaagccccca
aactcatgat ttatgaggtc agtaatcggc cctcaggggt ttctaatcgc 600
ttctctggct ccaagtctgg caacacggcc tccctgacca tctctgggct ccaggctgag
660 gacgaggctg attattactg cagctcatat acaagcagca gcactcttgt
cttcggaact 720 gggaccaagg tcaccgtcct aggt 744
[0362] SC06-325 Amino Acid Sequence (SEQ ID NO: 368)
TABLE-US-00147 EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGG
IIPMFGTTNYAQKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGD
KGIYYYYMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTQSALTQPASVSGS
PGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNR
FSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLG
[0363] SC06-325 VH Amino Acid Sequence (SEQ ID NO: 365)
TABLE-US-00148 EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGG
IIPMFGTTNYAQKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGD
KGIYYYYMDVWGKGTTVTVSS
[0364] SC06-325 VL Amino Acid Sequence (SEQ ID NO: 366)
TABLE-US-00149 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLV FGTGTKVTVLG
[0365] The SC06-327 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 369) and a light chain
variable region (SEQ ID NO: 370) encoded by the nucleic acid
sequence shown in SEQ ID NO: 371 and the amino acid sequence shown
in SEQ ID NO: 372. The VH-locus is VH1 (1-69) and the VL locus is
VL3 (V2-14).
[0366] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-327
antibody have the following CDR sequences: THAIS (SEQ ID NO: 637),
GIIAIFGTANYAQKFQG (SEQ ID NO: 638) and GSGYHISTPFDN (SEQ ID NO:
639). The light chain CDRs of the SC06-327 antibody have the
following CDR sequences: GGNNIGSKGVH (SEQ ID NO: 640), DDSDRPS (SEQ
ID NO: 622) and QVWDSSSDHVV (SEQ ID NO: 642).
[0367] SC06-327 Nucleotide Sequence (SEQ ID NO: 371)
TABLE-US-00150 gaggtgcagc tggtggagac cggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cctctggagg caccttcagg
acccatgcta tcagttgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcgcta tcttcggaac agcaaactac 180 gcacagaagt
tccagggcag aatcacgatt accgcggacg aatccacgag tacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt atttctgtgc gagaggcagt
300 ggttatcata tatcgacacc ctttgacaac tggggccagg gaaccctggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gtcctatgtg 420 ctgactcagc caccctcggt gtcagtggcc
ccaggacaga cggccaggat tacctgtggg 480 ggaaacaaca ttggaagtaa
aggtgtgcac tggtaccagc agaagcctgg ccaggcccct 540 gtgctggtcg
tctatgatga tagcgaccgg ccctcaggga tccctgagcg attctctggc 600
tccaactctg ggaacacggc caccctgacc atcagcaggg tcgaagccgg ggatgaggcc
660 gactattact gtcaggtgtg ggatagtagt agtgatcatg tggtattcgg
cggagggacc 720 aagctgaccg tcctaggt 738
[0368] SC06-327 Amino Acid Sequence (SEQ ID NO: 372)
TABLE-US-00151 EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGG
IIAIFGTANYAQKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGS
GYHISTPFDNWGQGTLVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSVSVA
PGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG
SNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG
[0369] SC06-327 VH Amino Acid Sequence (SEQ ID NO: 369)
TABLE-US-00152 EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGG
IIAIFGTANYAQKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGS
GYHISTPFDNWGQGTLVTVSS
[0370] SC06-327 VL Amino Acid Sequence (SEQ ID NO: 370)
TABLE-US-00153 SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFG GGTKLTVLG
[0371] The SC06-328 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 373) and a light chain
variable region (SEQ ID NO: 374) encoded by the nucleic acid
sequence shown in SEQ ID NO: 375 and the amino acid sequence shown
in SEQ ID NO: 376. The VH-locus is VH1 (1-69) and the VL locus is
VKIII (A27).
[0372] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-328
antibody have the following CDR sequences: GYAIS (HCDR1, SEQ ID NO:
643), GIIPIFGTTNYAQKFQG (HCDR2, SEQ ID NO: 644) and
VKDGYCTLTSCPVGWYFDL (HCDR3, SEQ ID NO: 645). The light chain CDRs
of the SC06-328 antibody have the following CDR sequences:
RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), GASSRAT (LCDR2, SEQ ID NO:
631) and QQYGSSLT (LCDR3, SEQ ID NO: 648).
[0373] SC06-328 Nucleotide Sequence (SEQ ID NO: 375)
TABLE-US-00154 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggaca catcttcagc
ggctatgcaa tcagttgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcccta tctttggtac aacaaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacc aatccacgag cacagcctac 240
atggacctga gcaacttgag atctgaggac acggccgtct attactgtgc gagagtgaaa
300 gatggatatt gtactcttac cagctgccct gtcggctggt acttcgatct
ctggggccgt 360 ggcaccctgg tcactgtctc gagcggtacg ggcggttcag
gcggaaccgg cagcggcact 420 ggcgggtcga cggaaattgt gatgacgcag
tctccaggca ccctgtcttt gtctccaggg 480 gaaagagcca ccctctcgtg
cagggccagt cagagtgtta gcagcagcta cttagcctgg 540 taccagcaga
aacctggcca ggctcccagg ctcctcatct ttggtgcctc cagcagggcc 600
actggcatcc cagacaggtt cagtggcagt gggtctggga cagacttcac tctcaccatc
660 agcagactgg agcctgaaga ttttgcagtg tattactgtc agcagtatgg
tagctcactc 720 actttcggcg gagggaccaa gctggagatc aaacgt 756
[0374] SC06-328 Amino Acid Sequence (SEQ ID NO: 376)
TABLE-US-00155 EVALVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGG
IIPIFGTTNYAQKFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVK
DGYCTLTSCPVGWYFDLWGRGTLVTVSSGTGGSGGTGSGTGGSTEIVMTQ
SPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGASSRA
TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGGGTKLEI KR
[0375] SC06-328 VH Amino Acid Sequence (SEQ ID NO: 373)
TABLE-US-00156 EVALVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGG
IIPIFGTTNYAQKFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVK
DGYCTLTSCPVGWYFDLWGRGTLVTVSS
[0376] SC06-328 VL Amino Acid Sequence (SEQ ID NO: 374)
TABLE-US-00157 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIF
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGG GTKLEIKR
[0377] The SC06-329 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 377) and a light chain
variable region (SEQ ID NO: 378) encoded by the nucleic acid
sequence shown in SEQ ID NO: 379 and the amino acid sequence shown
in SEQ ID NO: 380. The VH-locus is VH1 (1-69) and the VL locus is
VKIII (A27).
[0378] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-329
antibody have the following CDR sequences: SNSIS (HCDR1, SEQ ID NO:
649), GIFALFGTTDYAQKFQG (HCDR2, SEQ ID NO: 650) and GSGYTTRNYFDY
(HCDR3, SEQ ID NO: 651). The light chain CDRs of the SC06-329
antibody have the following CDR sequences: RASQSVSSNYLG (LCDR1, SEQ
ID NO: 652), GASSRAS (LCDR2, SEQ ID NO: 653) and QQYGSSPLT (LCDR3,
SEQ ID NO: 654).
[0379] SC06-329 Nucleotide Sequence (SEQ ID NO: 379)
TABLE-US-00158 gaggtccagc tggtacagtc tggggctgag gttaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg catcttcaga
agcaattcta tcagttgggt gcgacaggcc 120 cctgggcaag ggcttgagtg
gatgggaggg atcttcgctc ttttcggaac aacagactac 180 gcgcagaagt
tccagggcag agtcacgatt accgcggacg aatcttcgac cacagtctac 240
ctggagctga gtagcctgac atctgaggac acggccgttt attactgtgc gagaggcagt
300 ggctacacca cacgcaacta ctttgactac tggggccagg gcaccctggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac ggaaattgtg 420 ctgactcagt ctccaggcac cctgtctttg
tctccagggg aaagagccac actctcctgc 480 agggccagtc agagtgttag
cagcaactac ttaggctggt accagcagaa acctggccag 540 gctcccaggc
tcctgatcta tggtgcatcc agcagggcca gtggcatccc agacaggttc 600
agtggcggtg ggtctgggac agacttcact ctcaccatca gcagactgga gcctgaagat
660 tttgcagtgt attactgtca gcagtatggt agctcacccc tcactttcgg
cggagggacc 720 aaggtggaga tcaaacgt 738
[0380] SC06-329 Amino Acid Sequence (SEQ ID NO: 380)
TABLE-US-00159 EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGG
IFALFGTTDYAQKFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGS
GYTTRNYFDYWGQGTLVTVSSGTGGSGGTGSGTGGSTEIVLTQSPGTLSL
SPGERATLSCRASQSVSSNYLGWTQQKPGQAPRLLIYGASSRASGIPDRF
SGGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR
[0381] SC06-329 VH Amino Acid Sequence (SEQ ID NO: 377)
TABLE-US-00160 EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGG
IFALFGTTDYAQKFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGS
GYTTRNYFDYWGQGTLVTVSS
[0382] SC06-329 VL Amino Acid Sequence (SEQ ID NO: 378)
TABLE-US-00161 EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWTQQKPGQAPRLLIY
GASSRASGIPDRFSGGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFG GGTKVEIKR
[0383] The SC06-331 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 381) and a light chain
variable region (SEQ ID NO: 382) encoded by the nucleic acid
sequence shown in SEQ ID NO: 383 and the amino acid sequence shown
in SEQ ID NO: 384. The VH-locus is VH1 (1-69) and the VL locus is
VL3 (V2-14).
[0384] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-331
lantibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID
NO: 571), GIIGMFGTANYAQKFQG (HCDR2, SEQ ID NO: 655) and GNYYYESSLDY
(HCDR3, SEQ ID NO: 656). The light chain CDRs of the SC06-331
antibody have the following CDR sequences: GGNNIGSKSVH (LCDR1, SEQ
ID NO: 621), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDH (LCDR3,
SEQ ID NO: 657).
[0385] SC06-331 Nucleotide Sequence (SEQ ID NO: 383)
TABLE-US-00162 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcggta tgttcggtac agcaaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aatttacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggaaat
300 tattactatg agagtagtct cgactactgg ggccagggaa ccctggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgca gtctgtcgtg 420 acgcagccgc cctcggtgtc agtggcccca
ggacagacgg ccaggattac ctgtggggga 480 aacaacattg gaagtaaaag
tgtgcactgg taccagcaga agccaggcca ggcccctgtg 540 ctggtcgtct
atgatgatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc 600
aactctggga acacggccac cctgaccatc agcagggtcg aagccgggga tgaggccgac
660 tattactgtc aggtgtggga tagtagtagt gatcattatg tcttcggaac
tgggaccaag 720 gtcaccgtcc taggt 735
[0386] SC06-331 Amino Acid Sequence (SEQ ID NO: 384)
TABLE-US-00163 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIGMFGTANYAQKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGN
YYYESSLDYWGQGTLVTVSSGTGGSGGTGSGTGGSTQSVVTQPPSVSVAP
GQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGS
NSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG
[0387] SC06-331 VH Amino Acid Sequence (SEQ ID NO: 381)
TABLE-US-00164 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG
GIIGMFGTANYAQKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCAR
GNYYYESSLDYWGQGTLVTVSS
[0388] SC06-331 VL Amino Acid Sequence (SEQ ID NO: 382)
TABLE-US-00165 QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFG TGTKVTVLG
[0389] The SC06-332 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 385) and a light chain
variable region (SEQ ID NO: 386) encoded by the nucleic acid
sequence shown in SEQ ID NO: 387 and the amino acid sequence shown
in SEQ ID NO: 388. The VH-locus is VH1 (1-69) and the VL locus is
VKI (A20).
[0390] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-332
antibody have the following CDR sequences: NFAIN (HCDR1, SEQ ID NO:
658), GIIAVFGTTKYAHKFQG (HCDR2, SEQ ID NO: 659) and GPHYYSSYMDV
(HCDR3, SEQ ID NO: 660). The light chain CDRs of the SC06-332
antibody have the following CDR sequences: RASQGISTYLA (LCDR1, SEQ
ID NO: 661), AASTLQS (LCDR2, SEQ ID NO: 662) and QKYNSAPS (LCDR3,
SEQ ID NO: 663).
[0391] SC06-332 Nucleotide Sequence (SEQ ID NO: 387)
TABLE-US-00166 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtaaaggtc 60 tcctgcaagg cttctggagg ccccttccgc
aattttgcta tcaactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcgctg tctttgggac gacaaagtac 180 gcacataagt
tccagggcag agtcaccatc accgcggacg actccacaaa tacagcttac 240
atggagctgg gcagcctgaa atctgaggac acggccgtgt attactgtgc gagaggtccc
300 cactactact cctcctacat ggacgtctgg ggcgaaggga ccacggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgga catccagttg 420 acccagtctc catcctccct gtctgcatct
gtaggagaca gagtcaccat cacttgccgg 480 gcgagtcagg gcattagcac
ttatttagcc tggtatcagc agaaacccgg gaaagttcct 540 aaactcctga
tctatgctgc atccactttg caatcagggg tcccatctcg gttcagtggc 600
agtggatctg ggacagattt cactctcacc atcagcagcc tgcagcctga agatgttgca
660 acttattact gtcaaaagta taacagtgcc ccttctttcg gccctgggac
caaagtggat 720 atcaaacgt 729
[0392] SC06-332 Amino Acid Sequence (SEQ ID NO: 388)
TABLE-US-00167 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGG
IIAVFGTTKYAHKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGP
HYYSSYMDVWGEGTTVTVSSGTGGDGGTGSGTGGSTDIQLTQSPSSLSAS
VGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSG
SGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPGTKVDIKR
[0393] SC06-332 VH Amino Acid Sequence (SEQ ID NO: 385)
TABLE-US-00168 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMG
GIIAVFGTTKYAHKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCAR
GPHYYSSYMDVWGEGTTVTVSS
[0394] SC06-332 VL Amino Acid Sequence (SEQ ID NO: 386)
TABLE-US-00169 DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGP GTKVDIKR
[0395] The SC06-334 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 389) and a light chain
variable region (SEQ ID NO: 390) encoded by the nucleic acid
sequence shown in SEQ ID NO: 391 and the amino acid sequence shown
in SEQ ID NO: 392. The VH-locus is VH1 (1-69) and the VL locus is
VL3 (V2-14).
[0396] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-334
antibody have the following CDR sequences: SNAVS (HCDR1, SEQ ID NO:
664), GILGVFGSPSYAQKFQG (HCDR2, SEQ ID NO: 665) and GPTYYYSYMDV
(HCDR3, SEQ ID NO: 666). The light chain CDRs of the SC06-334
antibody have the following CDR sequences: GGNNIGRNSVH (LCDR1, SEQ
ID NO: 667), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWHSSSDHYV
(LCDR3, SEQ ID NO: 669).
[0397] SC06-334 Nucleotide Sequence (SEQ ID NO: 391)
TABLE-US-00170 gaggtgcagc tggtggagac tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 ccctgcaaat cttctggaag ccccttcagg
agtaatgctg tcagctgggt gcgacaggcc 120 cccggacaag ggcttgagtg
ggtgggagga atcctcggtg tctttggttc accaagctac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aatccaccaa cacagtccac 240
atggagctga gaggtttgag atctgaggac acggccgtgt attattgtgc gagaggtcct
300 acctactact actcctacat ggacgtctgg ggcaaaggga ccacggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgtc ctatgtgctg 420 actcagccac cctcggagtc agtggcccca
ggacagacgg ccaggattac ctgtggggga 480 aataacattg gaagaaatag
tgtgcactgg tatcagcaga agccaggcca ggcccctgtg 540 ctggtcgtgt
atgatgatag cgaccggccc tcagggatcc ctgagcgatt ttctggctcc 600
aagtctggga acacggccac cctgattatc agcagggtcg aagtcgggga tgaggccgac
660 tactactgtc aggtgtggca tagtagtagt gatcattatg tcttcggaac
tgggaccaag 720 gtcaccgtcc taggt 735
[0398] SC06-334 Amino Acid Sequence (SEQ ID NO: 392)
TABLE-US-00171 EVALVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGG
ILGVFGSPSYAQKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGP
TYYYSYMDVWGKGTTVTVSSGTGGSGGTGSGTGGSTSYVLTQPPSESVAP
GQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGS
KSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFGTGTKVTVLG
[0399] SC06-334 VH Amino Acid Sequence (SEQ ID NO: 389)
TABLE-US-00172 EVALVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVG
GILGVFGSPSYAQKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCAR
GPTYYYSYMDVWGKGTTVTVSS
[0400] SC06-334 VL Amino Acid Sequence (SEQ ID NO: 390)
TABLE-US-00173 SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFG TGTKVTVLG
[0401] The SC06-336 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 393) and a light chain
variable region (SEQ ID NO: 394) encoded by the nucleic acid
sequence shown in SEQ ID NO: 395 and the amino acid sequence shown
in SEQ ID NO: 396. The VH-locus is VH1 (1-69) and the VL locus is
VKIII (A27).
[0402] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-336
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
670), GIFGMFGTANYAQKFQG (HCDR2, SEQ ID NO: 671) and SSGYYPQYFQD
(HCDR3, SEQ ID NO: 672). The light chain CDRs of the SC06-336
antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ
ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSSLT (LCDR3,
SEQ ID NO: 308).
[0403] SC06-336 Nucleotide Sequence (SEQ ID NO: 395)
TABLE-US-00174 cagatgcagc tggtacaatc tggagctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcttcggta tgtttgggac agcaaactac 180 gcgcagaagt
tccagggcag agtcacgatt accgcggacg aattcacgag cgcggcctac 240
atggagctga gcagcctggg atctgaggac acggccatgt attactgtgc gaggtctagt
300 ggttattacc cccaatactt ccaggactgg ggccagggca ccctggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgga aattgtgatg 420 acacagtctc caggcaccct gtctttgtct
ccagggcaaa gagccaccct ctcctgcagg 480 gccagtcaga gtgttagcag
cagctactta gcctggtacc agcagaaacc tggccaggct 540 cccagactcc
tcatgtatgg tgcatccagc agggccactg gcatcccaga caggttcagt 600
ggcagtgggt ctgggacaga cttcactctc accatcagca gactggagcc tgaagatttt
660 gcagtgtatt actgtcagca gtatggtagc tcatcgctca ctttcggcgg
agggaccaag 720 ctggagatca aacgt 735
[0404] SC06-336 Amino Acid Sequence (SEQ ID NO: 396)
TABLE-US-00175 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IFGMFGTANYAQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSS
GYYPQYFQDWGQGTLVTVSSGTGGSGGTGSGTGGSTEIVMTQSPGTLSLS
PGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYGASSRATGIPDRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFGGGTKLEIKR
[0405] SC06-336 VH Amino Acid Sequence (SEQ ID NO: 393)
TABLE-US-00176 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IFGMFGTANYAQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSS
GYYPQYFQDWGQGTLVTVSS
[0406] SC06-336 VL Amino Acid Sequence (SEQ ID NO: 394)
TABLE-US-00177 EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFG GGTKLEIKR
[0407] The SC06-339 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 397) and a light chain
variable region (SEQ ID NO: 398) encoded by the nucleic acid
sequence shown in SEQ ID NO: 399 and the amino acid sequence shown
in SEQ ID NO: 400. The VH-locus is VH1 (1-69) and the VL locus is
VL3 (V2-14).
[0408] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-339
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
303), GIIAIFHTPKYAQKFQG (HCDR2, SEQ ID NO: 306) and GSTYDFSSGLDY
(HCDR3, SEQ ID NO: 725). The light chain CDRs of the SC06-339
antibody have the following CDR sequences: GGNNIGSKSVH (LCDR1, SEQ
ID NO: 621), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDHVV
(LCDR3, SEQ ID NO: 642).
[0409] SC06-339 Nucleotide Sequence (SEQ ID NO: 399)
TABLE-US-00178 gaggtgcagc tggtggagtc cggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg catcttcaac
agttatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggc atcatcgcta tctttcatac accaaagtac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aatccacgaa cacagcctac 240
atggaactga gaagcctgaa atctgaggac acggccctgt attactgtgc gagagggtcc
300 acttacgatt tttcgagtgg ccttgactac tggggccagg gaaccctggt
caccgtctcg 360 agcggtacgg gcggttcagg cggaaccggc agcggcactg
gcgggtcgac gcaggcaggg 420 ctgactcagc caccctcggt gtcagtggcc
ccaggacaga cggccaggat tacctgtggg 480 ggaaacaaca ttggaagtaa
aagtgtgcac tggtaccagc agaagccagg ccaggcccct 540 gtcctagtcg
tctatgatga tagcgaccgg ccctcaggga tccctgagcg attctctggc 600
tccaactctg ggaacacggc caccctgacc atcagcaggg tcgaagccgg ggatgaggcc
660 gactattact gtcaggtgtg ggatagtagt agtgatcatg tggtattcgg
cggagggacc 720 aagctgaccg tcctaggt 738
[0410] SC06-339 Amino Acid Sequence (SEQ ID NO: 400)
TABLE-US-00179 EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGG
IIAIFHTPKYAQKFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGS
TYDFSSGLDYWGQGTLVTVSSGTGGSGGTGSGTGGSTQAGLTQPPSVSVA
PGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSG
SNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG
[0411] SC06-339 VH Amino Acid Sequence (SEQ ID NO: 397)
TABLE-US-00180 EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGG
IIAIFHTPKYAQKFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGS
TYDFSSGLDYWGQGTLVTVSS
[0412] SC06-339 VL Amino Acid Sequence (SEQ ID NO: 398)
TABLE-US-00181 QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFG GGTKLTVLG
[0413] The SC06-342 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 401) and a light chain
variable region (SEQ ID NO: 402) encoded by the nucleic acid
sequence shown in SEQ ID NO: 403 and the amino acid sequence shown
in SEQ ID NO: 404. The VH-locus is VH1 (1-69) and the VL locus is
VKIV (B3).
[0414] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-342
antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO:
251), GVIPIFRTANYAQNFQG (HCDR2, SEQ ID NO: 249) and
LNYHDSGTYYNAPRGWFDP (HCDR3, SEQ ID NO: 246). The light chain CDRs
of the SC06-342 antibody have the following CDR sequences:
KSSQSILNSSNNKNYLA (LCDR1, SEQ ID NO: 245), WASTRES (LCDR2, SEQ ID
NO: 570) and QQYYSSPPT (LCDR3, SEQ ID NO: 250).
[0415] SC06-342 Nucleotide Sequence (SEQ ID NO: 403)
TABLE-US-00182 caggtccagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg cttcttcagc
agctatgcta tcagctgggt gcgccaggcc 120 cctggacaag gacttgagtg
gatggggggg gtcatcccta tctttcgtac agcaaactac 180 gcacagaact
tccagggcag agtcaccatt accgcggacg aattcacatc gtatatggag 240
ctgagcagcc tgagatctga cgacacggcc gtgtattact gtgcgaggtt gaattaccat
300 gattcgggga cttattataa cgccccccgg ggctggttcg acccctgggg
ccagggaacc 360 ctggtcaccg tctcgagcgg tacgggcggt tcaggcggaa
ccggcagcgg cactggcggg 420 tcgacggaca tccagatgac ccagtctcca
gactccctgg ctgtgtctct gggcgagaag 480 gccaccatca actgcaagtc
cagccagagt attttaaaca gctccaacaa taagaactac 540 ttagcttggt
accagcagaa accaggacag cctcctaagc tgctcattta ctgggcatct 600
acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact
660 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca
gcaatattat 720 agtagtccgc cgacgttcgg ccaagggacc aaggtggaaa tcaaacgt
768
[0416] SC06-342 Amino Acid Sequence (SEQ ID NO: 404)
TABLE-US-00183 QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGG
VIPIFRTANYAQNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYH
DSGTYYNAPRGWFDPWGQGTLVTVSSGTGGSGGTGSGTGGSTDIQMTQSP
DSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPPKLLIYWAS
TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPPTFGQGT KVEIKR
[0417] SC06-342 VH Amino Acid Sequence (SEQ ID NO: 401)
TABLE-US-00184 QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGG
VIPIFRTANYAQNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYH
DSGTYYNAPRGWFDPWGQGTLVTVSS
[0418] SC06-342 VL Amino Acid Sequence (SEQ ID NO: 402)
TABLE-US-00185 DIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSS
PPTFGQGTKVEIKR
[0419] The SC06-343 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 405) and a light chain
variable region (SEQ ID NO: 406) encoded by the nucleic acid
sequence shown in SEQ ID NO: 407 and the amino acid sequence shown
in SEQ ID NO: 408. The VH-locus is VH1 (1-69) and the VL locus is
VL3 (V2-14).
[0420] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-343
antibody have the following CDR sequences: YYAMS (HCDR1, SEQ ID NO:
242), GISPMFGTTTYAQKFQG (HCDR2, SEQ ID NO: 307) and SSNYYDSVYDY
(HCDR3, SEQ ID NO: 290). The light chain CDRs of the SC06-343
antibody have the following CDR sequences: GGHNIGSNSVH (LCDR1, SEQ
ID NO: 224), DNSDRPS (LCDR2, SEQ ID NO: 223) and QVWGSSSDH (LCDR3,
SEQ ID NO: 227).
[0421] SC06-343 Nucleotide Sequence (SEQ ID NO: 407)
TABLE-US-00186 caggtccagc tggtgcagtc tggagctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagt caccttcagt
tactatgcta tgagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggagga atcagcccta tgtttgggac aacaacctac 180 gcacagaagt
tccagggcag agtcacgatt actgcggacg actccacgag tacagcctac 240
atggaggtga ggagcctgag atctgaggac acggccgtgt attactgtgc gagatcttcg
300 aattactatg atagtgtata tgactactgg ggccagggaa ccctggtcac
cgtctcgagc 360 ggtacgggcg gttcaggcgg aaccggcagc ggcactggcg
ggtcgacgca gtctgtcgtg 420 acgcagccgc cctcggagtc agtggcccca
ggacagacgg ccaggattac ctgtggggga 480 cataacattg gaagtaatag
tgtgcactgg taccagcaga agccaggcca ggcccctgtg 540 ctggtcgtgt
atgataatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc 600
aactctggga acacggccac cctgaccatc agcagggtcg aagccgggga tgaggccgac
660 tattactgtc aggtgtgggg tagtagtagt gaccattatg tcttcggaac
tgggaccaag 720 gtcaccgtcc taggt 735
[0422] SC06-343 Amino Acid Sequence (SEQ ID NO: 408)
TABLE-US-00187 QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGG
ISPMFGTTTYAQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSS
NYYDSVYDYWGQGTLVTVSSGTGGSGGTGSGTGGSTQSVVTQPPSESVAP
GQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDNSDRPSGIPERFSGS
NSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFGTGTKVTVLG
[0423] SC06-343 VH Amino Acid Sequence (SEQ ID NO: 405)
TABLE-US-00188 QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGG
ISPMFGTTTYAQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSS
NYYDSVYDYWGQGTLVTVSS
[0424] SC06-343 VL Amino Acid Sequence (SEQ ID NO: 406)
TABLE-US-00189 QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVLVVYDN
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWGSSSDHYVFG TGTKVTVLG
[0425] The SC06-344 HA-specific single-chain Fv antibody includes a
heavy chain variable region (SEQ ID NO: 409) and a light chain
variable region (SEQ ID NO: 410) encoded by the nucleic acid
sequence shown in SEQ ID NO: 411 and the amino acid sequence shown
in SEQ ID NO: 412. The VH-locus is VH1 (1-69) and the VL locus is
VL1 (V1-13).
[0426] The amino acids encompassing the CDRs are highlighted in
bold in the sequences below. The heavy chain CDRs of the SC06-344
antibody have the following CDR sequences: NYAMS (HCDR1, SEQ ID NO:
222), GIIAIFGTPKYAQKFQG (HCDR2, SEQ ID NO: 221) and IPHYNFGSGSYFDY
(HCDR3, SEQ ID NO: 220). The light chain CDRs of the SC06-344
antibody have the following CDR sequences: TGSSSNIGAGYDVH (LCDR1,
SEQ ID NO: 219), GNSNRPS (LCDR2, SEQ ID NO: 231) and GTWDSSLSAYV
(LCDR3, SEQ ID NO: 280).
[0427] SC06-344 Nucleotide Sequence (SEQ ID NO: 411)
TABLE-US-00190 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgagagtc 60 tcctgcaagg cttctggaag catcttcaga
aactatgcta tgagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcgcta tttttgggac accaaagtac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aatcgacgag cactgtctac 240
atggaactga gcggactgag atctgaggac acggccatgt attactgtgc gaggattccc
300 cactataatt ttggttcggg gagttatttc gactactggg gccagggaac
cctggtcacc 360 gtctcgagcg gtacgggcgg ttcaggcgga accggcagcg
gcactggcgg gtcgacgact 420 gtgttgacac agccgccctc agtgtctggg
gccccagggc agagggtcac catctcctgc 480 actgggagca gctccaacat
cggggcaggt tatgatgtac actggtacca gcagcttcca 540 ggaacagccc
ccaaactcct catctatggt aacagcaatc ggccctcagg ggtccctgac 600
cgattctctg gctccaagtc tggcacgtca gccaccctgg gcatcaccgg actccagact
660 ggggacgagg ccgattatta ctgcggaaca tgggatagca gcctgagtgc
ttatgtcttc 720 ggaactggga ccaaggtcac cgtcctaggt 750
[0428] SC06-344 Amino Acid Sequence (SEQ ID NO: 412)
TABLE-US-00191 QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGG
IIAIFGTPKYAQKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIP
HYNFGSGSYFDYWGQGTLVTVSSGTGGSGGTGSGTGGSTTVLTQPPSVSG
APGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPD
RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVLG
[0429] SC06-344 VH Amino Acid Sequence (SEQ ID NO: 409)
TABLE-US-00192 QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQGLEWMGG
IIAIFGTPKYAQKFQGRVTITADESTSTVYMELSGLRSEDTAMYYCARIP
HYNFGSGSYFDYWGQGTLVTVSS
[0430] SC06-344 VL Amino Acid Sequence (SEQ ID NO: 410)
TABLE-US-00193 TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIY
GNSNRPSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYV FGTGTKVTVLG
IgG HA Antibodies
[0431] The CR6141 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 199) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 279 and the heavy chain
amino acid sequence shown in SEQ ID NO: 413. The CR6141 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 414) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 415 and the light chain amino acid sequence shown in SEQ
ID NO: 416.
[0432] CR6141 Heavy Chain Nucleotide Sequence (SEQ ID NO: 279)
TABLE-US-00194 gaggtccagc tggtgcagtc tggggctgag gtgaagaagc
ctggggcctc agtgaaggtc 60 tcctgcaagg cttctgggta caccttcacc
ggctactatg tgtactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 gcacagaagt
tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagaagtaga
300 tccctggacg tctggggcca agggaccacg gtcaccgtct cgagtgctag
caccaagggc 360 cccagcgtgt tccccctggc ccccagcagc aagagcacca
gcggcggcac agccgccctg 420 ggctgcctgg tgaaggacta cttccccgag
cccgtgaccg tgagctggaa cagcggcgcc 480 ttgaccagcg gcgtgcacac
cttccccgcc gtgctgcaga gcagcggcct gtacagcctg 540 agcagcgtgg
tgaccgtgcc cagcagcagc ctgggcaccc agacctacat ctgcaacgtg 600
aaccacaagc ccagcaacac caaggtggac aaacgcgtgg agcccaagag ctgcgacaag
660 acccacacct gccccccctg ccctgccccc gagctgctgg gcggaccctc
cgtgttcctg 720 ttccccccca agcccaagga caccctcatg atcagccgga
cccccgaggt gacctgcgtg 780 gtggtggacg tgagccacga ggaccccgag
gtgaagttca actggtacgt ggacggcgtg 840 gaggtgcaca acgccaagac
caagccccgg gaggagcagt acaacagcac ctaccgggtg 900 gtgagcgtgc
tcaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaag 960
gtgagcaaca aggccctgcc tgcccccatc gagaagacca tcagcaaggc caagggccag
1020 ccccgggagc cccaggtgta caccctgccc cccagccggg aggagatgac
caagaaccag 1080 gtgtccctca cctgtctggt gaagggcttc taccccagcg
acatcgccgt ggagtgggag 1140 agcaacggcc agcccgagaa caactacaag
accacccccc ctgtgctgga cagcgacggc 1200 agcttcttcc tgtacagcaa
gctcaccgtg gacaagagcc ggtggcagca gggcaacgtg 1260 ttcagctgca
gcgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgagc 1320
ctgagccccg gcaag 1335
[0433] CR6141 Heavy Chain Amino Acid Sequence (SEQ ID NO: 413)
TABLE-US-00195 EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGW
ISAYNGNTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSR
SLDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0434] CR6141 VH Amino Acid Sequence (SEQ ID NO: 199)
TABLE-US-00196 EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVYWVRQAPGQGLEWMGW
ISAYNGNTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSR
SLDVWGQGTTVTVSS
[0435] CR6141 Light Chain Nucleotide Sequence (SEQ ID NO: 415)
TABLE-US-00197 gatgttgtga tgactcagtc tccagactcc ctggctgtgt
ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta
tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccagg
acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg
tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240
atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtact
300 cctctcactt tcggcggagg gaccaaagtg gatatcaaac gtgcggccgc
acccagcgtg 360 ttcatcttcc ccccctccga cgagcagctg aagagcggca
ccgccagcgt ggtgtgcctg 420 ctgaacaact tctacccccg ggaggccaag
gtgcagtgga aggtggacaa cgccctgcag 480 agcggcaaca gccaggagag
cgtgaccgag caggacagca aggactccac ctacagcctg 540 agcagcaccc
tcaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600
gtgacccacc agggcctgag cagccccgtg accaagagct tcaaccgggg cgagtgt
657
[0436] CR6141 Light Chain Amino Acid Sequence (SEQ ID NO: 416)
TABLE-US-00198 DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST
PLTFGGGTKVDIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC
[0437] CR6141 VL Amino Acid Sequence (SEQ ID NO: 414)
TABLE-US-00199 DVVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST
PLTFGGGTKVDIKR
[0438] The CR6255 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 417) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 418 and the heavy chain
amino acid sequence shown in SEQ ID NO: 419. The CR6255 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 420) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 421 and the light chain amino acid sequence shown in SEQ
ID NO: 422.
[0439] CR6255 Heavy Chain Nucleotide Sequence (SEQ ID NO: 418)
TABLE-US-00200 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0440] CR6255 Heavy Chain Amino Acid Sequence (SEQ ID NO: 419)
TABLE-US-00201 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0441] CR6255 VH Amino Acid Sequence (SEQ ID NO: 417)
TABLE-US-00202 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0442] CR6255 Light Chain Nucleotide Sequence (SEQ ID NO: 421)
TABLE-US-00203 tcctatgtgc tgactcagcc accctcagcg tctgggaccc
ccgggcagag ggtcaccatc 60 tcttgttctg gaagcacgtt caacatcgga
agtaatgctg tagactggta ccggcagctc 120 ccaggaacgg cccccaaact
cctcatctat agtaataatc agcggccctc aggggtccct 180 gaccgattct
ctggctccag gtctggcacc tcagcctccc tggccatcag tgggctccag 240
tctgaggatg aggctgatta ttactgtgca gcatgggatg acatcctgaa tgttccggta
300 ttcggcggag ggaccaagct gaccgtccta ggtgcggccg caggccagcc
caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc
aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc
gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt
ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca
gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600
tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660
[0443] CR6255 Light Chain Amino Acid Sequence (SEQ ID NO: 422)
TABLE-US-00204 SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIY
SNNQRPSGVPDRFSGSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPV
FGGGTKLTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG
AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS
CQVTHEGSTVEKTVAPTECS
[0444] CR6255 VL Amino Acid Sequence (SEQ ID NO: 420)
TABLE-US-00205 SYVLTQPPSASGTPGQRVTISCSGSTFNIGSNAVDWYRQLPGTAPKLLIY
SNNQRPSGVPDRFSGSRSGTSASLAISGLQSEDEADYYCAAWDDILNVPV FGGGTKLTVLG
[0445] The CR6257 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 423) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 424 and the heavy chain
amino acid sequence shown in SEQ ID NO: 425. The CR6257 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 426) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 427 and the light chain amino acid sequence shown in SEQ
ID NO: 428.
[0446] CR6257 Heavy Chain Nucleotide Sequence (SEQ ID NO: 424)
TABLE-US-00206 caggtccagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0447] CR6257 Heavy Chain Amino Acid Sequence (SEQ ID NO: 425)
TABLE-US-00207 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0448] CR6257 VH Amino Acid Sequence (SEQ ID NO: 423)
TABLE-US-00208 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0449] CR6257 Light Chain Nucleotide Sequence (SEQ ID NO: 427)
TABLE-US-00209 cagtctgccc tgactcagcc tgccgccgtg tctgggtctc
ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt
ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180 tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cacttatgtc
300 ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc
caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc
aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc
gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt
ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca
gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600
tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660
[0450] CR6257 Light Chain Amino Acid Sequence (SEQ ID NO: 428)
TABLE-US-00210 QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYV
FGTGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG
AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS
CQVTHEGSTVEKTVAPTECS
[0451] CR6257 VL Amino Acid Sequence (SEQ ID NO: 426)
TABLE-US-00211 QSALTQPAAVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYV FGTGTKVTVLG
[0452] 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.
[0453] CR6260 Heavy Chain Nucleotide Sequence (SEQ ID NO: 430)
TABLE-US-00212 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0454] CR6260 Heavy Chain Amino Acid Sequence (SEQ ID NO: 431)
TABLE-US-00213 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0455] CR6260 VH Amino Acid Sequence (SEQ ID NO: 429)
TABLE-US-00214 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0456] CR6260 Light Chain Nucleotide Sequence (SEQ ID NO: 433)
TABLE-US-00215 tcctatgtgc tgactcagcc accctcagtc tctgggaccc
ccgggcagag ggtcaccatc 60 tcttgctctg gaagccgctc caacgtcgga
gataattctg tatattggta tcaacacgtc 120 ccagaaatgg cccccaaact
cctcgtctat aagaatactc aacggccctc aggagtccct 180 gcccggtttt
ccggctccaa gtctggcact tcagcctccc tggccatcat tggcctccag 240
tccggcgatg aggctgatta ttattgtgtg gcatgggatg acagcgtaga tggctatgtc
300 ttcggatctg ggaccaaggt caccgtccta ggtgcggccg caggccagcc
caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc
aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc
gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt
ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca
gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600
tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660
[0457] CR6260 Light Chain Amino Acid Sequence (SEQ ID NO: 434)
TABLE-US-00216 SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVY
KNTQRPSGVPARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYV
FGSGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG
AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS
CQVTHEGSTVEKTVAPTECS
[0458] CR6260 VL Amino Acid Sequence (SEQ ID NO: 432)
TABLE-US-00217 SYVLTQPPSVSGTPGQRVTISCSGSRSNVGDNSVYWYQHVPEMAPKLLVY
KNTQRPSGVPARFSGSKSGTSASLAIIGLQSGDEADYYCVAWDDSVDGYV FGSGTKVTVLG
[0459] The CR6261 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 435) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 436 and the heavy chain
amino acid sequence shown in SEQ ID NO: 437. The CR6261 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 438) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 439 and the light chain amino acid sequence shown in SEQ
ID NO: 440.
[0460] CR6261 Heavy Chain Nucleotide Sequence (SEQ ID NO: 436)
TABLE-US-00218 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0461] CR6261 Heavy Chain Amino Acid Sequence (SEQ ID NO: 437)
TABLE-US-00219 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0462] CR6261 VH Amino Acid Sequence (SEQ ID NO: 435)
TABLE-US-00220 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0463] CR6261 Light Chain Nucleotide Sequence (SEQ ID NO: 439)
TABLE-US-00221 cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc
caggacagaa ggtcaccatc 60 tcctgctctg gaagcagctc caacattggg
aatgattatg tatcctggta ccagcagctc 120 ccaggaacag cccccaaact
cctcatttat gacaataata agcgaccctc agggattcct 180 gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240
actggggacg aggccaacta ttactgcgca acatgggatc gccgcccgac tgcttatgtt
300 gtcttcggcg gagggaccaa gctgaccgtc ctaggtgcgg ccgcaggcca
gcccaaggcc 360 gctcccagcg tgaccctgtt ccccccctcc tccgaggagc
tgcaggccaa caaggccacc 420 ctggtgtgcc tcatcagcga cttctaccct
ggcgccgtga ccgtggcctg gaaggccgac 480 agcagccccg tgaaggccgg
cgtggagacc accaccccca gcaagcagag caacaacaag 540 tacgccgcca
gcagctacct gagcctcacc cccgagcagt ggaagagcca ccggagctac 600
agctgccagg tgacccacga gggcagcacc gtggagaaga ccgtggcccc caccgagtgc
660
[0464] CR6261 Light Chain Amino Acid Sequence (SEQ ID NO: 440)
TABLE-US-00222 QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIY
DNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYV
VFGGGTKLTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYP
GAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSY
SCQVTHEGSTVEKTVAPTECS
[0465] CR6261 VL Amino Acid Sequence (SEQ ID NO: 438)
TABLE-US-00223 QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIY
DNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYV VFGGGTKLTVLG
[0466] The CR6262 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 441) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 442 and the heavy chain
amino acid sequence shown in SEQ ID NO: 443. The CR6262 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 444) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 445 and the light chain amino acid sequence shown in SEQ
ID NO: 446.
[0467] CR6262 Heavy Chain Nucleotide Sequence (SEQ ID NO: 442)
TABLE-US-00224 caggtacagc tgcagcagtc aggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg tttccggagt cattttcagc
ggcagtgcga tcagctgggt gcgacaggcc 120 cctggacaag gccttgagtg
gatgggaggg atcagccctc tctttggcac aacaaattac 180 gcacaaaagt
tccagggcag agtcacgatt accgcggacc aatccacgaa cacaacctac 240
atggaggtga acagcctgag atatgaggac acggccgtgt atttctgtgc gcgaggtcca
300 aaatattaca gtgagtacat ggacgtctgg ggcaaaggga ccacggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0468] CR6262 Heavy Chain Amino Acid Sequence (SEQ ID NO: 443)
TABLE-US-00225 QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGG
ISPLFGTTNYAQKFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGP
KYYSEYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0469] CR6262 VH Amino Acid Sequence (SEQ ID NO: 441)
TABLE-US-00226 QVQLQQSGAEVKKPGSSVKVSCKVSGVIFSGSAISWVRQAPGQGLEWMGG
ISPLFGTTNYAQKFQGRVTITADQSTNTTYMEVNSLRYEDTAVYFCARGP
KYYSEYMDVWGKGTTVTVSS
[0470] CR6262 Light Chain Nucleotide Sequence (SEQ ID NO: 445)
TABLE-US-00227 gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60 atcacttgcc gggcgagtca gggcattagc
agttatttag cctggtatca gcagaagcca 120 gggaaagttc ctacactcct
gatctatgat gcatccactt tgcgatcagg ggtcccatct 180 cgcttcagtg
gcagtggatc tgcgacagat ttcactctca ccatcagcag cctgcagcct 240
gaagatgttg caacttatta ctgtcaaagg tataacagtg cccccccgat caccttcggc
300 caagggacac gactggagat taaacgtgcg gccgcaccca gcgtgttcat
cttccccccc 360 tccgacgagc agctgaagag cggcaccgcc agcgtggtgt
gcctgctgaa caacttctac 420 ccccgggagg ccaaggtgca gtggaaggtg
gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga
cagcaaggac tccacctaca gcctgagcag caccctcacc 540 ctgagcaagg
ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642
[0471] CR6262 Light Chain Amino Acid Sequence (SEQ ID NO: 446)
TABLE-US-00228 DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYD
ASTLRSGVPSRFSGSGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFG
QGTRLEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0472] CR6262 VL Amino Acid Sequence (SEQ ID NO: 444)
TABLE-US-00229 DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKVPTLLIYD
ASTLRSGVPSRFSGSGSATDFTLTISSLQPEDVATYYCQRYNSAPPITFG QGTRLEIKR
[0473] The CR6268 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 447) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 448 and the heavy chain
amino acid sequence shown in SEQ ID NO: 449. The CR6268 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 450) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 451 and the light chain amino acid sequence shown in SEQ
ID NO: 452.
[0474] CR6268 Heavy Chain Nucleotide Sequence (SEQ ID NO: 448)
TABLE-US-00230 caggtccagc tggtacagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagt
agttatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggagga atcatgggta tgtttggcac aactaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aattcacgag cgcagcctac 240
atggagctga ggagcctgag atctgaggac acggccgtct actactgtgc gaggtctagt
300 ggttattacc ccgaatactt ccaggactgg ggccagggca ccctggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0475] CR6268 Heavy Chain Amino Acid Sequence (SEQ ID NO: 449)
TABLE-US-00231 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IMGMFGTTNYAQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCARSS
GYYPEYFQDWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0476] CR6268 VH Amino Acid Sequence (SEQ ID NO: 447)
TABLE-US-00232 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IMGMFGTTNYAQKFQGRVTITADEFTSAAYMELRSLRSEDTAVYYCARSS
GYYPEYFQDWGQGTLVTVSS
[0477] CR6268 Light Chain Nucleotide Sequence (SEQ ID NO: 451)
TABLE-US-00233 cagtctgtgc tgactcagcc accctcagag tccgtgtccc
caggacagac agccagcgtc 60 acctgctctg gacataaatt gggggataaa
tatgtttcgt ggtatcagca gaagccaggc 120 cagtcccctg tattactcat
ctatcaagat aacaggcggc cctcagggat ccctgagcga 180 ttcataggct
ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctctg 240
gatgaggctg actattactg tcaggcgtgg gacagcagca ctgcggtttt cggcggaggg
300 accaagctga ccgtcctagg tgcggccgca ggccagccca aggccgctcc
cagcgtgacc 360 ctgttccccc cctcctccga ggagctgcag gccaacaagg
ccaccctggt gtgcctcatc 420 agcgacttct accctggcgc cgtgaccgtg
gcctggaagg ccgacagcag ccccgtgaag 480 gccggcgtgg agaccaccac
ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540 tacctgagcc
tcacccccga gcagtggaag agccaccgga gctacagctg ccaggtgacc 600
cacgagggca gcaccgtgga gaagaccgtg gcccccaccg agtgcagc 648
[0478] CR6268 Light Chain Amino Acid Sequence (SEQ ID NO: 452)
TABLE-US-00234 QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQD
NRRPSGIPERFIGSNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGG
TKLTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV
AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT
HEGSTVEKTVAPTECS
[0479] CR6268 VL Amino Acid Sequence (SEQ ID NO: 450)
TABLE-US-00235 QSVLTQPPSESVSPGQTASVTCSGHKLGDKYVSWYQQKPGQSPVLLIYQD
NRRPSGIPERFIGSNSGNTATLTISGTQALDEADYYCQAWDSSTAVFGGG TKLTVLG
[0480] The CR6272 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 453) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 454 and the heavy chain
amino acid sequence shown in SEQ ID NO: 455. The CR6272 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 456) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 457 and the light chain amino acid sequence shown in SEQ
ID NO: 458.
[0481] CR6272 Heavy Chain Nucleotide Sequence (SEQ ID NO: 454)
TABLE-US-00236 cagatgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttctcc
agttatgcta tcacctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcggta tgtttggttc aacaaactac 180 gcacagaact
tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240
atggagctga gcagcctcag atctgaggac acggccgtgt attactgtgc gagaagtact
300 ggttattacc ctgcatacct ccaccactgg ggccagggca ccctggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0482] CR6272 Heavy Chain Amino Acid Sequence (SEQ ID NO: 455)
TABLE-US-00237 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGG
IIGMFGSTNYAQNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARST
GYYPAYLHHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0483] CR6272 VH Amino Acid Sequence (SEQ ID NO: 453)
TABLE-US-00238 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGG
IIGMFGSTNYAQNFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARST
GYYPAYLHHWGQGTLVTVSS
[0484] CR6272 Light Chain Nucleotide Sequence (SEQ ID NO: 457)
TABLE-US-00239 cagtctgccc tgactcagcc tcgctcagtg tccgggtctc
ctggacagtc agtcaccatc 60 tcctgcactg gaaccagcag tgatgttggt
ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa
actcatgatt tatgatgtca gtaagcggcc ctcaggggtc 180 cctgatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg atgaggctga ttattactgc agctcatata caagcagcag cactcatgtc
300 ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc
caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc
aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc
gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgtg
gagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca
gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600
tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660
[0485] CR6272 Light Chain Amino Acid Sequence (SEQ ID NO: 458)
TABLE-US-00240 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHV
FGTGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG
AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS
CQVTHEGSTVEKTVAPTECS
[0486] CR6272 VL Amino Acid Sequence (SEQ ID NO: 456)
TABLE-US-00241 GSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTHV FGTGTKVTVLG
[0487] The CR696 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 459) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 460 and the heavy chain
amino acid sequence shown in SEQ ID NO: 461. The CR6296 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 462) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 463 and the light chain amino acid sequence shown in SEQ
ID NO: 464.
[0488] CR6296 Heavy Chain Nucleotide Sequence (SEQ ID NO: 460)
TABLE-US-00242 gaggtgcagc tggtggagac cggggctgag gtgaagaagc
ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc
agctactata tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggatgg atcaacccta acagtggtgg cacaaactat 180 gcacagaagt
ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240
atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagagagggg
300 aaatggggac ctcaagcggc ttttgatatc tggggccaag ggacaatggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0489] CR6296 Heavy Chain Amino Acid Sequence (SEQ ID NO: 461)
TABLE-US-00243 EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREG
KWGPQAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK
[0490] CR6296 VH Amino Acid Sequence (SEQ ID NO: 459)
TABLE-US-00244 EVQLVETGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREG
KWGPQAAFDIWGQGTMVTVSS
[0491] CR6296 Light Chain Nucleotide Sequence (SEQ ID NO: 463)
TABLE-US-00245 gaaattgtga tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc
agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct
cctcatctat gatgcatcca gcagggccac tgacatccca 180 gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcactttg gacgttcggc
300 caagggacca aggtggagat caaacgtgcg gccgcaccca gcgtgttcat
cttccccccc 360 tccgacgagc agctgaagag cggcaccgcc agcgtggtgt
gcctgctgaa caacttctac 420 ccccgggagg ccaaggtgca gtggaaggtg
gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga
cagcaaggac tccacctaca gcctgagcag caccctcacc 540 ctgagcaagg
ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642
[0492] CR6296 Light Chain Amino Acid Sequence (SEQ ID NO: 464)
TABLE-US-00246 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY
DASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFG
QGTKVEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0493] CR6296 VL Amino Acid Sequence (SEQ ID NO: 462)
TABLE-US-00247 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY
DASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLWTFG QGTKVEIKR
[0494] The CR6301 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 465) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 466 and the heavy chain
amino acid sequence shown in SEQ ID NO: 467. The CR6301 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 468) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 469 and the light chain amino acid sequence shown in SEQ
ID NO: 470.
[0495] CR6301 Heavy Chain nucleotide sequence (SEQ ID NO: 466)
TABLE-US-00248
gaggtgcagctggtagagtctgggggaggcttggtacagcctggggggtccctgagactc 60
tcctgtgcagcctctggattcacctttagcatctatgccatgagctgggtccgccaggca 120
ccagggaaggggctggagtgggtctcagctattagtagtagtggtgatagcacatactac 180
gcagactccgtgaagggccggttcaccatctccagagacaacgccaggaacacgctgtat 240
ctgcaaatgaacagtctgagagccgaggacacggctgtgtattactgtgcgagagcgtat 300
ggctacacgttcgacccctggggccagggaaccctggtcaccgtctcgagtgctagcacc 360
aagggccccagcgtgttccccctggcccccagcagcaagagcaccagcggcggcacagcc 420
gccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgagctggaacagc 480
ggcgccttgaccagcggcgtgcacaccttccccgccgtgctgcagagcagcggcctgtac 540
agcctgagcagcgtggtgaccgtgcccagcagcagcctgggcacccagacctacatctgc 600
aacgtgaaccacaagcccagcaacaccaaggtggacaaacgcgtggagcccaagagctgc 660
gacaagacccacacctgccccccctgccctgcccccgagctgctgggcggaccctccgtg 720
ttcctgttcccccccaagcccaaggacaccctcatgatcagccggacccccgaggtgacc 780
tgcgtggtggtggacgtgagccacgaggaccccgaggtgaagttcaactggtacgtggac 840
ggcgtggaggtgcacaacgccaagaccaagccccgggaggagcagtacaacagcacctac 900
cgggtggtgagcgtgctcaccgtgctgcaccaggactggctgaacggcaaggagtacaag 960
tgcaaggtgagcaacaaggccctgcctgcccccatcgagaagaccatcagcaaggccaag 1020
ggccagccccgggagccccaggtgtacaccctgccccccagccgggaggagatgaccaag 1080
aaccaggtgtccctcacctgtctggtgaagggcttctaccccagcgacatcgccgtggag 1140
tgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagc 1200
gacggcagcttcttcctgtacagcaagctcaccgtggacaagagccggtggcagcagggc 1260
aacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagc 1320
ctgagcctgagccccggcaag 1341
[0496] CR6301 Heavy Chain Amino Acid Sequence (SEQ ID NO: 467)
TABLE-US-00249 EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSA
ISSSGDSTYYADSVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAY
GYTFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0497] CR6301 VH Amino Acid Sequence (SEQ ID NO: 465)
TABLE-US-00250 EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMSWVRQAPGKGLEWVSA
ISSSGDSTYYADSVKGRFTISRDNARNTLYLQMNSLRAEDTAVYYCARAY
GYTFDPWGQGTLVTVSS
[0498] CR6301 Light Chain Nucleotide Sequence (SEQ ID NO: 469)
TABLE-US-00251 gaaattgtgc tgactcagtc tccactctcc ctgcccgtca
cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg
catagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggca
gtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccc
300 ctcactttcg gcggagggac caaggtggag atcaaacgtg cggccgcacc
cagcgtgttc 360 atcttccccc cctccgacga gcagctgaag agcggcaccg
ccagcgtggt gtgcctgctg 420 aacaacttct acccccggga ggccaaggtg
cagtggaagg tggacaacgc cctgcagagc 480 ggcaacagcc aggagagcgt
gaccgagcag gacagcaagg actccaccta cagcctgagc 540 agcaccctca
ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 600
acccaccagg gcctgagcag ccccgtgacc aagagcttca accggggcga gtgt 654
[0499] CR6301 Light Chain Amino Acid Sequence (SEQ ID NO: 470)
TABLE-US-00252 EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP
LTFGGGTKVEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC
[0500] CR6301 VL Amino Acid Sequence (SEQ ID NO:468)
TABLE-US-00253 EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP
LTFGGGTKVEIKR
[0501] The CR6307 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 471) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 472 and the heavy chain
amino acid sequence shown in SEQ ID NO: 473. The CR6307 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 474) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 475 and the light chain amino acid sequence shown in SEQ
ID NO: 476.
[0502] CR6307 Heavy Chain nucleotide sequence (SEQ ID NO: 472)
TABLE-US-00254 caggtccagc tggtgcagtc tgggggaggc ctggtcaagc
ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt
agctatagca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg
ggtctcatcc attagtagta gtagtagtta catatactac 180 gtagactcag
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagaggtggt
300 gggagctacg gggcctacga aggctttgac tactggggcc agggcaccct
ggtcaccgtc 360 tcgagtgcta gcaccaaggg ccccagcgtg ttccccctgg
cccccagcag caagagcacc 420 agcggcggca cagccgccct gggctgcctg
gtgaaggact acttccccga gcccgtgacc 480 gtgagctgga acagcggcgc
cttgaccagc ggcgtgcaca ccttccccgc cgtgctgcag 540 agcagcggcc
tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc 600
cagacctaca tctgcaacgt gaaccacaag cccagcaaca ccaaggtgga caaacgcgtg
660 gagcccaaga gctgcgacaa gacccacacc tgccccccct gccctgcccc
cgagctgctg 720 ggcggaccct ccgtgttcct gttccccccc aagcccaagg
acaccctcat gatcagccgg 780 acccccgagg tgacctgcgt ggtggtggac
gtgagccacg aggaccccga ggtgaagttc 840 aactggtacg tggacggcgt
ggaggtgcac aacgccaaga ccaagccccg ggaggagcag 900 tacaacagca
cctaccgggt ggtgagcgtg ctcaccgtgc tgcaccagga ctggctgaac 960
ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ctgcccccat cgagaagacc
1020 atcagcaagg ccaagggcca gccccgggag ccccaggtgt acaccctgcc
ccccagccgg 1080 gaggagatga ccaagaacca ggtgtccctc acctgtctgg
tgaagggctt ctaccccagc 1140 gacatcgccg tggagtggga gagcaacggc
cagcccgaga acaactacaa gaccaccccc 1200 cctgtgctgg acagcgacgg
cagcttcttc ctgtacagca agctcaccgt ggacaagagc 1260 cggtggcagc
agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1320
tacacccaga agagcctgag cctgagcccc ggcaag 1356
[0503] CR6307 Heavy Chain Amino Acid Sequence (SEQ ID NO: 473)
TABLE-US-00255 QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSS
ISSSSSYIYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGG
GSYGAYEGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK
[0504] CR6307 VH Amino Acid Sequence (SEQ ID NO: 471)
TABLE-US-00256 QVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSS
ISSSSSYIYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGG
GSYGAYEGFDYWGQGTLVTVSS
[0505] CR6307 Light Chain Nucleotide Sequence (SEQ ID NO: 475)
TABLE-US-00257 gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60 ctctcctgca gggccagtca gcgtgttagc
agctacttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct
catctatggt gcatccacca gggccgctgg catcccagac 180 aggttcagtg
gcagtgggtc tgggacagac ttcactctca ccatcagcag actggagcct 240
gaagattctg cagtgtatta ctgtcagcag tatggtagga caccgctcac tttcggcgga
300 gggaccaagg tggagatcaa acgtgcggcc gcacccagcg tgttcatctt
ccccccctcc 360 gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc
tgctgaacaa cttctacccc 420 cgggaggcca aggtgcagtg gaaggtggac
aacgccctgc agagcggcaa cagccaggag 480 agcgtgaccg agcaggacag
caaggactcc acctacagcc tgagcagcac cctcaccctg 540 agcaaggccg
actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600
agcagccccg tgaccaagag cttcaaccgg ggcgagtgt 639
[0506] CR6307 Light Chain Amino Acid Sequence (SEQ ID NO: 476)
TABLE-US-00258 EIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYG
ASTRAAGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGG
GTKVEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[0507] CR6307 VL Amino Acid Sequence (SEQ ID NO: 474)
TABLE-US-00259 EIVLYQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYG
ASTRAAGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYGRTPLTFGG GTKVEIKR
[0508] The CR6310 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 477) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 478 and the heavy chain
amino acid sequence shown in SEQ ID NO: 479. The CR6310 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 480) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 481 and the light chain amino acid sequence shown in SEQ
ID NO: 482.
[0509] CR6310 Heavy Chain nucleotide sequence (SEQ ID NO: 478)
TABLE-US-00260 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0510] CR6310 Heavy Chain Amino Acid Sequence (SEQ ID NO: 479)
TABLE-US-00261 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0511] CR6310 VH Amino Acid Sequence (SEQ ID NO: 477)
TABLE-US-00262 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0512] CR6310 Light Chain Nucleotide Sequence (SEQ ID NO: 481)
TABLE-US-00263 tcctatgtgc tgactcagcc accctcggtg tcagtggccc
caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa
agtgtgcact ggtaccagca gaagccaggc 120 caggcccctg tgctggtcgt
ctatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct
ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240
gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgc tgtgttcgga
300 ggaggcaccc agctgaccgt cctcggtgcg gccgcaggcc agcccaaggc
cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca
acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg
accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac
caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc
tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600
gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654
[0513] CR6310 Light Chain Amino Acid Sequence (SEQ ID NO: 482)
TABLE-US-00264 SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFG
GGTQLTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS
[0514] CR6310 VL Amino Acid Sequence (SEQ ID NO: 480)
TABLE-US-00265 SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHAVFG GGTQLTVLG
[0515] The CR6314 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 483) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 484 and the heavy chain
amino acid sequence shown in SEQ ID NO: 485. The CR6314 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 486) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 487 and the light chain amino acid sequence shown in SEQ
ID NO: 488.
[0516] CR6314 Heavy Chain Nucleotide Sequence (SEQ ID NO: 484)
TABLE-US-00266 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaagtc 60 tcttgcaagg cttctggagg ccccttccgc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcctgagtg
gatgggaggg atcatcccta tttttggtac aacaaaatac 180 gcaccgaagt
tccagggcag agtcacgatt accgcggacg atttcgcggg cacagtttac 240
atggagctga gcagcctgcg atctgaggac acggccatgt actactgtgc gaaacatatg
300 gggtaccagg tgcgcgaaac tatggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0517] CR6314 Heavy Chain Amino Acid Sequence (SEQ ID NO: 485)
TABLE-US-00267 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0518] CR6314 VH Amino Acid Sequence (SEQ ID NO: 483)
TABLE-US-00268 EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGG
IIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHM
GYQVRETMDVWGKGTTVTVSS
[0519] CR6314 Light Chain Nucleotide Sequence (SEQ ID NO: 487)
TABLE-US-00269 tcctatgtgc tgactcagcc accctcagcg tctgggaccc
ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc caacatcgga
agtaattatg tatactggta ccagcagctc 120 ccaggcacgg cccccaaact
cctcatctat agggatggtc agcggccctc aggggtccct 180 gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tggactccgg 240
tccgatgatg aggctgatta ttactgtgca acatgggatg acaacctgag tggtccagta
300 ttcggcggag ggaccaagct gaccgtccta ggtgcggccg caggccagcc
caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc
aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc
gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt
ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca
gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600
tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660
[0520] CR6314 Light Chain Amino Acid Sequence (SEQ ID NO: 488)
TABLE-US-00270 SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIY
RDGQRPSGVPDRFSGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPV
FGGGTKLTVLGAAAQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA
VTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPGQWKSHRSYSC
QVTHEGSTVEKTVAPTECSG
[0521] CR6314 VL Amino Acid Sequence (SEQ ID NO: 486)
TABLE-US-00271 SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIY
RDGQRPSGVPDRFSGSKSGTSASLAISGLRSDDEADYYCATWDDNLSGPV FGGGTKLTVLG
[0522] The CR6323 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 489) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 490 and the heavy chain
amino acid sequence shown in SEQ ID NO: 491. The CR6323 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 492) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 493 and the light chain amino acid sequence shown in SEQ
ID NO: 494.
[0523] CR6323 Heavy Chain nucleotide sequence (SEQ ID NO: 490)
TABLE-US-00272 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
cagggtcctc ggtgaaggtc 60 tcctgtaagg cctctggagg caccttctcc
agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggagac atcatcggta tgtttggttc aacaaactac 180 gcacagaact
tccagggcag actcacgatt accgcggacg aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaagtagt
300 ggttattacc ctgcatacct cccccactgg ggccagggca ccttggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0524] CR6323 Heavy Chain Amino Acid Sequence (SEQ ID NO: 491)
TABLE-US-00273 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGD
IIGMFGSTNYAQNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSS
GYYPAYLPHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0525] CR6323 VH Amino Acid Sequence (SEQ ID NO: 489)
TABLE-US-00274 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGD
IIGMFGSTNYAQNFQGRLTITADESTSTAYMELSSLRSEDTAVYYCARSS
GYYPAYLPHWGQGTLVTVSS
[0526] CR6323 Light Chain Nucleotide Sequence (SEQ ID NO: 493)
TABLE-US-00275 gaaattgtgt tgacccagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc
agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct
cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacccag aactttcggc
300 ggagggacca aggtggagat caaacgtgcg gccgcaccca gcgtgttcat
cttccccccc 360 tccgacgagc agctgaagag cggcaccgcc agcgtggtgt
gcctgctgaa caacttctac 420 ccccgggagg ccaaggtgca gtggaaggtg
gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga
cagcaaggac tccacctaca gcctgagcag caccctcacc 540 ctgagcaagg
ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642
[0527] CR6323 Light Chain Amino Acid Sequence (SEQ ID NO: 494)
TABLE-US-00276 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFG
GGTKVEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0528] CR6323 VL Amino Acid Sequence (SEQ ID NO: 492)
TABLE-US-00277 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFG GGTKVEIKR
[0529] The CR6325 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 495) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 496 and the heavy chain
amino acid sequence shown in SEQ ID NO: 497. The CR6325 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 498) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 499 and the light chain amino acid sequence shown in SEQ
ID NO: 500.
[0530] CR6325 Heavy Chain nucleotide sequence (SEQ ID NO: 496)
TABLE-US-00278 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
cggggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc
ttctattcta tgagctgggt gcgacaggcc 120 cctggacaag gacttgagtg
gatgggaggg atcatcccta tgtttggtac aacaaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggtcg aatccacgag cacagcctac 240
atggaggtga gcagcctgag atctgaggac acggccgttt attactgtgc gagaggtgat
300 aagggtatct actactacta catggacgtc tggggcaaag ggaccacggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0531] CR6325 Heavy Chain Amino Acid Sequence (SEQ ID NO: 497)
TABLE-US-00279 EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGG
IIPMFGTTNYAQKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGD
KGIYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0532] CR6325 VH Amino Acid Sequence (SEQ ID NO: 495)
TABLE-US-00280 EVQLVESGAEVKKPGSSVKVSCKASGGTFSFYSMSWVRQAPGQGLEWMGG
IIPMFGTTNYAQKFQGRVTITAVESTSTAYMEVSSLRSEDTAVYYCARGD
KGIYYYYMDVWGKGTTVTVSS
[0533] CR6325 Light Chain Nucleotide Sequence (SEQ ID NO: 499)
TABLE-US-00281 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc
ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt
ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180 tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cactcttgtc
300 ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc
caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc
aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc
gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt
ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca
gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600
tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660
[0534] CR6325 Light Chain Amino Acid Sequence (SEQ ID NO: 500)
TABLE-US-00282 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLV
FGTGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG
AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS
CQVTHEGSTVEKTVAPTECS
[0535] CR6325 VL Amino Acid Sequence (SEQ ID NO: 498)
TABLE-US-00283 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI
YEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLV FGTGTKVTVLG
[0536] The CR6327 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 501) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 502 and the heavy chain
amino acid sequence shown in SEQ ID NO: 503. The CR6327 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 504) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 505 and the light chain amino acid sequence shown in SEQ
ID NO: 506.
[0537] CR6327 Heavy Chain nucleotide sequence (SEQ ID NO: 502)
TABLE-US-00284 gaggtgcagc tggtggagac cggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cctctggagg caccttcagg
acccatgcta tcagttgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcgcta tcttcggaac agcaaactac 180 gcacagaagt
tccagggcag aatcacgatt accgcggacg aatccacgag tacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt atttctgtgc gagaggcagt
300 ggttatcata tatcgacacc ctttgacaac tggggccagg gaaccctggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0538] CR6327 Heavy Chain Amino Acid Sequence (SEQ ID NO: 503)
TABLE-US-00285 EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGG
IIAIFGTANYAQKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGS
GYHISTPFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0539] CR6327 VH Amino Acid Sequence (SEQ ID NO: 501)
TABLE-US-00286 EVQLVETGAEVKKPGSSVKVSCKASGGTFRTHAISWVRQAPGQGLEWMGG
IIAIFGTANYAQKFQGRITITADESTSTAYMELSSLRSEDTAVYFCARGS
GYHISTPFDNWGQGTLVTVSS
[0540] CR6327 Light Chain Nucleotide Sequence (SEQ ID NO: 505)
TABLE-US-00287 tcctatgtgc tgactcagcc accctcggtg tcagtggccc
caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa
ggtgtgcact ggtaccagca gaagcctggc 120 caggcccctg tgctggtcgt
ctatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct
ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240
gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgt ggtattcggc
300 ggagggacca agctgaccgt cctaggtgcg gccgcaggcc agcccaaggc
cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca
acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg
accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac
caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc
tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600
gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654
[0541] CR6327 Light Chain Amino Acid Sequence (SEQ ID NO: 506)
TABLE-US-00288 SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFG
GGTKLTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS
[0542] CR6327 VL Amino Acid Sequence (SEQ ID NO: 504)
TABLE-US-00289 SYVLTQPPSVSVAPGQTARITCGGNNIGSKGVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFG GGTKLTVLG
[0543] The CR6328 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 507) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 508 and the heavy chain
amino acid sequence shown in SEQ ID NO: 509. The CR6328 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 510) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 511 and the light chain amino acid sequence shown in SEQ
ID NO: 512.
[0544] CR6328 Heavy Chain nucleotide sequence (SEQ ID NO: 508)
TABLE-US-00290 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggaca catcttcagc
ggctatgcaa tcagttgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcccta tctttggtac aacaaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacc aatccacgag cacagcctac 240
atggacctga gcaacttgag atctgaggac acggccgtct attactgtgc gagagtgaaa
300 gatggatatt gtactcttac cagctgccct gtcggctggt acttcgatct
ctggggccgt 360 ggcaccctgg tcactgtctc gagtgctagc accaagggcc
ccagcgtgtt ccccctggcc 420 cccagcagca agagcaccag cggcggcaca
gccgccctgg gctgcctggt gaaggactac 480 ttccccgagc ccgtgaccgt
gagctggaac agcggcgcct tgaccagcgg cgtgcacacc 540 ttccccgccg
tgctgcagag cagcggcctg tacagcctga gcagcgtggt gaccgtgccc 600
agcagcagcc tgggcaccca gacctacatc tgcaacgtga accacaagcc cagcaacacc
660 aaggtggaca aacgcgtgga gcccaagagc tgcgacaaga cccacacctg
ccccccctgc 720 cctgcccccg agctgctggg cggaccctcc gtgttcctgt
tcccccccaa gcccaaggac 780 accctcatga tcagccggac ccccgaggtg
acctgcgtgg tggtggacgt gagccacgag 840 gaccccgagg tgaagttcaa
ctggtacgtg gacggcgtgg aggtgcacaa cgccaagacc 900 aagccccggg
aggagcagta caacagcacc taccgggtgg tgagcgtgct caccgtgctg 960
caccaggact ggctgaacgg caaggagtac aagtgcaagg tgagcaacaa ggccctgcct
1020 gcccccatcg agaagaccat cagcaaggcc aagggccagc cccgggagcc
ccaggtgtac 1080 accctgcccc ccagccggga ggagatgacc aagaaccagg
tgtccctcac ctgtctggtg 1140 aagggcttct accccagcga catcgccgtg
gagtgggaga gcaacggcca gcccgagaac 1200 aactacaaga ccaccccccc
tgtgctggac agcgacggca gcttcttcct gtacagcaag 1260 ctcaccgtgg
acaagagccg gtggcagcag ggcaacgtgt tcagctgcag cgtgatgcac 1320
gaggccctgc acaaccacta cacccagaag agcctgagcc tgagccccgg caag
1374
[0545] CR6328 Heavy Chain Amino Acid Sequence (SEQ ID NO: 509)
TABLE-US-00291 EVQLVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGG
IIPIFGTTNYAQKFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVK
DGYCTLTSCPVGWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
[0546] CR6328 VH Amino Acid Sequence (SEQ ID NO: 507)
TABLE-US-00292 EVQLVESGAEVKKPGSSVKVSCKASGHIFSGYAISWVRQAPGQGLEWMGG
IIPIFGTTNYAQKFQGRVTITADQSTSTAYMDLSNLRSEDTAVYYCARVK
DGYCTLTSCPVGWYFDLWGRGTLVTVSS
[0547] CR6328 Light Chain Nucleotide Sequence (SEQ ID NO: 511)
TABLE-US-00293 gaaattgtga tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60 ctctcgtgca gggccagtca gagtgttagc
agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct
cctcatcttt ggtgcctcca gcagggccac tggcatccca 180 gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcactcac tttcggcgga
300 gggaccaagc tggagatcaa acgtgcggcc gcacccagcg tgttcatctt
ccccccctcc 360 gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc
tgctgaacaa cttctacccc 420 cgggaggcca aggtgcagtg gaaggtggac
aacgccctgc agagcggcaa cagccaggag 480 agcgtgaccg agcaggacag
caaggactcc acctacagcc tgagcagcac cctcaccctg 540 agcaaggccg
actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600
agcagccccg tgaccaagag cttcaaccgg ggcgagtgt 639
[0548] CR6328 Light Chain Amino Acid Sequence (SEQ ID NO: 512)
TABLE-US-00294 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIF
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGG
GTKLEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[0549] CR6328 VL Amino Acid Sequence (SEQ ID NO: 510)
TABLE-US-00295 EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIF
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSLTFGG GTKLEIKR
[0550] The CR6329 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 513) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 514 and the heavy chain
amino acid sequence shown in SEQ ID NO: 515. The CR6329 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 516) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 517 and the light chain amino acid sequence shown in SEQ
ID NO: 518.
[0551] CR6329 Heavy Chain nucleotide sequence (SEQ ID NO: 514)
TABLE-US-00296 gaggtccagc tggtacagtc tggggctgag gttaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg catcttcaga
agcaattcta tcagttgggt gcgacaggcc 120 cctgggcaag ggcttgagtg
gatgggaggg atcttcgctc ttttcggaac aacagactac 180 gcgcagaagt
tccagggcag agtcacgatt accgcggacg aatcttcgac cacagtctac 240
ctggagctga gtagcctgac atctgaggac acggccgttt attactgtgc gagaggcagt
300 ggctacacca cacgcaacta ctttgactac tggggccagg gcaccctggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0552] CR6329 Heavy Chain Amino Acid Sequence (SEQ ID NO: 515)
TABLE-US-00297 EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGG
IFALFGTTDYAQKFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGS
GYTTRNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK
[0553] CR6329 VH Amino Acid Sequence (SEQ ID NO: 513)
TABLE-US-00298 EVQLVQSGAEVKKPGSSVKVSCKASGGIFRSNSISWVRQAPGQGLEWMGG
IFALFGTTDYAQKFQGRVTITADESSTTVYLELSSLTSEDTAVYYCARGS
GYTTRNYFDYWGQGTLVTVSS
[0554] CR6329 Light Chain Nucleotide Sequence (SEQ ID NO: 517)
TABLE-US-00299 gaaattgtgc tgactcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccaca 60 ctctoctgca gggccagtca gagtgttagc
agcaactact taggctggta ccagcagaaa 120 cctggccagg ctcccaggct
cctgatctat ggtgcatcca gcagggccag tggcatccca 180 gacaggttca
gtggcggtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacccct cactttcggc
300 ggagggacca aggtggagat caaacgtgcg gccgcaggcc agcccaaggc
cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca
acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg
accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac
caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc
tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600
gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654
[0555] CR6329 Light Chain Amino Acid Sequence (SEQ ID NO: 518)
TABLE-US-00300 EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWYQQKPGQAPRLLIY
GASSRASGIPDRFSGGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFG
GGTKVEIKRAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS
[0556] CR6329 VL Amino Acid Sequence (SEQ ID NO: 516)
TABLE-US-00301 EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLGWYQQKPGQAPRLLIY
GASSRASGIPDRFSGGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFG GGTKVEIKR
[0557] The CR6331 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 519) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 520 and the heavy chain
amino acid sequence shown in SEQ ID NO: 521. The CR6331 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 522) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 523 and the light chain amino acid sequence shown in SEQ
ID NO: 524.
[0558] CR6331 Heavy Chain Nucleotide Sequence (SEQ ID NO: 520)
TABLE-US-00302 gaggtgcagc tggtggagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcggta tgttcggtac agcaaactac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aatttacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggaaat
300 tattactatg agagtagtct cgactactgg ggccagggaa ccctggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0559] CR6331 Heavy Chain Amino Acid Sequence (SEQ ID NO: 521)
TABLE-US-00303 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIGMFGTANYAQKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGN
YYYESSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0560] CR6331 VH Amino Acid Sequence (SEQ ID NO: 519)
TABLE-US-00304 EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIGMFGTANYAQKFQGRVTITADEFTSTAYMELSSLRSEDTAVYYCARGN
YYYESSLDYWGQGTLVTVSS
[0561] CR6331 Light Chain Nucleotide Sequence (SEQ ID NO: 523)
TABLE-US-00305 cagtctgtcg tgacgcagcc gccctcggtg tcagtggccc
caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa
agtgtgcact ggtaccagca gaagccaggc 120 caggcccctg tgctggtcgt
ctatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct
ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240
gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatta tgtcttcgga
300 actgggacca aggtcaccgt cctaggtgcg gccgcaggcc agcccaaggc
cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca
acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg
accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac
caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc
tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600
gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654
[0562] CR6331 Light Chain Amino Acid Sequence (SEQ ID NO: 524)
TABLE-US-00306 QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFG
TGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS
[0563] CR6331 VL Amino Acid Sequence (SEQ ID NO: 522)
TABLE-US-00307 QSVVTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFG TGTKVTVLG
[0564] The CR6332 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 525) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 526 and the heavy chain
amino acid sequence shown in SEQ ID NO: 527. The CR6332 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 528) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 529 and the light chain amino acid sequence shown in SEQ
ID NO: 530.
[0565] CR6332 Heavy Chain Nucleotide Sequence (SEQ ID NO: 526)
TABLE-US-00308 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtaaaggtc 60 tcctgcaagg cttctggagg ccccttccgc
aattttgcta tcaactgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcgctg tctttgggac gacaaagtac 180 gcacataagt
tccagggcag agtcaccatc accgcggacg actccacaaa tacagcttac 240
atggagctgg gcagcctgaa atctgaggac acggccgtgt attactgtgc gagaggtccc
300 cactactact cctcctacat ggacgtctgg ggcgaaggga ccacggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0566] CR6332 Heavy Chain Amino Acid Sequence (SEQ ID NO: 527)
TABLE-US-00309 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGG
IIAVFGTTKYAHKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGP
HYYSSYMDVWGEGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0567] CR6332 VH Amino Acid Sequence (SEQ ID NO: 525)
TABLE-US-00310 QVQLVQSGAEVKKPGSSVKVSCKASGGPFRNFAINWVRQAPGQGLEWMGG
IIAVFGTTKYAHKFQGRVTITADDSTNTAYMELGSLKSEDTAVYYCARGP
HYYSSYMDVWGEGTTVTVSS
[0568] CR6332 Light Chain Nucleotide Sequence (SEQ ID NO: 529)
TABLE-US-00311 gacatccagt tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60 atcacttgcc gggcgagtca gggcattagc
acttatttag cctggtatca gcagaaaccc 120 gggaaagttc ctaaactcct
gatctatgct gcatccactt tgcaatcagg ggtcccatct 180 cggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccttcttt cggccctggg
300 accaaagtgg atatcaaacg tgcggccgca cccagcgtgt tcatcttccc
cccctccgac 360 gagcagctga agagcggcac cgccagcgtg gtgtgcctgc
tgaacaactt ctacccccgg 420 gaggccaagg tgcagtggaa ggtggacaac
gccctgcaga gcggcaacag ccaggagagc 480 gtgaccgagc aggacagcaa
ggactccacc tacagcctga gcagcaccct caccctgagc 540 aaggccgact
acgagaagca caaggtgtac gcctgcgagg tgacccacca gggcctgagc 600
agccccgtga ccaagagctt caaccggggc gagtgt 636
[0569] CR6332 Light Chain Amino Acid Sequence (SEQ ID NO: 530)
TABLE-US-00312 DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPG
TKVDIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC
[0570] CR6332 VL Amino Acid Sequence (SEQ ID NO: 528)
TABLE-US-00313 DIQLTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKVPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPSFGPG TKVDIKR
[0571] The CR6334 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 531) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 532 and the heavy chain
amino acid sequence shown in SEQ ID NO: 533. The CR6334 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 534) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 535 and the light chain amino acid sequence shown in SEQ
ID NO: 536.
[0572] CR6334 Heavy Nucleotide Sequence (SEQ ID NO: 532)
TABLE-US-00314 gaggtgcagc tggtggagac tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 ccctgcaaat cttctggaag ccccttcagg
agtaatgctg tcagctgggt gcgacaggcc 120 cccggacaag ggcttgagtg
ggtgggagga atcctcggtg tctttggttc accaagctac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aatccaccaa cacagtccac 240
atggagctga gaggtttgag atctgaggac acggccgtgt attattgtgc gagaggtcct
300 acctactact actcctacat ggacgtctgg ggcaaaggga ccacggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0573] CR6334 Heavy Chain Amino Acid Sequence (SEQ ID NO: 533)
TABLE-US-00315 EVQLVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGG
ILGVFGSPSYAQKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGP
TYYYSYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0574] CR6334 VH Amino Acid Sequence (SEQ ID NO: 531)
TABLE-US-00316 EVQLVETGAEVKKPGSSVKVPCKSSGSPFRSNAVSWVRQAPGQGLEWVGG
ILGVFGSPSYAQKFQGRVTITADESTNTVHMELRGLRSEDTAVYYCARGP
TYYYSYMDVWGKGTTVTVSS
[0575] CR6334 Light Chain Nucleotide Sequence (SEQ ID NO: 535)
TABLE-US-00317 tcctatgtgc tgactcagcc accctcggag tcagtggccc
caggacagac ggccaggatt 60 acctgtgggg gaaataacat tggaagaaat
agtgtgcact ggtatcagca gaagccaggc 120 caggcccctg tgctggtcgt
gtatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttttctggct
ccaagtctgg gaacacggcc accctgatta tcagcagggt cgaagtcggg 240
gatgaggccg actactactg tcaggtgtgg catagtagta gtgatcatta tgtcttcgga
300 actgggacca aggtcaccgt cctaggtgcg gccgcaggcc agcccaaggc
cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca
acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg
accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac
caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc
tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600
gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654
[0576] CR6334 Light Chain Amino Acid Sequence (SEQ ID NO: 536)
TABLE-US-00318 SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFG
TGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS
[0577] CR6334 VL Amino Acid Sequence (SEQ ID NO: 534)
TABLE-US-00319 SYVLTQPPSESVAPGQTARITCGGNNIGRNSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSKSGNTATLIISRVEVGDEADYYCQVWHSSSDHYVFG TGTKVTVLG
[0578] The CR6336 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 537) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 538 and the heavy chain
amino acid sequence shown in SEQ ID NO: 539. The CR6336 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 540) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 541 and the light chain amino acid sequence shown in SEQ
ID NO: 542.
[0579] CR6336 Heavy Chain Nucleotide Sequence (SEQ ID NO: 538)
TABLE-US-00320 cagatgcagc tggtacaatc tggagctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc
agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcttcggta tgtttgggac agcaaactac 180 gcgcagaagt
tccagggcag agtcacgatt accgcggacg aattcacgag cgcggcctac 240
atggagctga gcagcctggg atctgaggac acggccatgt attactgtgc gaggtctagt
300 ggttattacc cccaatactt ccaggactgg ggccagggca ccctggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0580] CR6336 Heavy Chain Amino Acid Sequence (SEQ ID NO: 539)
TABLE-US-00321 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IFGMFGTANYAQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSS
GYYPQYFQDWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0581] CR6336 VH Amino Acid Sequence (SEQ ID NO: 537)
TABLE-US-00322 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IFGMFGTANYAQKFQGRVTITADEFTSAAYMELSSLGSEDTAMYYCARSS
GYYPQYFQDWGQGTLVTVSS
[0582] CR6336 Light Chain Nucleotide Sequence (SEQ ID NO: 541)
TABLE-US-00323 gaaattgtga tgacacagtc tccaggcacc ctgtctttgt
ctccagggca aagagccacc 60 ctctcctgca gggccagtca gagtgttagc
agcagctact tagcctggta ccagcagaaa 120 cctggccagg ctcccagact
cctcatgtat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcatcgct cactttcggc
300 ggagggacca agctggagat caaacgtgcg gccgcaccca gcgtgttcat
cttccccccc 360 tccgacgagc agctgaagag cggcaccgcc agcgtggtgt
gcctgctgaa caacttctac 420 ccccgggagg ccaaggtgca gtggaaggtg
gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga
cagcaaggac tccacctaca gcctgagcag caccctcacc 540 ctgagcaagg
ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgagcagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642
[0583] CR6336 Light Chain Amino Acid Sequence (SEQ ID NO: 542)
TABLE-US-00324 EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFG
GGTKLEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0584] CR6336 VL Amino Acid Sequence (SEQ ID NO: 540)
TABLE-US-00325 EIVMTQSPGTLSLSPGQRATLSCRASQSVSSSYLAWYQQKPGQAPRLLMY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSLTFG GGTKLEIKR
[0585] The CR6339 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 543) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 545 and the heavy chain
amino acid sequence shown in SEQ ID NO: 546. The CR6339 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 547) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 548 and the light chain amino acid sequence shown in SEQ
ID NO: 549.
[0586] CR6339 Heavy Chain Nucleotide Sequence (SEQ ID NO: 545)
TABLE-US-00326 gaggtgcagc tggtggagtc cggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg catcttcaac
agttatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggc atcatcgcta tctttcatac accaaagtac 180 gcacagaagt
tccagggcag agtcacgatt accgcggacg aatccacgaa cacagcctac 240
atggaactga gaagcctgaa atctgaggac acggccctgt attactgtgc gagagggtcc
300 acttacgatt tttcgagtgg ccttgactac tggggccagg gaaccctggt
caccgtctcg 360 agtgctagca ccaagggccc cagcgtgttc cccctggccc
ccagcagcaa gagcaccagc 420 ggcggcacag ccgccctggg ctgcctggtg
aaggactact tccccgagcc cgtgaccgtg 480 agctggaaca gcggcgcctt
gaccagcggc gtgcacacct tccccgccgt gctgcagagc 540 agcggcctgt
acagcctgag cagcgtggtg accgtgccca gcagcagcct gggcacccag 600
acctacatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa acgcgtggag
660 cccaagagct gcgacaagac ccacacctgc cccccctgcc ctgcccccga
gctgctgggc 720 ggaccctccg tgttcctgtt cccccccaag cccaaggaca
ccctcatgat cagccggacc 780 cccgaggtga cctgcgtggt ggtggacgtg
agccacgagg accccgaggt gaagttcaac 840 tggtacgtgg acggcgtgga
ggtgcacaac gccaagacca agccccggga ggagcagtac 900 aacagcacct
accgggtggt gagcgtgctc accgtgctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt gagcaacaag gccctgcctg cccccatcga gaagaccatc
1020 agcaaggcca agggccagcc ccgggagccc caggtgtaca ccctgccccc
cagccgggag 1080 gagatgacca agaaccaggt gtccctcacc tgtctggtga
agggcttcta ccccagcgac 1140 atcgccgtgg agtgggagag caacggccag
cccgagaaca actacaagac caccccccct 1200 gtgctggaca gcgacggcag
cttcttcctg tacagcaagc tcaccgtgga caagagccgg 1260 tggcagcagg
gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320
acccagaaga gcctgagcct gagccccggc aag 1353
[0587] CR6339 Heavy Chain Amino Acid Sequence (SEQ ID NO: 546)
TABLE-US-00327 EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGG
IIAIFHTPKYAQKFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGS
TYDFSSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[0588] CR6339 VH Amino Acid Sequence (SEQ ID NO: 543)
TABLE-US-00328 EVQLVESGAEVKKPGSSVKVSCKASGGIFNSYAISWVRQAPGQGLEWMGG
IIAIFHTPKYAQKFQGRVTITADESTNTAYMELRSLKSEDTALYYCARGS
TYDFSSGLDYWGQGTLVTVSS
[0589] CR6339 Light Chain Nucleotide Sequence (SEQ ID NO: 548)
TABLE-US-00329 caggcagggc tgactcagcc accctcggtg tcagtggccc
caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa
agtgtgcact ggtaccagca gaagccaggc 120 caggcccctg tcctagtcgt
ctatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct
ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240
gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgt ggtattcggc
300 ggagggacca agctgaccgt cctaggtgcg gccgcaggcc agcccaaggc
cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca
acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg
accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac
caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc
tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600
gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654
[0590] CR6339 Light Chain Amino Acid Sequence (SEQ ID NO: 549)
TABLE-US-00330 QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFG
GGTKLTVLGAAAGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ
VTHEGSTVEKTVAPTECS
[0591] CR6339 VL Amino Acid Sequence (SEQ ID NO: 547)
TABLE-US-00331 QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDD
SDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFG GGTKLTVLG
[0592] The CR6342 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 550) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 551 and the heavy chain
amino acid sequence shown in SEQ ID NO: 552. The CR6342 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 553) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 554 and the light chain amino acid sequence shown in SEQ
ID NO: 555.
[0593] CR6342 Heavy Chain nucleotide sequence (SEQ ID NO: 551)
TABLE-US-00332 caggtccagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg cttcttcagc
agctatgcta tcagctgggt gcgccaggcc 120 cctggacaag gacttgagtg
gatggggggg gtcatcccta tctttcgtac agcaaactac 180 gcacagaact
tccagggcag agtcaccatt accgcggacg aattcacatc gtatatggag 240
ctgagcagcc tgagatctga cgacacggcc gtgtattact gtgcgaggtt gaattaccat
300 gattcgggga cttattataa cgccccccgg ggctggttcg acccctgggg
ccagggaacc 360 ctggtcaccg tctcgagtgc tagcaccaag ggccccagcg
tgttccccct ggcccccagc 420 agcaagagca ccagcggcgg cacagccgcc
ctgggctgcc tggtgaagga ctacttcccc 480 gagcccgtga ccgtgagctg
gaacagcggc gccttgacca gcggcgtgca caccttcccc 540 gccgtgctgc
agagcagcgg cctgtacagc ctgagcagcg tggtgaccgt gcccagcagc 600
agcctgggca cccagaccta catctgcaac gtgaaccaca agcccagcaa caccaaggtg
660 gacaaacgcg tggagcccaa gagctgcgac aagacccaca cctgcccccc
ctgccctgcc 720 cccgagctgc tgggcggacc ctccgtgttc ctgttccccc
ccaagcccaa ggacaccctc 780 atgatcagcc ggacccccga ggtgacctgc
gtggtggtgg acgtgagcca cgaggacccc 840 gaggtgaagt tcaactggta
cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 900 cgggaggagc
agtacaacag cacctaccgg gtggtgagcg tgctcaccgt gctgcaccag 960
gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct gcctgccccc
1020 atcgagaaga ccatcagcaa ggccaagggc cagccccggg agccccaggt
gtacaccctg 1080 ccccccagcc gggaggagat gaccaagaac caggtgtccc
tcacctgtct ggtgaagggc 1140 ttctacccca gcgacatcgc cgtggagtgg
gagagcaacg gccagcccga gaacaactac 1200 aagaccaccc cccctgtgct
ggacagcgac ggcagcttct tcctgtacag caagctcacc 1260 gtggacaaga
gccggtggca gcagggcaac gtgttcagct gcagcgtgat gcacgaggcc 1320
ctgcacaacc actacaccca gaagagcctg agcctgagcc ccggcaag 1368
[0594] CR6342 Heavy Chain Amino Acid Sequence (SEQ ID NO: 552)
TABLE-US-00333 QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGG
VIPIFRTANYAQNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYH
DSGTYYNAPRGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK
[0595] CR6342 VH Amino Acid Sequence (SEQ ID NO: 550)
TABLE-US-00334 QVQLVQSGAEVKKPGSSVKVSCKASGGFFSSYAISWVRQAPGQGLEWMGG
VIPIFRTANYAQNFQGRVTITADEFTSYMELSSLRSDDTAVYYCARLNYH
DSGTYYNAPRGWFDPWGQGTLVTVSS
[0596] CR6342 Light Chain Nucleotide Sequence (SEQ ID NO: 554)
TABLE-US-00335 gacatccaga tgacccagtc tccagactcc ctggctgtgt
ctctgggcga gaaggccacc 60 atcaactgca agtccagcca gagtatttta
aacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccagg
acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg
tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240
atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtagt
300 ccgccgacgt tcggccaagg gaccaaggtg gaaatcaaac gtgcggccgc
acccagcgtg 360 ttcatcttcc ccccctccga cgagcagctg aagagcggca
ccgccagcgt ggtgtgcctg 420 ctgaacaact tctacccccg ggaggccaag
gtgcagtgga aggtggacaa cgccctgcag 480 agcggcaaca gccaggagag
cgtgaccgag caggacagca aggactccac ctacagcctg 540 agcagcaccc
tcaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600
gtgacccacc agggcctgag cagccccgtg accaagagct tcaaccgggg cgagtgt
657
[0597] CR6342 Light Chain Amino Acid Sequence (SEQ ID NO: 555)
TABLE-US-00336 DIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSS
PPTFGQGTKVEIKRAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC
[0598] CR6342 VL Amino Acid Sequence (SEQ ID NO: 553)
TABLE-US-00337 DIQMTQSPDSLAVSLGEKATINCKSSQSILNSSNNKNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSS
PPTFGQGTKVEIKR
[0599] The CR6343 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 556) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 557 and the heavy chain
amino acid sequence shown in SEQ ID NO: 558. The CR6343 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 559) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 560 and the light chain amino acid sequence shown in SEQ
ID NO: 561.
[0600] CR6343 Heavy Chain nucleotide sequence (SEQ ID NO: 557)
TABLE-US-00338 caggtccagc tggtgcagtc tggagctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagt caccttcagt
tactatgcta tgagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggagga atcagcccta tgtttgggac aacaacctac 180 gcacagaagt
tccagggcag agtcacgatt actgcggacg actccacgag tacagcctac 240
atggaggtga ggagcctgag atctgaggac acggccgtgt attactgtgc gagatcttcg
300 aattactatg atagtgtata tgactactgg ggccagggaa ccctggtcac
cgtctcgagt 360 gctagcacca agggccccag cgtgttcccc ctggccccca
gcagcaagag caccagcggc 420 ggcacagccg ccctgggctg cctggtgaag
gactacttcc ccgagcccgt gaccgtgagc 480 tggaacagcg gcgccttgac
cagcggcgtg cacaccttcc ccgccgtgct gcagagcagc 540 ggcctgtaca
gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 600
tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaaacg cgtggagccc
660 aagagctgcg acaagaccca cacctgcccc ccctgccctg cccccgagct
gctgggcgga 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc
tcatgatcag ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgagc
cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 agcacctacc
gggtggtgag cgtgctcacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ccatcgagaa gaccatcagc
1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgccccccag
ccgggaggag 1080 atgaccaaga accaggtgtc cctcacctgt ctggtgaagg
gcttctaccc cagcgacatc 1140 gccgtggagt gggagagcaa cggccagccc
gagaacaact acaagaccac cccccctgtg 1200 ctggacagcg acggcagctt
cttcctgtac agcaagctca ccgtggacaa gagccggtgg 1260 cagcagggca
acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1320
cagaagagcc tgagcctgag ccccggcaag 1350
[0601] CR6343 Heavy Chain Amino Acid Sequence (SEQ ID NO: 558)
TABLE-US-00339 QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGG
ISPMFGTTTYAQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSS
NYYDSVYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0602] CR6343 VH Amino Acid Sequence (SEQ ID NO: 556)
TABLE-US-00340 QVQLVQSGAEVKKPGSSVKVSCKASGVTFSYYAMSWVRQAPGQGLEWMGG
ISPMFGTTTYAQKFQGRVTITADDSTSTAYMEVRSLRSEDTAVYYCARSS
NYYDSVYDYWGQGTLVTVSS
[0603] CR6343 Light Chain Nucleotide Sequence (SEQ ID NO: 560)
TABLE-US-00341 cagtctgtcg tgacgcagcc gccctcggag tcagtggccc
caggacagac ggccaggatt 60 acctgtgggg gacataacat tggaagtaat
agtgtgcact ggtaccagca gaagccaggc 120 caggcccctg tgctggtcgt
gtatgataat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct
ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240
gatgaggccg actattactg tcaggtgtgg ggtagtagta gtgaccatta tgtcttcgga
300 actgggacca aggtcaccgt cctaggtgcg gccgcaggcc agcccaaggc
cgctcccagc 360 gtgaccctgt tccccccctc ctccgaggag ctgcaggcca
acaaggccac cctggtgtgc 420 ctcatcagcg acttctaccc tggcgccgtg
accgtggcct ggaaggccga cagcagcccc 480 gtgaaggccg gcgtggagac
caccaccccc agcaagcaga gcaacaacaa gtacgccgcc 540 agcagctacc
tgagcctcac ccccgagcag tggaagagcc accggagcta cagctgccag 600
gtgacccacg agggcagcac cgtggagaag accgtggccc ccaccgagtg cagc 654
[0604] CR6343 Light Chain Amino Acid Sequence (SEQ ID NO: 561)
TABLE-US-00342 QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVL
VVYDNSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW
GSSSDHYVFGTGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANK
ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA
SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
[0605] CR6343 VL Amino Acid Sequence (SEQ ID NO: 559)
TABLE-US-00343 QSVVTQPPSESVAPGQTARITCGGHNIGSNSVHWYQQKPGQAPVL
VVYDNSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW
GSSSDHYVFGTGTKVTVLG
[0606] The CR6344 HA-specific IgG antibody includes a heavy chain
variable region (SEQ ID NO: 562) encoded by the heavy chain
nucleotide sequence shown in SEQ ID NO: 563 and the heavy chain
amino acid sequence shown in SEQ ID NO: 564. The CR6344 HA-specific
IgG antibody also includes a light chain variable region (SEQ ID
NO: 565) encoded by the light chain nucleotide sequence shown in
SEQ ID NO: 566 and the light chain amino acid sequence shown in SEQ
ID NO: 567.
[0607] CR6344 Heavy Chain nucleotide sequence (SEQ ID NO: 563)
TABLE-US-00344 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgagagtc 60 tcctgcaagg cttctggaag catcttcaga
aactatgcta tgagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg
gatgggaggg atcatcgcta tttttgggac accaaagtac 180 gcacagaagt
tccagggcag agccacgatt accgcggacg aatcgacgag cactgtctac 240
atggaactga gcggactgag atctgaggac acggccatgt attactgtgc gaggattccc
300 cactataatt ttggttcggg gagttatttc gactactggg gccagggaac
cctggtcacc 360 gtctcgagtg ctagcaccaa gggccccagc gtgttccccc
tggcccccag cagcaagagc 420 accagcggcg gcacagccgc cctgggctgc
ctggtgaagg actacttccc cgagcccgtg 480 accgtgagct ggaacagcgg
cgccttgacc agcggcgtgc acaccttccc cgccgtgctg 540 cagagcagcg
gcctgtacag cctgagcagc gtggtgaccg tgcccagcag cagcctgggc 600
acccagacct acatctgcaa cgtgaaccac aagcccagca acaccaaggt ggacaaacgc
660 gtggagccca agagctgcga caagacccac acctgccccc cctgccctgc
ccccgagctg 720 ctgggcggac cctccgtgtt cctgttcccc cccaagccca
aggacaccct catgatcagc 780 cggacccccg aggtgacctg cgtggtggtg
gacgtgagcc acgaggaccc cgaggtgaag 840 ttcaactggt acgtggacgg
cgtggaggtg cacaacgcca agaccaagcc ccgggaggag 900 cagtacaaca
gcacctaccg ggtggtgagc gtgctcaccg tgctgcacca ggactggctg 960
aacggcaagg agtacaagtg caaggtgagc aacaaggccc tgcctgcccc catcgagaag
1020 accatcagca aggccaaggg ccagccccgg gagccccagg tgtacacccc
gccccccagc 1080 cgggaggaga tgaccaagaa ccaggtgtcc ctcacctgtc
tggtgaaggg cttctacccc 1140 agcgacatcg ccgtggagtg ggagagcaac
ggccagcccg agaacaacta caagaccacc 1200 ccccctgtgc tggacagcga
cggcagcttc ttcctgtaca gcaagctcac cgtggacaag 1260 agccggtggc
agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 1320
cactacaccc agaagagcct gagcctgagc cccggcaag 1359
[0608] CR6344 Heavy Chain Amino Acid Sequence (SEQ ID NO: 564)
TABLE-US-00345 QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQ
GLEWMGGIIAIFGTPKYAQKFQGRVTITADESTSTVYMELSGL
RSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK
[0609] CR6344 VH Amino Acid Sequence (SEQ ID NO: 562)
TABLE-US-00346 QVQLVQSGAEVKKPGSSVRVSCKASGSIFRNYAMSWVRQAPGQ
GLEWMGGIIAIFGTPKYAQKFQGRVTITADESTSTVYMELSGL
RSEDTAMYYCARIPHYNFGSGSYFDYWGQGTLVTVSS
[0610] CR6344 Light Chain Nucleotide Sequence (SEQ ID NO: 566)
TABLE-US-00347 actgtgttga cacagccgcc ctcagtgtct ggggccccag
ggcagagggt caccatctcc 60 tgcactggga gcagctccaa catcggggca
ggttatgatg tacactggta ccagcagctt 120 ccaggaacag cccccaaact
cctcatctat ggtaacagca atcggccctc aggggtccct 180 gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240
actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgcttatgtc
300 ttcggaactg ggaccaaggt caccgtccta ggtgcggccg caggccagcc
caaggccgct 360 cccagcgtga ccctgttccc cccctcctcc gaggagctgc
aggccaacaa ggccaccctg 420 gtgtgcctca tcagcgactt ctaccctggc
gccgtgaccg tggcctggaa ggccgacagc 480 agccccgtga aggccggcgt
ggagaccacc acccccagca agcagagcaa caacaagtac 540 gccgccagca
gctacctgag cctcaccccc gagcagtgga agagccaccg gagctacagc 600
tgccaggtga cccacgaggg cagcaccgtg gagaagaccg tggcccccac cgagtgcagc
660
[0611] CR6344 Light Chain Amino Acid Sequence (SEQ ID NO: 567)
TABLE-US-00348 TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK
LLIYGNSNRPSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGTW
DSSLSAYVFGTGTKVTVLGAAAGQPKAAPSVTLFPPSSEELQANKA
TLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS
YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
[0612] CR6344 VL Amino Acid Sequence (SEQ ID NO: 565)
TABLE-US-00349 TVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK
LLIYGNSNRPSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGTW
DSSLSAYVFGTGTKVTVLG
HA Antibody Epitopes
[0613] The invention relates to an isolated human HA antibody that
is able to recognize and bind to an epitope in the HA2 subunit of
the influenza haemagglutinin protein (HA) (also known as
hemagglutinin (HA)), characterized in that the HA antibody has
neutralizing activity against an influenza virus 5 including HA of
the H5 subtype. Examples of influenza strains that contain such a
HA of the H5 subtype and that are important strains in view of
pandemic threats are H5N1, H5N2, H5N8, and H5N9. Particularly
preferred are HA antibodies that at least neutralize the H5N1
influenza strain. Preferably, an HA antibody of the invention does
not depend on an epitope in the HA1 subunit of the HA protein for
binding to said HA protein.
[0614] A number of the antibodies of the invention (such as CR6307
and CR6323) do not depend on conformational epitopes and recognize
the HA2 epitope even in a reduced form (when used in
western-blotting). This is an advantage over the antibodies from
the art because when a conformational change is induced in the HA
protein due to whatever mutation in another part of the protein,
such conformational change will not most likely hamper the binding
of the antibodies of the present invention to the HA2 epitope,
whereas antibodies that do depend on conformation might very well
be unable to bind when such mutations occur.
[0615] In another preferred embodiment, an HA antibody of the
invention also has neutralizing activity against an influenza virus
comprising HA of the H1 subtype, and preferably wherein the HA
antibody also has neutralizing activity against influenza virus
comprising HA of the H2, H6 and/or H9 subtype. The HA antibodies of
the invention interact with an epitope present in the HA2 epitopes
present in the H5, H1, H2, H6, and H9 subtypes (see, International
Patent Application PCT/EP2007/059356, published as WO 2008/028946,
the contents of which are incorporated by reference in their
entirety), and it has been shown that the HA antibodies of the
invention cross-neutralize between influenza subtypes because of
this epitope-sharing.
[0616] In another preferred aspect of the invention an HA antibody
of the invention binds to an epitope that is selected from the
group consisting of the amino acid sequence: GVTNKVNSIIDK (SEQ ID
NO: 198), GVTNKVNSIINK (SEQ ID NO: 283), GVTNKENSIIDK (SEQ ID NO:
202), GVTNKVNRIIDK (SEQ ID NO: 201), GITNKVNSVIEK (SEQ ID NO: 281),
GITNKENSVIEK (SEQ ID NO: 257), GITNKVNSIIDK (SEQ ID NO: 225), and
KITSKVNNIVDK (SEQ ID NO: 216). Certain HA antibodies of the
invention, CR6261, CR6325, and CR6329 interact with the
GVTNKVNSIIDK (SEQ ID NO: 198) epitope present in H5N1, and are not
hampered by a mutation in the TGLRN (SEQ ID NO: 200) epitope in HA1
that do influence the binding of C179. Moreover, some HA
antibodies, such as CR6307 and CR6323 are not even hampered by a
escape mutant, as disclosed in Okuno et al. (1993) with a
valine->glutamic acid mutation at position 6 (exemplified by
GVTNKENSIIDK (SEQ ID NO: 202)). This epitope is part of an extended
alpha helix in the HA2 region. The residues in this putative
epitope that are predicted to be most solvent exposed are
underlined in bold: GVTNKENSIIDK (SEQ ID NO: 202). These amino
acids would be most accessible to an HA antibody and thus may form
the most important region of the epitope. Consistent with this
notion the highlighted (bolded) amino acids are absolutely
conserved in identity and position in all the sequences presented.
This knowledge could be used to predict binding epitopes in
influenza subtypes that do not carry the same sequence as above
(i.e. H3, H7 and B strains).
HA Antibodies II
[0617] The invention provides neutralizing human monoclonal
antibodies that bind influenza A virus and inhibit the influenza A
virus from infecting a cell. Although neutralizing human monoclonal
antibodies of the invention bind epitopes within proteins that are
exposed on the surface of an influenza virus, the invention focuses
on the relatively invariant Influenza hemagglutinin (HA) protein. A
neutralizing MAb raised against an Influenza HA protein, which is
maintained in its native conformation, provides a superior therapy
for all Influenza A strains because it is not dependent upon small
changes to the amino acid sequence.
[0618] The Influenza hemagglutinin (HA) protein is responsible for
allowing the virus to recognize target cells through binding the
monosaccharide sialic acid-containing receptors on the surface of
the target cell prior to infection. Moreover, the Influenza HA
protein is responsible for allowing entry of the viral genome into
the target cell by fusing the host endosomal membrane with the
viral membrane.
[0619] The Influenza hemagglutinin (HA) protein is a homotrimeric
integral membrane glycoprotein found on the surface of the
Influenza virus. Using the host cell's protein synthesis machinery,
the Influenza HA protein is first synthesized as a single-chain
precursor polypeptide (HA0) in the endoplasmic reticulum, where it
is also assembled as a homotrimer. The resulting HA homotrimer is
subsequently exported to the cell surface via the Golgi network. HA
homotrimers located on a cell surface are cleaved by a
host-produced protease into two smaller peptide subunits: HA1 and
HA2. The HA2 subunit forms a long helical chain anchored to the
viral membrane whereas the HA1 subunit tops the HA2 subunit to form
a large globule. The cleavage step, which converts the HA0
precursor into the mature HA protein containing HA1 and HA2
subunits, is essential for the viral pathogenicity of Influenza.
Structurally, the mature HA protein contains a central
.alpha.-helix coil resulting in an overall cylindrical shape with
three spherical heads. The HA protein, and specifically, the HA1
subunit of the mature HA protein, binds receptors containing
glycans with terminal sialic acids on host cells. The way in which
sialic acid is connected to galactose, for example, .alpha.2-3
linkages as in avian serotypes versus .alpha.2-6 linkages as in
human serotypes, not only determine species specificity of an
Influenza virus, but also prevents cross-species infection.
However, within certain serotypes of HA, such as H1 and H3, only
two amino acid mutations in the framework sequence are required to
convert species specificity from avian to human.
[0620] To mediate infection, the Influenza HA protein first binds
sialic acid-containing receptors present on the surface of the
target cell. Consequently, the target cell membrane endocytoses or
engulfs the Influenza virus. Once inside the endosome, and upon the
host cell's acidification of that compartment, the Influenza HA
protein partially unfolds revealing a very hydrophobic fusion
peptide that inserts itself into the endosomal membrane. As the
rest of the Influenza HA protein refolds, the fusion protein
retracts and fuses the endosomal membrane with the viral membrane.
Upon fusion of the cellular and viral membranes, the contents of
the virus, including the viral genome, are released in the
cytoplasm of the target cell.
[0621] At least 16 different Influenza A hemagglutinin serotypes or
antigens have been identified: H1-H16. Only HA serotypes H1-H3
normally mediate human Influenza infection. However, Influenza
strains thought to infect only certain avian or mammalian species
can mutate to infect humans. As described above, only a few amino
acids need to change along the length of the entire protein to
enable Influenza to cross a species barrier. For instance, a single
amino acid change in the sequence of the H5 subtype allowed an
avian-specific Influenza strain to become infectious in humans
(H5N1). A pandemic arose when an Influenza strain common to swine
species, became lethal to humans (H1N1). In contrast to Influenza
A, Influenza B and C viruses each contain only one form of HA
protein.
[0622] Specifically, the invention provides an isolated fully human
monoclonal antibody, wherein said monoclonal antibody has the
following characteristics: a) binds to an influenza A virus; b)
binds to a cell contacted with influenza A; c) binds to an epitope
of an influenza A viral protein; and, optionally, d) neutralizes
influenza A virus infection. An antibody that does not neutralize
influenza A virus infection may be used, for instance, for a
conjugate therapy. In certain aspects, this antibody binds to a
eukaryotic cell. Moreover, the cell is optionally a human cell.
[0623] In another aspect, this antibody is isolated from a B-cell
from a human donor. Isolation of a fully human monoclonal antibody
of the invention from a B-cell is performed using recombinant
methods. Alternatively, or in addition, the isolated fully human
monoclonal antibody of the invention is isolated from the
supernatant of a plasma cell cultured either in vitro or ex vivo.
Plasma cells also known as a differentiated B-cells, plasma
B-cells, plasmacytes, or effector B-cells. The fully human
monoclonal antibody isolated from either a B-cell or a plasma cell
demonstrates neutralizing activity.
[0624] Antibodies of the invention bind to an epitope of influenza
A viral hemagglutinin (HA) protein. Exemplary HA epitopes to which
the antibodies of the invention bind include a hemagglutinin
precursor peptide (HA0), a HA1 subunit, a HA2 subunit, a mature
protein containing HA1 and HA2, and a recombinant HA polypeptide.
Alternatively, antibodies of the invention bind to an epitope
within a hemagglutinin precursor peptide (HA0), a HA1 subunit, a
HA2 subunit, a mature protein containing HA1 and HA2, or a
recombinant HA polypeptide. Recombinant HA polypeptides are
encoded, for example, by the sequence of SEQ ID NO: 727, 728, 729,
730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742,
743, or 744.
[0625] Antibodies of the invention bind to an epitope that is
linear or non-linear. In certain aspects of the invention, a
non-linear epitope is a discontinuous epitope.
[0626] An antibody of the invention is TCN-522 (3212_I12), TCN-521
(3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526
(5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529
(5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532
(5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535
(5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538
(3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541
(5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544
(5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547
(5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550
(5096_F06), TCN-551 (5243_D01), TCN-552 (5249.sub.--1123), TCN-553
(5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556
(5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559
(5097_G08), TCN-560 (5084_P10), or TCN-504 (3251_K17).
[0627] The invention further encompasses an antibody that binds the
same epitope as TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523
(5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527
(5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530
(5248_H10a), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533
(5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536
(5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539
(5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542
(5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545
(5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548
(5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551
(5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554
(5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557
(5081_A04), TCN-558 (5248_H10b), TCN-559 (5097_G08), TCN-560
(5084_P10), or TCN-504 (3251_K17).
[0628] The invention provides an isolated fully human monoclonal
anti-HA antibody or fragment thereof, wherein said antibody
includes a variable heavy chain (V.sub.H) region comprising CDR1
and CDR2, wherein the V.sub.H region is encoded by a human IGHV1
(or specifically, IGHV1-18, IGHV1-2, IGHV1-69, IGHV1-8), IGHV2 (or
specifically, IGHV2-5), IGHV3 (or specifically, IGHV3-30, IGHV3-33,
IGHV3-49, IGHV3-53, 66, IGHV3-7), IGHV4 (or specifically, IGHV4-31,
IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61), or IGHV5 (or specifically,
IGHV5-51) V.sub.H germline sequence or an allele thereof, or a
nucleic acid sequence that is homologous to the IGHV1, IGHV2,
IGHV3, IGHV4, or IGHV5 V.sub.H germline gene sequence or an allele
thereof. In one aspect, the nucleic acid sequence that is
homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 V.sub.H
germline sequence is at least 75% homologous to the IGHV1, IGHV2,
IGHV3, IGHV4, or IGHV5 V.sub.H germline sequence or an allele
thereof. Exemplary alleles include, but are not limited to,
IGHV1-18*01, IGHV1-2*02, IGHV1-2*04, IGHV1-69*01, IGHV1-69*05,
IGHV1-69*06, IGHV1-69*12, IGHV1-8*01, IGHV2-5*10, IGHV3-30-3*01,
IGHV3-30*03, IGHV3-30*18, IGHV3-33*05, IGHV3-49*04, IGHV3-53*01,
IGHV3-66*03, IGHV3-7*01, IGHV4-31*03, IGHV4-31*06, IGHV4-34*01,
IGHV4-34*02, IGHV4-34*03, IGHV4-34*12, IGHV4-39*01, IGHV4-59*01,
IGHV4-59*03, IGHV4-61*01, IGHV4-61*08, and IGHV5-51*01.
[0629] An antibody of the invention, or specifically, any antibody
described herein, may be operably-linked to a therapeutic agent or
a detectable label.
[0630] The invention further provides a pharmaceutical composition
including an antibody described herein and a pharmaceutical
carrier. This composition optionally includes an anti-viral drug, a
viral entry inhibitor or a viral attachment inhibitor. Exemplary
anti-viral drugs include, but are not limited to, a neuraminidase
inhibitor, a HA inhibitor, a sialic acid inhibitor and an M2 ion
channel inhibitor. In one embodiment of the composition, the M2 ion
channel inhibitor is amantadine or rimantadine. Alternatively, or
in addition, the neuraminidase inhibitor zanamivir or oseltamivir
phosphate. The composition may also include a second anti-Influenza
A antibody. The second anti-Influenza A antibody is optionally an
antibody described herein.
[0631] The invention provides a method for stimulating an immune
response in a subject, including administering to the subject the
pharmaceutical composition described herein.
[0632] Moreover, the invention provides a method for the treatment
of an Influenza virus infection in a subject, including
administering to the subject the pharmaceutical composition
described herein. This method further includes administering an
anti-viral drug, a viral entry inhibitor or a viral attachment
inhibitor.
[0633] The invention also provides a method for the prevention of
an Influenza virus infection in a subject, including administering
to the subject the pharmaceutical composition described herein
prior to exposure of the subject to Influenza virus or infection.
This method further includes administering an anti-viral drug, a
viral entry inhibitor or a viral attachment inhibitor. This method
may be a method of vaccination.
[0634] The subject of these methods may have an Influenza infection
or is predisposed to developing an Influenza virus infection.
Subjects predisposed to developing an Influenza infection, or who
may be at elevated risk for contracting an infection, are those
subjects with compromised immune systems because of autoimmune
disease, those persons receiving immunosuppressive therapy (for
example, following organ transplant), those persons afflicted with
human immunodeficiency syndrome (HIV) or acquired immune deficiency
syndrome (AIDS), certain forms of anemia that deplete or destroy
white blood cells, those persons receiving radiation or
chemotherapy, or those persons afflicted with an inflammatory
disorder. Additionally, subject of extreme young or old age are at
increased risk. Any person who comes into physical contact or close
physical proximity with an infected individual has an increased
risk of developing an Influenza virus infection. Moreover, a
subject is at risk of contracting an influenza infection due to
proximity to an outbreak of the disease, e.g. subject resides in a
densely-populated city or in close proximity to subjects having
confirmed or suspected infections of Influenza virus, or choice of
employment, e.g. hospital worker, pharmaceutical researcher,
traveler to infected area, or frequent flier.
[0635] According to the methods described herein, exemplary
anti-viral drugs include, but are not limited to, a neuraminidase
inhibitor, a HA inhibitor, a sialic acid inhibitor and an M2 ion
channel. In one aspect of these methods, the M2 ion channel
inhibitor is amantadine or rimantadine. Alternatively, or in
addition, the neuraminidase inhibitor is zanamivir or oseltamivir
phosphate.
[0636] These methods optionally include administering a second
anti-Influenza A antibody. For example, the antibody is
administered prior to or after exposure to Influenza virus. In
certain aspects of these methods, the antibody is administered at a
dose sufficient to promote viral clearance or to eliminate
Influenza A infected cells. The second antibody is optionally an
antibody described herein.
[0637] The invention further provides a method for determining the
presence of a Influenza virus infection in a subject, including the
steps of: (a) contacting a biological sample obtained from the
subject with an antibody described herein or the pharmaceutical
composition described herein; (b) detecting an amount of the
antibody that binds to the biological sample; and (c) comparing the
amount of antibody that binds to the biological sample to a control
value, and therefrom determining the presence of the Influenza
virus in the subject.
[0638] The invention provides a vaccine composition including an
antibody described herein. This composition optionally contains a
pharmaceutical carrier.
[0639] Alternatively, the invention provides a vaccine composition
including an epitope of an antibody described herein. This
composition optionally contains a pharmaceutical carrier.
[0640] Vaccines of the invention are multivalent vaccines. The term
"multivalent vaccine" is meant to describe a single vaccine that
elicits an immune response either to more than one infectious
agent, e.g. recombinant homotrimeric HA0 proteins or fragments
thereof derived from multiple strains of Influenza A (see, Table
9), or to several different epitopes of a molecule, e.g. a linear
and a discontinuous epitope of the same recombinant homotrimeric
HA0 protein or fragment thereof derived from a single strain of
Influenza A. Alternatively, or in addition, the term multivalent
vaccine is meant to describe the administration of a combination of
human antibodies raised against more than one infectious agent,
e.g. a combination of HuMHA antibodies raised against recombinant
homotrimeric HA0 proteins or fragments thereof derived from
multiple strains of Influenza A (see, Table 9).
[0641] The invention provides a diagnostic kit including an
antibody described herein.
[0642] The invention provides a prophylactic kit including an
antibody described herein or an epitope of an antibody described
herein. Alternatively, or in addition, the invention provides a
prophylactic kit including a vaccine composition described
herein.
[0643] In a preferred embodiment, the present invention provides
fully human monoclonal antibodies specifically directed against the
Influenza hemagglutinin glycoprotein, which neutralize influenza
infection. Optionally, the antibody is isolated from a B-cell from
a mammalian donor, and preferably, a human donor. In certain
embodiments of the invention, the antibody is identified for its
ability to bind an intact or whole Influenza virus. Alternatively,
or in addition, the antibody is identified isolated for its ability
to bind to an epitope of a recombinant homotrimeric Influenza HA0
protein or HA protein(s) isolated from multiple Influenza strains,
or made as recombinant proteins such as those influenza A virus
strains provided in Table 9. Alternatively, or in addition, the
antibody is identified for its ability to inhibit or neutralize
virus infection of susceptible eukaryotic cells. Exemplary
neutralizing antibodies of this profile include, but are not
limited to, those antibodies listed in Table 10. Alternatively, the
monoclonal antibody is an antibody that binds to the same epitope
as the antibodies provided in Table 10. In certain embodiments,
neutralizing human monoclonal antibodies of the invention are
anti-HA antibodies. A monoclonal anti-HA antibody of the invention
has one or more of the following characteristics: a) binds to an
epitope in an HA1 subunit of an Influenza hemagglutinin (HA)
protein; b) binds to an epitope in the HA2 subunit of Influenza
hemagglutinin (HA) protein; c) binds to an epitope in the
extracellular domain of an Influenza hemagglutinin (HA) protein,
consisting of an HA1 subunit and an HA2 subunit; d) binds to an
epitope of a recombinant homotrimeric Influenza HA0 protein; e)
binds to an epitope of an Influenza HA protein expressed on an
infected cell; f) binds to an epitope of an Influenza HA protein
expressed on a modified cell; g) binds to an Influenza virus; or h)
inhibits virus infection of susceptible eukaryotic cells.
[0644] Modified cells of the invention are transfected or
transformed with a polynucleotide that encodes an Influenza HA
protein, or any fragment thereof. The term "Influenza HA protein
fragment" is meant to describe any portion of the protein that is
smaller or less than the entire protein. Polynucleotides and
polypeptides of the invention do not always encode a functional
Influenza HA protein.
[0645] Infected cells of the invention are mammalian, and
preferably human in origin. Specifically, mammalian cells are
infected with Influenza A virus in vivo, in vitro, in situ, ex
vivo, in culture, and any combination thereof. Cells are infected
with active or inactive virions. Exemplary inactive virions display
the HA protein on their surfaces, however, they are
replication-defective, and therefore, unable to propagate within
the cell or subject.
[0646] Epitopes of the human monoclonal antibodies of the invention
include a transmembrane or integral membrane Influenza A protein.
Specifically, epitopes of the human monoclonal antibodies of the
invention comprise Influenza hemagglutinin (HA) protein.
[0647] Epitopes of the human monoclonal antibodies of the invention
include one or more subunits of an influenza hemagglutinin (HA)
protein. HA proteins of the invention include hemagglutinin
precursor proteins (HA0), the HA1 subunit, the HA2 subunit, the
mature protein containing the HA1 and HA2 subunits, and a
recombinant HA protein. Recombinant HA proteins contain SEQ ID NO:
726. Exemplary recombinant proteins include but, are not limited
to, those proteins described by SEQ ID NO: 727-744.
[0648] Epitopes of the human monoclonal antibodies of the invention
are linear or non-linear. For instance, a non-linear epitope is
discontinuous. Discontinuous epitopes are available for antibody
binding only when the Influenza HA protein is maintained in its
native homotrimeric conformation. When an antibody binds to a
discontinuous epitope, the antibody binds to a three-dimensional
surface of the target protein, i.e. the Influenza HA protein, upon
which juxtaposed amino acids are alternatively exposed or
masked.
[0649] Recombinant homotrimeric HA0 proteins of the invention are
encoded by, for instance, sequences described by any one of SEQ ID
NO: 727-744. In certain embodiments of the invention, the human
monoclonal antibodies, or monoclonal anti-HA antibodies, described
herein bind membrane-bound or soluble recombinant homotrimeric
Influenza HA proteins. Alternatively, the monoclonal anti-HA
antibodies described herein bind membrane-bound and soluble
recombinant homotrimeric Influenza HA proteins. In certain
embodiments of the invention, the monoclonal anti-HA antibodies
described herein bind and neutralize Influenza virus subtypes H1,
H2, and H3. In other embodiments of the invention, the monoclonal
anti-HA antibodies bind Influenza virus subtypes H1, H2, and H3,
and neutralize one of these subtypes, such as H1, H2, or H3. In a
specific embodiment, the monoclonal anti-HA antibodies bind
Influenza subtypes H1N1, H2N2, and H3N2, and neutralize H1N1.
[0650] In one aspect, the HA precursor polypeptide (HA0) of the
soluble and recombinant homotrimeric Influenza HA protein contains
a trimerization domain (foldon) encoded in the phage T4 fibritin.
An exemplary trimerization domain isolated from the phage T4
fibritin has the following sequence wherein a thrombin cleavage
site is italicized and bolded, a T4 trimerization domain or
sequence is underlined, a V5 tag is boxed, and a hexa-histidine
(His) tag is bolded:
TABLE-US-00350 (SEQ ID NO: 726) SGR PGSGYIPEAPRDGQAYVRKDGEWVLLSTFL
HHHHHH.
[0651] As used herein, the term "neutralizing antibody" is meant to
describe an antibody that inhibits or prevents influenza A
infection, inhibits or prevents Influenza A viral entry into a
cell, inhibits or prevents influenza replication, inhibits or
prevents influenza egress from a host cell, or reduces the
Influenza A titer in a cell, biological sample, or subject. In a
preferred embodiment, neutralizing antibodies of the invention
prevent viral entry into the cytoplasmic compartment of host
cells.
[0652] The present invention provides fully human monoclonal
antibodies that bind influenza virus and neutralize infection. In
certain embodiments, the present invention provides fully human
monoclonal neutralizing antibodies specific against the Influenza
hemagglutinin protein. The antibodies are respectively referred to
herein is human monoclonal anti-HA (huMHA) antibodies.
[0653] The Influenza hemagglutinin (HA) protein is a homotrimeric
integral membrane glycoprotein found on the surface of the
Influenza virus. To mimic the native conformation of this
homotrimeric protein, the methods of the invention provide an
isolated HA protein precursor that is operably-linked to a
trimerization or foldon domain from the phage T4 fibritin
protein
TABLE-US-00351 (SGR PGSGYIPEAPRDGQAYVRKDGEWVLLSTFL HHHHHH (SEQ ID
NO: 726)).
[0654] The resultant recombinant homotrimeric foldon HA protein not
only retains the native Influenza hemagglutinin homotrimeric
conformation, but also becomes soluble, i.e. the protein is no
longer bound to a viral or cellular membrane. Specifically, these
recombinant HA homotrimeric proteins lack an integral membrane or
transmembrane domain. In certain embodiments, these recombinant HA
homotrimeric proteins include HA1 and HA2 subunits as well as a
trimerization domain, the resultant recombinant HA homotrimeric
protein containing between 1-50, 50-100, 100-150, 150-200, 200-250,
250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600 amino
acids (aa) or any length of amino acids in between. Preferably,
these recombinant HA homotrimeric proteins contain between 565-575
amino acids (aa). Recombinant HA homotrimeric proteins further
include a signal cleavage site at the N-terminus containing between
15-25 aa. Alternatively, or in addition, recombinant HA
homotrimeric proteins further include a transmembrane domain
positioned between amino acids 525-535 of HA depending on the
influenza A virus subtype. In a preferred embodiment, the HA
protein is derived from one or more strains of an Influenza A
virus. Recombinant HA homotrimeric proteins of the invention retain
the native signal sequence to enable secretion. Moreover,
recombinant HA homotrimeric proteins of the invention contain a
same signal sequence, which is not derived from HA. Furthermore,
signal sequences used with recombinant HA homotrimeric proteins of
the invention include those signal sequences known in the art that
allow efficient secretion of proteins, such as the signal sequence
of the immunoglobulin light kappa chain. Alternatively, recombinant
HA homotrimeric proteins, or the HA0 precursors thereof, may have
the native signal sequences in the expression constructs used by
Immune Technology Corp. 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.
[0655] Recombinant HA homotrimeric proteins may retain a native
HA1/HA2 protease cleavage site, which is critical for viral
pathogenicity. In one aspect of the invention, recombinant HA
homotrimeric proteins contain a substituted HA1/HA2 protease
cleavage site. For example, the recombinant HA protein encoded by
SEQ ID NO: 737 does not have a native cleavage site, but rather a
cleavage site substituted from another HA protein. Furthermore,
these proteins optionally retain sialic acid-containing receptor
binding sites within the HA1 subunit.
[0656] According to the methods of the invention, human antibodies
obtained from blood, serum, plasma, or cerebral spinal fluid, are
contacted to recombinant and soluble HA homotrimers of the
invention in vitro, wherein the recombinant and soluble HA
homotrimers act as targets for human antibody binding to confirm
specificity of the isolated human antibody for an Influenza HA
homotrimer in its native conformation. In general, the methods
include obtaining serum or plasma samples from subjects or patients
that have been infected with or vaccinated against an infectious
agent. These serum or plasma samples are then screened to identify
those that contain antibodies specific for a particular polypeptide
associated with the infectious agent, such as, e.g. a polypeptide
specifically expressed on the surface of cells infected with the
infectious agent, but not uninfected cells. In particular
embodiments, the serum or plasma samples are screened by contacting
the samples with a cell that has been transfected with an
expression vector that expresses the polypeptide expressed on the
surface of infected cells. In particular embodiments the serum or
plasma samples are screened by contacting the samples with a
recombinant protein which represents a particular protein of the
infectious agent such as, e.g. hemagglutinin of the influenza A
virus. In particular embodiments the serum or plasma samples are
screened by contacting the samples with a purified form of the
infectious agent such as, e.g. intact whole virions of the
influenza A virus. In particular embodiments, the serum or plasma
samples are screened by contacting the samples with a live form of
the infectious agent such as, e.g. intact whole virions of the
influenza A virus to determine the presence of serum antibodies
that inhibit or neutralize infection of susceptible cells.
Exemplary susceptible cells are eukaryotic or mammalian cells, such
as MDCK cells.
[0657] Once a subject or patient is identified as having serum or
plasma containing an antibody specific for the infectious agent
polypeptide or virus of interest, mononuclear and/or B cells
obtained from the same subject or patient are used to identify a
cell or clone thereof that produces the antibody, using any of the
methods described herein or available in the art. Once a B cell
that produces the antibody is identified, cDNAs encoding the
variable regions or fragments thereof of the antibody may be cloned
using standard RT-PCR vectors and primers specific for conserved
antibody sequences, and subcloned into expression vectors used for
the recombinant production of monoclonal antibodies specific for
the infectious agent polypeptide of interest.
[0658] More specifically, B cells are collected from a particular
donor, i.e. a subject or patient is identified as having serum or
plasma containing an antibody specific for HA, cultured, and
antibody is secreted from these B cells into the culture medium.
The culture medium is separated from these B cells, the B cells are
lysed, and then frozen for storage. The culture medium is then
screened for antibody binding to various HA targets and/or
inhibition/neutralization of infection in vitro. When a culture
well is identified as having an antibody of the desired
specificity, reverse-transcriptase polymerase chain reaction
(RT-PCR) is applied to the B-cell lysate to amplify the antibody
variable regions and subsequently clone, express, and test for
binding and function of the recombinant antibody,
[0659] Human antibodies, such as the MAbs listed in Table 10, which
bind the recombinant and soluble HA homotrimer and/or bind whole
virions, and optionally inhibit or neutralize infection of live
virus are recombinantly reproduced and formulated into a
pharmaceutical composition for administration to a subject at risk
of contacting an Influenza virus. Furthermore, recombinant and
soluble HA homotrimers are derived from multiple strains of
Influenza viruses, including multiple strains of influenza A virus.
Exemplary human antibodies specifically bind Influenza A, and may
be selected for an inability to bind influenza B and C virus
strains.
[0660] The invention further provides a novel process whereby
full-length HA is expressed in mammalian cell lines, which allows
for the identification of human antibodies that bind this
cell-expressed HA. The huMHA antibodies have been shown to bind
conformational determinants on the HA-transfected cells, as well as
native HA, which can be isolated, or contacted to huMHA antibodies
when presented either on Influenza infected cells or on Influenza A
virus. Alternatively, or in addition, huMHA antibodies bind native
HA, recombinant homotrimeric HA, purified virus, infected cells,
linear peptide, synthetic HA peptide, HA transfected mammalian
cells, and HA expressed on the surface of genetically altered
bacteriophage virus, which are used for gene fragment display
assays. Thus, this invention has allowed for the identification and
production of human monoclonal antibodies that exhibit novel
specificity for a very broad range of Influenza A virus strains.
These antibodies may be used prophylactically to prevent Influenza
A infection, diagnostically to identify Influenza A infection and
therapeutically to treat Influenza A infection. Moreover, the
epitopes to which huMHA antibodies of the invention bind are used
as vaccines to prevent influenza A infection.
[0661] The huMHA antibodies of the invention has one or more of the
following characteristics: a) binds to an epitope in an HA1 subunit
of an Influenza hemagglutinin (HA) protein; b) binds to an epitope
in the HA2 subunit of Influenza hemagglutinin (HA) protein; c)
binds to an epitope in the extracellular domain of an Influenza
hemagglutinin (HA) protein, consisting of an HA1 subunit and an HA2
subunit; d) binds to an epitope of a recombinant homotrimeric
Influenza HA0 protein; e) binds to an epitope of an Influenza HA
protein expressed on an infected cell; 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. The huMHA antibodies of the invention eliminate
Influenza infected cells through immune effector mechanisms such as
ADCC and/or CDC and promote direct viral clearance by binding to
Influenza virions.
[0662] Exemplary Influenza A strains used for screening human
plasma samples, B Cell Culture supernatants (BCC SN), and
monoclonal transfection supernatants (MN are shown in Table 8
below). Live strains were used for the neutralization assays
described herein. Inactivated strains were used for the virus
binding assays described herein. Recombinant homotrimeric HA
protein was used in the trimeric HA binding assay.
TABLE-US-00352 TABLE 8 Virus Trimeric HA Virus Subtype
Neutralization binding binding A/California/4/09 H1 + A/Solomon
Islands/ H1 + + + 3/06 A/South Carolina/1/18 H1 + A/Japan/305/57 H2
+ + A/Wisconsin/67/05 H3 + + + A/swine/Ontario/ H4 + 01911-2/99
A/Vietnam/1203/04 H5 + A/Indonesia/5/05 H5 + A/Egypt/3300- H5 +
NAMRU3/08 A/common magpie/ H5 + Hong Kong/5052/07 A/Anhui/1/05 H5 +
A/chicken/Vietnam/ H5 + NCVD-016/08 A/Hong Kong/156/97 H5 +
A/northern shoveler/ H6 + California/HKWF115/ 07
A/Netherlands/219/03 H7 + A/duck/Yangzhou/ H8 + 02/05 A/Hong
Kong/2108/03 H9 + A/Hong Kong/1073/99 H9 +
[0663] Exemplary HA sequences include those sequences listed on
Table 9 below.
TABLE-US-00353 TABLE 9 GenBank Type Accession No. Subtype HA
Sequence from Strain SEQ ID NO: A ACP41105 H1
A/California/04/2009(H1N1) SEQ ID NO: 727 A ABU99109 H1 A/Solomon
Islands/3/2006(H1N1) SEQ ID NO: 728 A AFI17241 H1 A/South
Carolina/1/18(H1N1) SEQ ID NO: 729 A AAA43185 H2
A/Japan/305/1957(H2N2) SEQ ID NO: 730 A ACF54576 H3
A/Wisconsin/67/2005(H3N2) SEQ ID NO: 731 A AAG17427 H4
A/Swine/Ontario/01911-2/99 (H4N6) SEQ ID NO: 732 A AF028709 H5
A/Hong Kong/156/97 (H5N1) SEQ ID NO: 733 A AAT73274 H5
A/VietNam/1203/2004(H5N1) SEQ ID NO: 734 A ABW06108 H5
A/Indonesia/5/2005(H5N1) SEQ ID NO: 735 A ACI06185 H5
A/Egypt/3300-NAMRU3/2008(H5N1) SEQ ID NO: 736 A ACJ26242 H5
A/common magpie/Hong Kong/5052/2007(H5N1) SEQ ID NO: 737 A ABD28180
H5 A/Anhui/1/2005(H5N1) SEQ ID NO: 738 A ACO07033 H5
A/chicken/Vietnam/NCVD-016/2008(H5N1) SEQ ID NO: 739 A ACE81692 H6
A/northern shoveler/California/HKWF115/2007(H6N1) SEQ ID NO: 740 A
AAR02640 H7 A/Netherlands/219/03(H7N7) SEQ ID NO: 741 A ABK32094 H8
A/duck/Yangzhou/02/2005(H8N4) SEQ ID NO: 742 A ABB58945 H15
A/HK/2108/2003(H9N2) SEQ ID NO: 743 A NC_004908 H9 A/Hong
Kong/1073/99 (H9N2) SEQ ID NO: 744
[0664] In one embodiment, the huMHA antibodies of the invention
bind to an HA that wholly or partially includes the amino acid
residues from position 1 to position 525 of Influenza hemagglutinin
when numbered in accordance with SEQ ID NO: 727-744. Alternatively,
the monoclonal antibody is an antibody that binds to the same
epitope as the mAbs listed in Table 10.
TABLE-US-00354 TABLE 10 BCC Well ID Theraclone ID 3251_K17 TCN-504
3280_D18 TCN-521 3212_I12 TCN-522 5248_A17 TCN-523 5237_B21 TCN-524
5084_C17 TCN-526 5086_C06 TCN-527 5087_P17 TCN-528 5297_H01 TCN-529
5248_H10a TCN-530 5091_H13 TCN-531 5262_H18 TCN-532 5256_A17
TCN-533 5249_B02 TCN-534 5246_P19 TCN-535 5095_N01 TCN-536 3194_D21
TCN-537 3206_O17 TCN-538 5056_A08 TCN-539 5060_F05 TCN-540 5062_M11
TCN-541 5079_A16 TCN-542 5081_G23 TCN-543 5082_A19 TCN-544 5082_I15
TCN-545 5089_L08 TCN-546 5092_F11 TCN-547 5092_P01 TCN-548 5092_P04
TCN-549 5096_F06 TCN-550 5243_D01 TCN-551 5249_I23 TCN-552 5261_C18
TCN-553 5277_M05 TCN-554 5246_L16 TCN-555 5089_K12 TCN-556 5081_A04
TCN-557 5248_H10b TCN-558 5097_G08 TCN-559 5084_P10 TCN-560
[0665] The antibodies of the invention are able to neutralize
Influenza A. Monoclonal antibodies can be produced by known
procedures, e.g., as described by R. Kennet et al. in "Monoclonal
Antibodies and Functional Cell Lines; Progress and Applications".
Plenum Press (New York), 1984. Further materials and methods
applied are based on known procedures, e.g., such as described in
J. Virol. 67:6642-6647, 1993.
[0666] These antibodies can be used as prophylactic or therapeutic
agents upon appropriate formulation, or as a diagnostic tool.
[0667] A "neutralizing antibody" is one that can neutralize the
ability of that pathogen to initiate and/or perpetuate an infection
in a host and/or in target cells in vitro. The invention provides a
neutralizing monoclonal human antibody, wherein the antibody
recognizes an antigen from an Influenza virus, which is preferably
derived from the HA protein. Preferably an antibody according to
the invention is a novel monoclonal antibody referred to herein as
TCN-522 (corresponding to BCC plate and well location 3212_I12),
TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526
(5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529
(5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532
(5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535
(5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538
(3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541
(5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544
(5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547
(5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550
(5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553
(5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556
(5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559
(5097_G08), TCN-560 (5084_P10), and TCN-504 (3251_K17). These
antibodies were initially isolated from human samples and are
produced by the B cell cultures referred to as 3212_I12, 3280_D18,
5248_A17, 5237_B21, 5084_C17, 5086_C06, 5087_P17, 5297_H01,
5248_H10a, 5091_H13, 5262_H18, 5256_A17, 5249_B02, 5246_P19,
5095_N01, 3194_D21, 3206_O17, 5056_A08, 5060_F05, 5062_M11,
5079_A16, 5081_G23, 5082_A19, 5082_I15, 5089_L08, 5092_F11,
5092_P01, 5092_P04, 5096_F06, 5243_D01, 5249_I23, 5261_C18,
5277_M05, 5246_L16, 5089_K12, 5081_A04, 5248_H10b, 5097_G08,
5084_P10, and 3251_K17. These antibodies have broad neutralizing
activity or broad binding activity for Influenza A in vitro.
[0668] The CDRs of the antibody heavy chains are referred to as
CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of the
antibody light chains are referred to as CDRL1, CDRL2 and CDRL3,
respectively. The position of the CDR amino acids is defined
according to the IMGT numbering system as: CDR1--IMGT positions 27
to 38, CDR2--IMGT positions 56 to 65 and CDR3--IMGT positions 105
to 117. (Lefranc, M P. et al. 2003 IMGT unique numbering for
immunoglobulin and T cell receptor variable regions and Ig
superfamily V-like domains. Dev Comp Immunol. 27 (1):55-77;
Lefranc, M P. 1997. Unique database numbering system for
immunogenetic analysis. Immunology Today, 18:509; Lefranc, M P.
1999. The IMGT unique numbering for Immunoglobulins, T cell
receptors and Ig-like domains. The Immunologist, 7:132-136.)
[0669] The sequences of the antibodies were determined, including
the sequences of the variable regions of the Gamma heavy and Kappa
or Lambda light chains of the antibodies designated. In addition,
the sequence of each of the polynucleotides and polypeptides
encoding the antibody sequences was determined for TCN-522
(3212_I12), TCN-521 (3280_D18), TCN-523 (5248_A17), TCN-563
(5237_B21), TCN-526 (5084_C17), TCN-527 (5086_C06), TCN-528
(5087_P17), TCN-529 (5297_H01), TCN-530 (5248_H10a), TCN-531
(5091_H13), TCN-532 (5262_H18), TCN-533 (5256_A17), TCN-534
(5249_B02), TCN-535 (5246_P19), TCN-536 (5095_N01), TCN-537
(3194_D21), TCN-538 (3206_O17), TCN-539 (5056_A08), TCN-540
(5060_F05), TCN-541 (5062_M11), TCN-542 (5079_A16), TCN-543
(5081_G23), TCN-544 (5082_A19), TCN-545 (5082_I15), TCN-546
(5089_L08), TCN-547 (5092_F11), TCN-548 (5092_P01), TCN-549
(5092_P04), TCN-550 (5096_F06), TCN-551 (5243_D01), TCN-552
(5249.sub.--1123), TCN-553 (5261_C18), TCN-554 (5277_M05), TCN-555
(5246_L16), TCN-556 (5089_K12), TCN-557 (5081_A04), TCN-558
(5248_H10b), TCN-559 (5097_G08), TCN-560 (5084_P10), and TCN-504
(3251_K17).
[0670] 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.
[0671] TCN-504 (3251_K17) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00355 (SEQ ID NO: 745)
CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCG
GAGACCCTGTCCCTCACTTGCGCTGTCTCTGGTGTCTCCATCAGC
AATATTGATTTCTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAG
GGGCTAGAATGGATTGGCAATATCTATTATACGGGGATCACCTTC
TACAACCCGTCCCTCAGCAGTCGAGTCGCCATATCCATTGACACC
TCCAAGAACCAGTTCTCCCTGACTCTGACTTCTGTGACCGCCGCA
GACACGGCTATGTATTACTGTGCGAGACATTACGGTGACTCCGAG
GCAATAAACGATGCCTTTGACATCTGGGGCCAAGGGACAATGCTC ACCGTCTCGAGC
[0672] TCN-504 (3251_K17) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00356 (SEQ ID NO: 746)
QVQLQESGPGLVKPSETLSLTCAVSGVSISNIDFYWGWIRQPPGK
GLEWIGNIYYTGITFYNPSLSSRVAISIDTSKNQFSLTLTSVTAA
DTAMYYCARHYGDSEAINDAFDIWGQGTMLTVSS
[0673] TCN-504 (3251_K17) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00357 CDR 1: (SEQ ID NO: 747) NIDFYWG CDR 2: (SEQ ID NO:
748) NIYYTGITFYNPSLSS CDR 3: (SEQ ID NO: 749) HYGDSEAINDAFDI
[0674] TCN-504 (3251_K17) Gamma Heavy Chain Chothia CDRs
TABLE-US-00358 CDR 1: (SEQ ID NO: 750) GVSISN CDR 2: (SEQ ID NO:
751) NIYYTGITF CDR 3: (SEQ ID NO: 749) HYGDSEAINDAFDI
[0675] TCN-504 (3251_K17) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00359 (SEQ ID NO: 752)
GAGATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAATAGTTTAG
CCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGT
GCATCCACCAGGGCCACTGGTATCCCACCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGACTGAAGATTTTG
CAGTTTATTACTGTCAACAATATATTAACTGGCGTCCGCTCAGTTTTGGC
GGAGGGACCAAGGTGGAGATCAAA
[0676] TCN-504 (3251_K17) Light Chain Variable Region Amino Acid
Sequence (KabatCDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00360 (SEQ ID NO: 753)
EIVMTQSPATLSVSPGERATLSCRASQSVGNSLAWYQQRPGQAPRLLIYG
ASTRATGIPPRFSGSGSGTEFTLTISSLQTEDFAVYYCQQYINWRPLSFG GGTKVEIK
[0677] TCN-504 (3251_K17) Light Chain Kabat CDRs:
TABLE-US-00361 CDR 1: RASQSVGNSLA (SEQ ID NO: 754) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QQYINWRPLS (SEQ ID NO: 756)
[0678] TCN-504 (3251_K17) Light Chain Chothia CDRs:
TABLE-US-00362 CDR 1: RASQSVGNSLA (SEQ ID NO: 754) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QQYINWRPLS (SEQ ID NO: 756)
[0679] TCN-521 (3280_D18) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00363 (SEQ ID NO: 758)
GAAGTGCAGTTGGTGCAGTCTGGAGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCGCCTGTGTAGTCTCTGGGTTCACCGTCACCAGCAATTATA
TAACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT
ATTTATAGTCATGGTCGCGCATATTATTCAGCCTCCGTGAATGGCCGATT
CACCATCTCCAGACACACTTCCAAGAACACAGTTTATCTTGAAATGAACA
GCCTGAGACCTGAGGACACGGCCGTCTATTACTGTGCGGGCGGGGGCCTA
GTCGGTGGCTACGACGAATATTTCTTTGACTATTGGGGCCAGGGAACCCT
GGCCACCGTCTCCTCA
[0680] TCN-521 (3280_D18) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00364 (SEQ ID NO: 759)
EVQLVQSGGGLVQPGGSLRLACVVSGFTVTSNYITWVRQAPGKGLEWVSV
IYSHGRAYYSASVNGRFTISRHTSKNTVYLEMNSLRPEDTAVYYCAGGGL
VGGYDEYFFDYWGQGTLATVSS
[0681] TCN-521 (3280_D18) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00365 CDR 1: SNYIT (SEQ ID NO: 760) CDR 2:
VIYSHGRAYYSASVNG (SEQ ID NO: 761) CDR 3: GGLVGGYDEYFFDY (SEQ ID NO:
762)
[0682] TCN-521 (3280_D18) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00366 CDR 1: GFTVTS (SEQ ID NO: 763) CDR 2: VIYSHGRAY (SEQ
ID NO: 764) CDR 3: GGLVGGYDEYFFDY (SEQ ID NO: 762)
[0683] TCN-521 (3280_D18) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00367 (SEQ ID NO: 765)
GAAACTGTCTTGACGCAATCTCCAGGCACCTTGTCTTTGACTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGAGTCGGTCAGAGTGTTAGCGGCAGCCACT
TAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCGGTGGCAGTGT
GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT
CTGCAGTTTATTACTGTCAGCAGTATGGTGACTCACGATACACTTTTGGC
CAGGGGACCAAGCTGGAGATCAAA
[0684] TCN-521 (3280_D18) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00368 (SEQ ID NO: 766)
ETVLTQSPGTLSLTPGERATLSCRVGQSVSGSHLAWYQQKPGQAPRLLIY
GASSRATGIPDRFGGSVSGTDFTLTISRLEPEDSAVYYCQQYGDSRYTFG QGTKLEIK
[0685] TCN-521 (3280_D18) Light Chain Kabat CDRs:
TABLE-US-00369 CDR 1: RVGQSVSGSHLA (SEQ ID NO: 767) CDR 2: GASSRAT
(SEQ ID NO: 768) CDR 3: QQYGDSRYT (SEQ ID NO: 769)
[0686] TCN-521 (3280_D18) Light Chain Chothia CDRs:
TABLE-US-00370 CDR 1: RVGQSVSGSHLA (SEQ ID NO: 767) CDR 2: GASSRAT
(SEQ ID NO: 768) CDR 3: QQYGDSRYT (SEQ ID NO: 769)
[0687] TCN-522 (3212_I12) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00371 (SEQ ID NO: 770)
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTTTTGAAACCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTGTCTGGGGGGTCCCTCACTGATTACTCTT
GGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATCGGTGAC
ACCCTTCATAATGGCTACACCAACTACAACCCGTCCCTCAGGGGTCGAGT
TTCCATCTCAATAGACACGTCCAAGAACCAGGTCTCACTCAGGCTGACCT
CTGTGACCGCCGCGGACACGGCTCTTTATTACTGTGCGAGAGGCTCAGGT
GGATATGGTGGCTTCGATTATTTTGGCAAGCTCCGGACATGGGACTTCTG
GGGCCAGGGAACGCTGGTCACCGTCTCCTCA
[0688] TCN-522 (3212_I12) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00372 (SEQ ID NO: 771)
QVQLQQWGAGLLKPSETLSLTCTVSGGSLTDYSWNWIRQPPGKGLEWIGD
TLHNGYTNYNPSLRGRVSISIDTSKNQVSLRLTSVTAADTALYYCARGSG
GYGGFDYFGKLRTWDFWGQGTLVTVSS
[0689] TCN-522 (3212_I12) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00373 CDR 1: DYSWN (SEQ ID NO: 772) CDR 2:
DTLHNGYTNYNPSLRG (SEQ ID NO: 773) CDR 3: GSGGYGGFDYFGKLRTWDF (SEQ
ID NO: 774)
[0690] TCN-522 (3212_I12) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00374 CDR 1: GGSLTD (SEQ ID NO: 775) CDR 2: DTLHNGYTN (SEQ
ID NO: 776) CDR 3: GSGGYGGFDYFGKLRTWDF (SEQ ID NO: 774)
[0691] TCN-522 (3212_I12) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00375 (SEQ ID NO: 777)
GACATTCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAG
GCTGGTATCAGCAAAAACCAGGGAACGCCCCTAAGCGCCTGATCTTTGGT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGAGTTCACTCTCACAATCAGCAGCCTGCAGCCTGAGGACTTTG
CAACTTATTACTGTCTACAGCATAATAGTTACCCGTACACTTTTGGCCAG
GGGACCAAGCTGGAGATCAAG
[0692] TCN-522 (3212_I12) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00376 (SEQ ID NO: 778)
DIQLTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGNAPKRLIFG
ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPYTFGQ GTKLEIK
[0693] TCN-522 (3212_I12) Light Chain Kabat CDRs:
TABLE-US-00377 CDR 1: RASQGIRNDLG (SEQ ID NO: 779) CDR 2: GASSLQS
(SEQ ID NO: 780) CDR 3: LQHNSYPYT (SEQ ID NO: 781)
[0694] TCN-522 (3212_I12) Light Chain Chothia CDRs
TABLE-US-00378 CDR 1: RASQGIRNDLG (SEQ ID NO: 779) CDR 2: GASSLQS
(SEQ ID NO: 780) CDR 3: LQHNSYPYT (SEQ ID NO: 781)
[0695] TCN-523 (5248_A17) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00379 (SEQ ID NO: 782)
CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCAGCTTCAGCAACTATGCCT
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ACCATCCCTCTACTTGGTACAACAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTTCCGCGGACCAATTCACGAGCACAGCCTACATGGAGCTGG
GCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTACGAGACGGAAA
ATGACTACGGCTTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA
[0696] TCN-523 (5248_A17) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00380 (SEQ ID NO: 783)
QVQLVQSGAEVKKPGSSVKVSCKASGGSFSNYAFSWVRQAPGQGLEWMGG
TIPLLGTTNYAQKFQGRVTISADQFTSTAYMELGSLRSEDTAVYYCTRRK
MTTAFDSWGQGTLVTVSS
[0697] TCN-523 (5248_A17) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00381 CDR 1: NYAFS (SEQ ID NO: 784) CDR 2:
GTIPLLGTTNYAQKFQG (SEQ ID NO: 785) CDR 3: RKMTTAFDS (SEQ ID NO:
786)
[0698] TCN-523 (5248_A17) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00382 CDR 1: GGSFSN (SEQ ID NO: 787) CDR 2: GTIPLLGTTN
(SEQ ID NO: 788) CDR 3: RKMTTAFDS (SEQ ID NO: 786)
[0699] TCN-523 (5248_A17) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00383 (SEQ ID NO: 789)
CAGCCTGTTCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTC
GGTCACACTCACCTGCACCCTGAGCAGCGCCTACAGTAATTATAAAGTGG
ACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGCTTTGTGATGCGAGTG
GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTT
CTCAGTCTTGGGCTCAGGCCTGAATCGGTACCTGACCATCAAGAACATCC
AGGAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGG
AGCAACTTCGTGTCCCCTTACGTATTCGGCGGAGGGACCAAGCTGACCGT TCTA
[0700] TCN-523 (5248_A17) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00384 (SEQ ID NO: 790)
QPVLTQPPSASASLGASVTLTCTLSSAYSNYKVDWYQQRPGKGPRFVMRV
GTGGIVGSKGDGIPDRFSVLGSGLNRYLTIKNIQEEDESDYHCGADHGSG
SNFVSPYVFGGGTKLTVL
[0701] TCN-523 (5248_A17) Light Chain Kabat CDRs:
TABLE-US-00385 CDR 1: TLSSAYSNYKVD (SEQ ID NO: 791) CDR 2:
VGTGGIVGSKGD (SEQ ID NO: 792) CDR 3: GADHGSGSNFVSPYV (SEQ ID NO:
793)
[0702] TCN-523 (5248_A17) Light Chain Chothia CDRs:
TABLE-US-00386 CDR 1: TLSSAYSNYKVD (SEQ ID NO: 791) CDR 2:
VGTGGIVGSKGD (SEQ ID NO: 792) CDR 3: GADHGSGSNFVSPYV (SEQ ID NO:
793)
[0703] TCN-563 (5237_B21) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00387 (SEQ ID NO: 794)
CAGGTGCAGCTGGCGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTC
GGTGAAAGTCTCATGCACGGCTTCTGGAGGCATCTTCAGGAAGAATGCAA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG
ATCATCGCAGTCTTTAACACAGCAAATTACGCGCAGAAGTTTCAGGGCAG
AGTCAAAATTACCGCAGACGAATCCGGGAATACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTCACCCA
AAATATTTCTATGGTTCGGGGAGTTATCCGGACTTCTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGC
[0704] TCN-563 (5237_B21) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00388 (SEQ ID NO: 795)
QVQLAQSGAEVKRPGSSVKVSCTASGGIFRKNAISWVRQAPGQGLEWMGG
IIAVFNTANYAQKFQGRVKITADESGNTAYMELSSLRSDDTAVYYCASHP
KYFYGSGSYPDFWGQGTLVTVSS
[0705] TCN-563 (5237_B21) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00389 CDR 1: KNAIS (SEQ ID NO: 796) CDR 2:
GIIAVFNTANYAQKFQG (SEQ ID NO: 797) CDR 3: HPKYFYGSGSYPDF (SEQ ID
NO: 798)
[0706] TCN-563 (5237_B21) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00390 CDR 1: GGIFRK (SEQ ID NO: 799) CDR 2: GIIAVFNTAN
(SEQ ID NO: 800) CDR 3: HPKYFYGSGSYPDF (SEQ ID NO: 798)
[0707] TCN-563 (5237_B21) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00391 (SEQ ID NO: 801)
CAATCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTC
AGTCACCATCTCCTGCACTGGAAGCAGCAGTGATGTTGGTGCTTCTAACT
CTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCGTTATT
TATGATGTCACTGAGCGACCCTCAGGGGTCCCTCATCGCTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGCCTGAGG
ACGAGGCTGATTATTTCTGCTGCGCATATGGAGGCAAATATCTTGTGGTC
TTCGGCGGAGGGACCAAGGTGACCGTCCTC
[0708] TCN-563 (5237_B21) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00392 (SEQ ID NO: 802)
QSALTQPRSVSGSPGQSVTISCTGSSSDVGASNSVSWYQQHPGKAPKLVI
YDVTERPSGVPHRFSGSKSGNTASLTVSGLQPEDEADYFCCAYGGKYLVV FGGGTKVTVL
[0709] TCN-563 (5237_B21) Light Chain Kabat CDRs:
TABLE-US-00393 CDR 1: TGSSSDVGASNSVS (SEQ ID NO: 803) CDR 2:
DVTERPS (SEQ ID NO: 804) CDR 3: CAYGGKYLVV (SEQ ID NO: 805)
[0710] TCN-563 (5237_B21) Light Chain Chothia CDRs:
TABLE-US-00394 CDR 1: TGSSSDVGASNSVS (SEQ ID NO: 803) CDR 2:
DVTERPS (SEQ ID NO: 804) CDR 3: CAYGGKYLVV (SEQ ID NO: 805)
[0711] TCN-526 (5084_C17) heavy chain variable region nucleotide
sequence:
TABLE-US-00395 (SEQ ID NO: 806)
GAGGTGCTGATGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CGTGAGACTCTCCTGTGTAGCCTCTGGATTCAGTTTCAGTAGTCATTGGA
TGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC
ATAGAGGACGATGGAGGTGACAAGTACTATGTGGACTCTGTGAAGGGCCG
ATTCATTATCTCCAGAGACAACGCCAAGAATTCAGTGTATCTGCAAATGA
ACAGCCTAAGAGCCGAGGACACGGCTGTGTATTTCTGTGCGAGAGGTTCG
GGGAGCTCTGATAGAAGTGATTATGACCCCCACTACTACTACTACTTGGA
CGTCTGGGGCAAAGGGGCCACGGTCACCGTCTCCTCA
[0712] TCN-526 (5084_C17) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00396 (SEQ ID NO: 807)
EVLMVESGGGLVQFGGSVRLSCVASGFSFSSHWMTWVRQAPGKGLEWVAN
IEDDGGDKYYVDSVKGRFIISRDNAKNSVYLQMNSLRAEDTAVYFCARGS
GSSDRSDYDPHYYYYLDVWGKGATVTVSS
[0713] TCN-526 (5084_C17) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00397 CDR 1: SHWMT (SEQ ID NO: 808) CDR 2:
NIEDDGGDKYYVDSVKG (SEQ ID NO: 809) CDR 3: GSGSSDRSDYDPHYYYYLDV (SEQ
ID NO: 810)
[0714] TCN-526 (5084_C17) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00398 CDR 1: GFSFSS (SEQ ID NO: 811) CDR 2: NIEDDGGDKY
(SEQ ID NO: 812) CDR 3: GSGSSDRSDYDPHYYYYLDV (SEQ ID NO: 810)
[0715] TCN-526 (5084_C17) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00399 (SEQ ID NO: 813)
GACATCCAGCTGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGTAGGTATTTAA
ATTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGCTGTTTGCT
GCTTCTACTTTGCTAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACGGAATCACAGTCCCTCGTGGACGTTCGGCCAA
GGGACCAGGGTGGAAATCAAA
[0716] TCN-526 (5084_C17) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00400 (SEQ ID NO: 814)
DIQLTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLLFA
ASTLLDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQRNHSPSWTFGQ GTRVEIK
[0717] TCN-526 (5084_C17) Light Chain Kabat CDRs:
TABLE-US-00401 CDR 1: RASQSISRYLN (SEQ ID NO: 815) CDR 2: AASTLLD
(SEQ ID NO: 816) CDR 3: QRNHSPSWT (SEQ ID NO: 817)
[0718] TCN-526 (5084_C17) Light Chain Chothia CDRs:
TABLE-US-00402 CDR 1: RASQSISRYLN (SEQ ID NO: 815) CDR 2: AASTLLD
(SEQ ID NO: 816) CDR 3: QRNHSPSWT (SEQ ID NO: 817)
[0719] TCN-527 (5086_C06) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00403 (SEQ ID NO: 818)
CAGGTGCAGCTGCAAGAGTCGGGCCCGGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCAACTGCGCTGTCTCTGGAGGCTCCATCAGTAATTACTACT
GGAGCTGGATCCGGCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGCTAT
ATCTCTTACAATGGGAGGCCCAAGTACAACCCCTCCCTCACGAGTCGAGT
CACCATATCCGTCGACACGTCCAAGGACCAGTTCTCCCTGGAGCTGCGCT
CTGTGACCGCTGCGGACACGGCCCTTTATTACTGTGCGAGAGAAACGCGG
TTCGGGGAGTTATTATCTCCCTATGATGCTTTTGAAATCTGGGGCCAAGG
GACAATGGTCACCGTCTCCTCA
[0720] TCN-527 (5086_C06) Gamma Heavy Chain Variable Region Acid
Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00404 (SEQ ID NO: 819)
QVQLQESGPGLVKPSETLSLNCAVSGGSISNYYWSWIRQFPGKGLEWIGY
ISYNGRPKYNPSLTSRVTISVDTSKDQFSLELRSVTAADTALYYCARETR
FGELLSPYDAFEIWGQGTMVTVSS
[0721] TCN-527 (5086_C06) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00405 CDR 1: NYYWS (SEQ ID NO: 820) CDR 2:
YISYNGRPKYNPSLTS (SEQ ID NO: 821) CDR 3: ETRFGELLSPYDAFEI (SEQ ID
NO: 822)
[0722] TCN-527 (5086_C06) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00406 CDR 1: GGSISN (SEQ ID NO: 824) CDR 2: YISYNGRPK (SEQ
ID NO: 823) CDR 3: ETRFGELLSPYDAFEI (SEQ ID NO: 822)
[0723] TCN-527 (5086_C06) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00407 (SEQ ID NO: 825)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATGACTTGCCGGGCAAGTCAGAACATTAGAAGCTATTTAA
ATTGGTATCAGCAGAGACCAGGGACAGCCCCTAAACTCCTGATCTATGCT
GCATCCACTTTACACAGTGGGGTCCCATCAAGGTTCAGTGGCGGTGGGTC
TGGGACAGATTTCACTCTCACCATCAATAATCTGCAACCTGAAGATTTTG
CATCTTACTACTGTCAACAGAGTTACGATAACCCTCAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAA
[0724] TCN-527 (5086_C06) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00408 (SEQ ID NO: 826)
DIQMTQSPSSLSASVGDRVTMTCRASQNIRSYLNWYQQRPGTAPKLLIYA
ASTLHSGVPSRFSGGGSGTDFTLTINNLQPEDFASYYCQQSYDNPQTFGQ GTKVEIK
[0725] TCN-527 (5086_C06) Light Chain Kabat CDRs:
TABLE-US-00409 CDR 1: RASQNIRSYLN (SEQ ID NO: 827) CDR 2: AASTLHS
(SEQ ID NO: 828) CDR 3: QQSYDNPQT (SEQ ID NO: 829)
[0726] TCN-527 (5086_C06) Light Chain Chothia CDRs:
TABLE-US-00410 CDR 1: RASQNIRSYLN (SEQ ID NO: 827) CDR 2: AASTLHS
(SEQ ID NO: 828) CDR 3: QQSYDNPQT (SEQ ID NO: 829)
[0727] TCN-528 (5087_P17) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00411 (SEQ ID NO: 830)
CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAATTATGACA
TCAACTGGATTCGACAGGCCCCTGGTCAAGGACTTGAGTGGATGGGCTGG
ATAAATCCCAACAGTGGAACCACGGGCTCTGCACAGAGGTTCCAGGGCAG
AGTCACCATAACCGTGGACACCTCCATAACCACAGTCTACATGGAACTGA
GCAGCCTGAGATCTGACGACACGGCCATTTACTACTGCGCGAGAGGCCGT
GAGCTCCTCCGGCTTCAACATTTTTTGACTGACTCCCAGTCCGAGAGGAG
GGACTGCTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
[0728] TCN-528 (5087_P17) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in bold, Chothia CDRs underlined)
TABLE-US-00412 (SEQ ID NO: 831)
QVQLVQSGSEVKKPGASVKVSCKASGYTFTNYDINWIRQAPGQGLEWMGW
INPNSGTTGSAQRFQGRVTITVDTSITTVYMELSSLRSDDTAIYYCARGR
ELLRLQHFLTDSQSERRDCFDPWGQGTLVTVSS
[0729] TCN-528 (5087_P17) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00413 CDR 1: NYDIN (SEQ ID NO: 832) CDR 2:
WINPNSGTTGSAQRFQG (SEQ ID NO: 833) CDR 3: GRELLRLQHFLTDSQSERRDCFDP
(SEQ ID NO: 834)
[0730] TCN-528 (5087_P17) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00414 CDR 1: GYTFTN (SEQ ID NO: 835) CDR 2: WINPNSGTTG
(SEQ ID NO: 836) CDR 3: GRELLRLQHFLTDSQSERRDCFDP (SEQ ID NO:
834)
[0731] TCN-528 (5087_P17) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00415 (SEQ ID NO: 837)
GATATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAATCAAGACATTGGCATTTATTTAA
ATTGGTATCAACAGAATCCAGGGAAAGTCCCTAAACTCCTGCTCCATGGT
GCGTCCAGTTTGCAGGGCGGGGTCCCATCAAGGTTCAGTGCCAGTGGATC
TGGGACAGATTTCACTCTCACCATTCACAGTCTACAACCTGAAGATTTAG
CAACCTACTACTGTCAACAGAGTCGCCGTCTACCGTACACTTTTGGCCAG
GGGACCAGGGTGGAACTCAAA
[0732] TCN-528 (5087_P17) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00416 (SEQ ID NO: 838)
DIQMTQSPSSLSASVGDRVTITCRANQDIGIYLNWYQQNPGKVPKLLLHG
ASSLQGGVPSRFSASGSGTDFTLTIHSLQPEDLATYYCQQSRRLPYTFGQ GTRVELK
[0733] TCN-528 (5087_P17) Light Chain Kabat CDRs:
TABLE-US-00417 CDR 1: RANQDIGIYLN (SEQ ID NO: 839) CDR 2: GASSLQG
(SEQ ID NO: 840) CDR 3: QQSRRLPYT (SEQ ID NO: 841)
[0734] TCN-528 (5087_P17) Light Chain Chothia CDRs:
TABLE-US-00418 CDR 1: RANQDIGIYLN (SEQ ID NO: 839) CDR 2: GASSLQG
(SEQ ID NO: 840) CDR 3: QQSRRLPYT (SEQ ID NO: 841)
[0735] TCN-529 (5297_H01) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00419 (SEQ ID NO: 842)
CAGATCACCTTGAGGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGAC
CCTCACGCTGACCTGCACCTTCTCTGGGTTTTCACTCAGCACTAATGGAG
TGAATGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT
GCACTCATTTACTGGGATGATGATAAGCGCTACAGTCCGTCTCTGAAGAG
AAGGCTCACCATCACCAAGGACACCTCCAAAAACCAAGTGGTCCTTACAC
TGACCAACATGGACCCTGTAGATACAGCCACATATTACTGTGCACACAGA
CCCGACTTCTATGGTGACTTCGAGTACTGGGGCCCGGGAACCCTGGTCAC CGTCTCCTCA
[0736] TCN-529 (5297_H01) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00420 (SEQ ID NO: 843)
QITLRESGPTLVKPTQTLTLTCTFSGFSLSTNGVNVGWIRQPPGKALEWL
ALIYWDDDKRYSPSLKRRLTITKDTSKNQVVLTLTNMDPVDTATYYCAH
RPDFYGDFEYWGPGTLVTVSS
[0737] TCN-529 (5297_H01) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00421 CDR 1: TNGVNVG (SEQ ID NO: 844) CDR 2:
LIYWDDDKRYSPSLKR (SEQ ID NO: 845) CDR 3: RPDFYGDFEY (SEQ ID NO:
846)
[0738] TCN-529 (5297_H01) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00422 CDR 1: GFSLSTNG (SEQ ID NO: 847) CDR 2: LIYWDDDKR
(SEQ ID NO: 848) CDR 3: RPDFYGDFEY (SEQ ID NO: 846)
[0739] TCN-529 (5297_H01) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00423 (SEQ ID NO: 849)
CAGTCTGCACTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCCGGACAGTC
GATCACCATCTCCTGCACTGGAAGCAGCAGTGACATTGGTGGTTATAACT
ATGTCTCCTGGTACCAACAACACCCAGGCAAGGCCCCCAAACTCATGATT
TACGATGTCAATAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACTATCTCTGGGCTCCAGACTGACG
ACGAGGCTGATTATTACTGCGGCTCATATACAGGCAGTCCTCATTATGTC
TTCGGAACTGGGACCAAGGTCACCGTCCTA
[0740] TCN-529 (5297_H01) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00424 (SEQ ID NO: 850)
QSALTQPASVSGSPGQSITISCTGSSSDIGGYNYVSWYQQHPGKAPKLMI
YDVNNRPSGVSNRFSGSKSGNTASLTISGLQTDDEADYYCGSYTGSPHYV FGTGTKVTVL
[0741] TCN-529 (5297_H01) Light Chain Kabat CDRs:
TABLE-US-00425 CDR 1: TGSSSDIGGYNYVS (SEQ ID NO: 851) CDR 2:
DVNNRPS (SEQ ID NO: 852) CDR 3: GSYTGSPHYV (SEQ ID NO: 853)
[0742] TCN-529 (5297_H01) Light Chain Chothia CDRs:
TABLE-US-00426 CDR 1: TGSSSDIGGYNYVS (SEQ ID NO: 851) CDR 2:
DVNNRPS (SEQ ID NO: 852) CDR 3: GSYTGSPHYV (SEQ ID NO: 853)
[0743] TCN-530 (5248_H10a) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00427 (SEQ ID NO: 854)
CAGGTCCAACTGGTGCAATCTGGGGCTGAGGTGAGGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCCCCTTCATGAGTTATGCTA
TCGGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCAACCCTGTGTTTGGTAGACCGCACTACGCACAGAAGTTCCAGGGCAG
AGTCACCATCGCCACGGACGACTCCACGAAGACATCGTACATGGAACTGA
GTAGCCTGACGTCTGAGGACACGGGCATGTATTACTGTGCGAGTAGGTAT
AGTAGGTCGTCCCCAGGGACCTTTGAGTCCTGGGGCCAGGGAACCCTGGT
CACCGTCTCGAGC
[0744] TCN-530 (5248_H10a) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00428 (SEQ ID NO: 855)
QVQLVQSGAEVRKPGSSVKVSCKASGGPFMSYAIGWVRQAPGQGLEWMG
GINPVFGRPHYAQKFQGRVTIATDDSTKTSYMELSSLTSEDTGMYYCAS
RYSRSSPGTFESWGQGTLVTVSS
[0745] TCN-530 (5248_H10a) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00429 CDR 1: SYAIG (SEQ ID NO: 856) CDR 2:
GINPVFGRPHYAQKFQG (SEQ ID NO: 857) CDR 3: RYSRSSPGTFES (SEQ ID NO:
858)
[0746] TCN-530 (5248_H10a) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00430 CDR 1: GGPFMS (SEQ ID NO: 859) CDR 2: GINPVFGRPH
(SEQ ID NO: 860) CDR 3: RYSRSSPGTFES (SEQ ID NO: 858)
[0747] TCN-530 (5248_H10a) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00431 (SEQ ID NO: 861)
GAAATAGTGATGACGCAGTTTCCAGCCACCCTGTCTGTGTCTCCCGGGGA
ACGAGTCACCCTCTCCTGTAGGGCCAGTCAGAGTGTTAGCAACAATTTAG
CCTGGTACCAGCAAAAACCTGGCCAGCCTCCCAGGCTCCTCATCTATGAT
GCATCTACCAGGGCCACGGGTGTCCCAGCCAAGTTCAGTGGCACTGGGTC
TGGCACAGAGTTCACTCTCAGCATCAGCAGCCTGCAGTCCGAAGATTTTG
CAGTTTATTACTGTCAGCAGTATCACAACTGGCCTCCCTCGTACAGTTTT
GGCCTGGGGACCAAGCTGGAGATCAAA
[0748] TCN-530 (5248_H10a) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00432 (SEQ ID NO: 862)
EIVMTQFPATLSVSPGERVTLSCRASQSVSNNLAWYQQKPGQPPRLLIY
DASTRATGVPAKFSGTGSGTEFTLSISSLQSEDFAVYYCQQYHNWPPS YSFGLGTKLEIK
[0749] TCN-530 (5248_H10a) Light Chain Kabat CDRs:
TABLE-US-00433 CDR 1: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT
(SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865)
[0750] TCN-530 (5248_H10a) Light Chain Chothia CDRs:
TABLE-US-00434 CDR 1: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT
(SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865)
[0751] TCN-531 (5091_H13) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00435 (SEQ ID NO: 866)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTACAGCCAGGGCGGTC
CCTGAAACTCTCCTGCACAGGTTCTGGATTCACCTTTGGTGATTATGGTG
TGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTC
ATTAGAACCAGACCTTGGGGTGGGACAGCAGATACCGCCGCGTCTGTGAA
AGGCAGATTCACTATTTCAAGAGATGATTCCAAAAGTCTCGCCTATCTGC
AAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTTGTAGA
GATGCCCCTCCAAATGTGGAAGTGGCTTCTATGACCAACTGGTACTTCGA
TCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCCTCA
[0752] TCN-531 (5091_H13) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00436 (SEQ ID NO: 867)
EVQLVESGGDLVQPGRSLKLSCTGSGFTFGDYGVTWVRQAPGKGLEWVGF
IRTRPWGGTADTAASVKGRFTISRDDSKSLAYLQMNSLKTEDTAVYYCCR
DAPPNVEVASMTNWYFDLWGRGTLVTVSS
[0753] TCN-531 (5091_H13) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00437 CDR 1: DYGVT (SEQ ID NO: 868) CDR 2:
FIRTRPWGGTADTAASVKG (SEQ ID NO: 869) CDR 3: DAPPNVEVASMTNWYFDL (SEQ
ID NO: 870)
[0754] TCN-531 (5091_H13) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00438 CDR 1: GFTFGD (SEQ ID NO: 871) CDR 2: FIRTRPWGGTAD
(SEQ ID NO: 872) CDR 3: DAPPNVEVASMTNWYFDL (SEQ ID NO: 870)
[0755] TCN-531 (5091_H13) light chain variable region nucleotide
sequence:
TABLE-US-00439 (SEQ ID NO: 873)
GACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGA
CAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTCTCAATTGTTTAG
CCTGGTATCAGCAGAAACCGGGGAAAGTTCCTAACCTCCTGATGTATGCT
GCATCCACATTGCAGTCAGGGGTCCCATCTCGGTTCAGCGGCAGTGGATT
TGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTG
CAACTTATTACTGTCAAACGTATGGCGGTGTCTCTACTTTCGGCGGAGGG
ACCAAGGTGGAGATCAGA
[0756] TCN-531 (5091_H13) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00440 (SEQ ID NO: 874)
DIQLTQSPSSLSASVGDRVTITCRASQGILNCLAWYQQKPGKVPNLLMYA
ASTLQSGVPSRFSGSGFGTDFTLTISSLQPEDVATYYCQTYGGVSTFGGG TKVEIR
[0757] TCN-531 (5091_H13) Light Chain Kabat CDRs:
TABLE-US-00441 CDR 1: RASQGILNCLA (SEQ ID NO: 875) CDR 2: AASTLQS
(SEQ ID NO: 876) CDR 3: QTYGGVST (SEQ ID NO: 877)
[0758] TCN-531 (5091_H13) Light Chain Chothia CDRs:
TABLE-US-00442 CDR 1: RASQGILNCLA (SEQ ID NO: 875) CDR 2: AASTLQS
(SEQ ID NO: 876) CDR 3: QTYGGVST (SEQ ID NO: 877)
[0759] TCN-532 (5262_H18) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00443 (SEQ ID NO: 878)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CTTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGAGACTT
ACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTAGAGTGGATT
GGATATATCTATTACATTGGGAACACCGACTACAGGCCCTCCCTCAAGAG
TCGAGTCACCATATCACTGGACACGTCCAAGAACCAGTTCTCCCTGAAGC
TGAGCTCTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGAGGC
GCTTATTATGATAGTAGTGGTTACCCGGCTTTTTATATCTGGGGCCAAGG
GACAATGGTCACCGTCTCCTCA
[0760] TCN-532 (5262_H18) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00444 (SEQ ID NO: 879)
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSETYYWSWIRQPPGKGLEWI
GYIYYIGNTDYRPSLKSRVTISLDTSKNQFSLKLSSVTAADTAVYYCARG
AYYDSSGYPAFYIWGQGTMVTVSS
[0761] TCN-532 (5262_H18) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00445 CDR 1: SETYYWS (SEQ ID NO: 880) CDR 2:
YIYYIGNTDYRPSLKS (SEQ ID NO: 881) CDR 3: GAYYDSSGYPAFYI (SEQ ID NO:
882)
[0762] TCN-532 (5262_H18) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00446 CDR 1: GGSVSSET (SEQ ID NO: 883) CDR 2: YIYYIGNTD
(SEQ ID NO: 884) CDR 3: GAYYDSSGYPAFYI (SEQ ID NO: 882)
[0763] TCN-532 (5262_H18) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00447 (SEQ ID NO: 885)
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAG
GGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGTCAGATTATG
ATGTGCACTGGTACAAGCAACTTCCAGGAACAGCCCCCAAACTCCTCATC
TTTGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTC
CAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGG
ATGAGGCTGATTATTACTGCCAATCCTATGACAGCAGCCTGAGTGGTTTT
CATGTCTTCGGAAGTGGGACCAAGGTCACCGTCCTA
[0764] TCN-532 (5262_H18) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00448 (SEQ ID NO: 886)
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGSDYDVHWYKQLPGTAPKLLI
FGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGF HVFGSGTKVTVL
[0765] TCN-532 (5262_H18) Light Chain Kabat CDRs:
TABLE-US-00449 CDR 1: TGSSSNIGSDYDVH (SEQ ID NO: 887) CDR 2:
GNSNRPS (SEQ ID NO: 888) CDR 3: QSYDSSLSGFHV (SEQ ID NO: 889)
[0766] TCN-532 (5262_H18) Light Chain Chothia CDRs:
TABLE-US-00450 CDR 1: TGSSSNIGSDYDVH (SEQ ID NO: 887) CDR 2:
GNSNRPS (SEQ ID NO: 888) CDR 3: QSYDSSLSGFHV (SEQ ID NO: 889)
[0767] TCN-533 (5256_A17) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00451 (SEQ ID NO: 890)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGACGTGAAGAAGCCTGGGTCCTC
GGTGACGGTCTCCTGCAAGGCTTCTGGAGGCAGCTTCAGCAACTATGGAA
TCAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGGGA
ATCATCCCTCTCATTAATGCACCGAACTACGCACCGAAGTTCCAGGGCAG
AGTGACGATTACCGCGGACATGTTCTCGAATATAGTCTCCTTGCAGTTGA
CCAGCCTGAGAACTGACGACACGGCCGTGTATTATTGTGCGAGACGAAAA
ATGACTACGGCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA
[0768] TCN-533 (5256_A17) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00452 (SEQ ID NO: 891)
QVQLVQSGADVKKPGSSVTVSCKASGGSFSNYGINWVRQAPGQGLEWMGG
IIPLINAPNYAPKFQGRVTITADMFSNIVSLQLTSLRTDDTAVYYCARRK
MTTAIDYWGQGTLVTVSS
[0769] TCN-533 (5256_A17) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00453 CDR 1: NYGIN (SEQ ID NO: 892) CDR 2:
GIIPLINAPNYAPKFQG (SEQ ID NO: 893) CDR 3: RKMTTAIDY (SEQ ID NO:
894)
[0770] TCN-533 (5256_A17) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00454 CDR 1: GGSFSN (SEQ ID NO: 787) CDR 2: GIIPLINAPN
(SEQ ID NO: 895) CDR 3: RKMTTAIDY (SEQ ID NO: 894)
[0771] TCN-533 (5256_A17) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00455 (SEQ ID NO: 896)
CAGCCTGTGTTGAGTCAGCCACCTTCTGCATCGGCCTCCCTGGGAGCCTC
CGTCACACTCACCTGCACCCTGAGTAGCGGCTTCGATAATTATCAAGTGG
CCTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGCTTTGTGATGCGGGTG
GGCAATGGTGGGAATGTGGCTTCCAAGGGGGATGGCATTCCTGATCGTTT
CTCAGTCTCGGGCTCAGGCCTGAATCGGTACCTGACCATCAAGAACATCC
AGGAAGACGATGAGAGTGACTATTATTGTGGGGCAGACCATGGCAGTGGG
AACAACTTCGTGTCCCCTTATGTGTTTGGCGGAGGGACCAAGCTGACCGT TCTA
[0772] TCN-533 (5256_A17) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00456 (SEQ ID NO: 897)
QPVLSQPPSASASLGASVTLTCTLSSGFDNYQVAWYQQRPGKGPRFVMRV
GNGGNVASKGDGIPDRFSVSGSGLNRYLTIKNIQEDDESDYYCGADHGSG
NNFVSPYVFGGGTKLTVL
[0773] TCN-533 (5256_A17) Light Chain Kabat CDRs:
TABLE-US-00457 CDR 1: TLSSGFDNYQVA (SEQ ID NO: 898) CDR 2:
VGNGGNVASKGD (SEQ ID NO: 899) CDR 3: GADHGSGNNFVSPYV (SEQ ID NO:
900)
[0774] TCN-533 (5256_A17) Light chain Chothia CDRs:
TABLE-US-00458 CDR 1: TLSSGFDNYQVA (SEQ ID NO: 898) CDR 2:
VGNGGNVASKGD (SEQ ID NO: 899) CDR 3: GADHGSGNNFVSPYV (SEQ ID NO:
900)
[0775] TCN-534 (5249_B02) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00459 (SEQ ID NO: 901)
CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCAGGGTCCTC
GGTGAAGGTCTCCTGCAGGGAATCTGGAGGCACCTTCAACGGCTACACTA
TCACCTGGGTGCGACAGGCCCCTGGGCAAGGCCTTGAGTGGATGGGAGGG
ATCATCCCTATGATGGGGACAGTCAACTACGCACAGAAGTTGCAGGGCAG
AGTCACCATTACCACGGACTATTTCACGAAAACAGCCTACATGGATCTGA
ACAATTTAAGATCTGAAGACACGGCCATGTATTATTGTGTGAAAATCAGA
TATACTGGGCAGCAGCTGCTCTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA
[0776] TCN-534 (5249_B02) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00460 (SEQ ID NO: 902)
QVQLVQSGAEVKKPGSSVKVSCRESGGTFNGYTITWVRQAPGQGLEWMGG
IIPMMGTVNYAQKLQGRVTITTDYFTKTAYMDLNNLRSEDTAMYYCVKIR
YTGQQLLWGQGTLVTVSS
[0777] TCN-534 (5249_B02) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00461 CDR 1: GYTIT (SEQ ID NO: 903) CDR 2:
GIIPMMGTVNYAQKLQG (SEQ ID NO: 904) CDR 3: IRYTGQQLL (SEQ ID NO:
905)
[0778] TCN-534 (5249_B02) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00462 CDR 1: GGTFNG (SEQ ID NO: 906) CDR 2: GIIPMMGTVN
(SEQ ID NO: 907) CDR 3: IRYTGQQLL (SEQ ID NO: 905)
[0779] TCN-534 (5249_B02) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00463 (SEQ ID NO: 908)
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCGGCATCTATAGGAGA
CAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTGCAAGTTGGTTGG
CCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAG
GCAGTTAATTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCCGATGATTTTG
CAACTTATTTCTGCCAACATTATGGTACTATTTCTCAGACCTTCGGCGGA
GGGACCAAGGTGGAGATCAAA
[0780] TCN-534 (5249_B02) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00464 (SEQ ID NO: 909)
DIQMTQSPSTLSASIGDRVTITCRASQSIASWLAWYQQKPGKAPKLLIYE
AVNLESGVPSRFSGSGSGTDFTLTISSLQPDDFATYFCQHYGTISQTFGG GTKVEIK
[0781] TCN-534 (5249_B02) Light Chain Kabat CDRs:
TABLE-US-00465 CDR 1: RASQSIASWLA (SEQ ID NO: 910) CDR 2: EAVNLES
(SEQ ID NO: 911) CDR 3: QHYGTISQT (SEQ ID NO: 912)
[0782] TCN-534 (5249_B02) Light Chain Chothia CDRs:
TABLE-US-00466 CDR 1: RASQSIASWLA (SEQ ID NO: 910) CDR 2: EAVNLES
(SEQ ID NO: 911) CDR 3: QHYGTISQT (SEQ ID NO: 912)
[0783] TCN-535 (5246_P19) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00467 (SEQ ID NO: 913)
CAGGTCCAGCTGGTGCAATCTGGGAGTGAGGTGAAGAAGCCTGGGACCTC
GGTGAAGGTCTCCTGCACGGCCTCTGGAAGTGTCTTCACCAATTATGGAA
TTAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCATCCCTCTCTTTGGCGCAGCCAAGTACGCACAGAAATTCCAGGGCAG
AGTCACCATCACAGCGGACGAATCCACGAAGACAGTCTACATGGAGCTGA
GCAGGCTGACATCTAAAGACACGGCCATATATTTCTGTGCGAAGGCCCCC
CGTGTCTACGAGTACTACTTTGATCAGTGGGGCCAGGGAACCCCAGTCAC CGTCTCCTCA
[0784] TCN-535 (5246_P19) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00468 (SEQ ID NO: 914)
QVQLVQSGSEVKKPGTSVKVSCTASGSVFTNYGISWVRQAPGQGLEWMGG
IIPLFGAAKYAQKFQGRVTITADESTKTVYMELSRLTSKDTAIYFCAKAP
RVYEYYFDQWGQGTPVTVSS
[0785] TCN-535 (5246_P19) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00469 CDR 1: NYGIS (SEQ ID NO: 915) CDR 2:
GIIPLFGAAKYAQKFQG (SEQ ID NO: 916) CDR 3: APRVYEYYFDQ (SEQ ID NO:
917)
[0786] TCN-535 (5246_P19) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00470 CDR 1: GSVFTN (SEQ ID NO: 918) CDR 2: GIIPLFGAAK
(SEQ ID NO: 919) CDR 3: APRVYEYYFDQ (SEQ ID NO: 917)
[0787] TCN-535 (5246_P19) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00471 (SEQ ID NO: 920)
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGTCAAT
TAGCCTGGTACCAGCAAAAACCTGGCCAGGCTCCCAGGCTCATCATCTAT
GGTGCGTCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGAAGTGG
GTCTGGGACAGACTTCACTCTCACCATCGGCAGACTGGAGCCTGAAGATT
TTGCAGTGTTTTTCTGTCAGCAGTATAGTACCTCACCTCCGACGTTCGGC
CAAGGGACCAAGGTGGATTTCAAA
[0788] TCN-535 (5246_P19) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00472 (SEQ ID NO: 921)
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSQLAWYQQKPGQAPRLIIY
GASTRATGIPDRFSGSGSGTDFTLTIGRLEPEDFAVFFCQQYSTSPPTFG QGTKVDFK
[0789] TCN-535 (5246_P19) Light Chain Kabat CDRs:
TABLE-US-00473 CDR 1: RASQSVSSSQLA (SEQ ID NO: 922) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QQYSTSPPT (SEQ ID NO: 923)
[0790] TCN-535 (5246_P19) Light Chain Chothia CDRs:
TABLE-US-00474 CDR 1: RASQSVSSSQLA (SEQ ID NO: 922) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QQYSTSPPT (SEQ ID NO: 923)
[0791] TCN-536 (5095_N01) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00475 (SEQ ID NO: 924)
CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGTCAGTGGTT
ACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAGGGGGCTGGAGTGGATT
GGGGAAATCAGTCATGGTGGAAGCACCAACTACAACCCGTCCCTCAAGAG
TCGAGTCACCATATCAGTGGACACGACCAAGAACCAGTTCTCCCTGAGAC
TGAGCTCTGTGACCGCCGCGGACACGGCCGTCTATTACTGTGCGAGAGGG
ACAGACCCTGACACGGAAGTATATTGTCGTGTTGGTAACTGCGCGGCCTT
TGACTACTGGGGCCAGGGAAGCCTGGTCACCGTCTCCTCA
[0792] TCN-536 (5095_N01) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00476 (SEQ ID NO: 925)
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSVSGYYWSWIRQPPGRGLEWI
GEISHGGSTNYNPSLKSRVTISVDTTKNQFSLRLSSVTAADTAVYYCARG
TDPDTEVYCRVGNCAAFDYWGQGSLVTVSS
[0793] TCN-536 (5095_N01) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00477 CDR 1: VSGYYWS (SEQ ID NO: 926) CDR 2:
EISHGGSTNYNPSLKS (SEQ ID NO: 927) CDR 3: GTDPDTEVYCRVGNCAAFDY (SEQ
ID NO: 928)
[0794] TCN-536 (5095_N01) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00478 CDR 1: GGSFSVSG (SEQ ID NO: 929) CDR 2: EISHGGSTN
(SEQ ID NO: 930) CDR 3: GTDPDTEVYCRVGNCAAFDY (SEQ ID NO: 928)
[0795] TCN-536 (5095_N01) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00479 (SEQ ID NO: 931)
GAAATTATATTGGCGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
GAGAGCCACCCTCTCCTGCAGGGCCAGCCAGTTTGTTAGCACCAGATCCC
TGGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGACTCCTCATCTAT
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACGCTCACCATCAGCAGACTGGAGCCTGAAGATT
TTGCAGTGTATTACTGTCAGCACTATGGTTACTCACCTAGGTACGCTTTT
GGCCAGGGGTCCAAGGTTGAGATCAAA
[0796] TCN-536 (5095_N01) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00480 (SEQ ID NO: 932)
EIILAQSPGTLSLSPGERATLSCRASQFVSTRSLAWYQQRPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGYSPRYAF GQGSKVEIK
[0797] TCN-536 (5095_N01) Light Chain Kabat CDRs:
TABLE-US-00481 CDR 1: RASQFVSTRSLA (SEQ ID NO: 933) CDR 2: GASSRAT
(SEQ ID NO: 768) CDR 3: QHYGYSPRYA (SEQ ID NO: 934)
[0798] TCN-536 (5095_N01) Light Chain Chothia CDRs:
TABLE-US-00482 CDR 1: RASQFVSTRSLA (SEQ ID NO: 933) CDR 2: GASSRAT
(SEQ ID NO: 768) CDR 3: QHYGYSPRYA (SEQ ID NO: 934)
[0799] TCN-537 (3194_D21) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00483 (SEQ ID NO: 935)
CAGGTGCAGCTCCAACAGTGGGGCTCAGGACTGTTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGAGATGACTACT
GGACCTGGATTCGCCAGCCCCCAGGCAAGGGGCTGGAGTGGATTGGGGAA
ATCAATCATAGTGGAAGAACCAACTACAACCCGTCCCTCAAGAGTCGAGT
CACCATATCAGTAGACACGTCCCTGAAACAGTTCTCCTTGAAGGTGATTT
CTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAGAGGGACGAGC
CATGTTTCCCGGTATTTTGATTGGTTACCACCCACCAACTGGTTCGACCC
CTGGGGCCAGGGAACCCAGGTCACCGTCTCGAGC
[0800] TCN-537 (3194_D21) gamma heavy chain variable region amino
acid sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00484 (SEQ ID NO: 936)
QVQLQQWGSGLLKPSETLSLTCAVYGGSFRDDYWTWIRQPPGKGLEWIGE
INHSGRTNYNPSLKSRVTISVDTSLKQFSLKVISVTAADTAVYYCARGTS
HVSRYFDWLPPTNWFDPWGQGTQVTVSS
[0801] TCN-537 (3194_D21) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00485 CDR 1: DDYWT (SEQ ID NO: 937) CDR 2:
EINHSGRTNYNPSLKS (SEQ ID NO: 938) CDR 3: GTSHVSRYFDWLPPTNWFDP (SEQ
ID NO: 939)
[0802] TCN-537 (3194_D21) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00486 CDR 1: GGSFRD (SEQ ID NO: 940) CDR 2: EINHSGRTN (SEQ
ID NO: 941) CDR 3: GTSHVSRYFDWLPPTNWFDP (SEQ ID NO: 939)
[0803] TCN-537 (3194_D21) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00487 (SEQ ID NO: 942)
GACATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT
TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCGTCATGTAT
GGTGCAGCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGCCAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT
TTGCAATGTATTACTGTCAGCAGTATGGTAACTCACCGATCACCTTCGGC
CAAGGGACACGACTGGAGATCAAA
[0804] TCN-537 (3194_D21) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00488 (SEQ ID NO: 943)
DIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLVMY
GAATRATGIPDRFSGSGSGPDFTLTISRLEPEDFAMYYCQQYGNSPITFG QGTRLEIK
[0805] TCN-537 (3194_D21) Light Chain Kabat CDRs:
TABLE-US-00489 CDR 1: RASQSVSSSYLA (SEQ ID NO: 944) CDR 2: GAATRAT
(SEQ ID NO: 945) CDR 3: QQYGNSPIT (SEQ ID NO: 946)
[0806] TCN-537 (3194_D21) Light Chain Chothia CDRs:
TABLE-US-00490 CDR 1: RASQSVSSSYLA (SEQ ID NO: 944) CDR 2: GAATRAT
(SEQ ID NO: 945) CDR 3: QQYGNSPIT (SEQ ID NO: 946)
[0807] TCN-538 (3206_O17) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00491 (SEQ ID NO: 947)
CAGATCACCTTGAAGGAGTCTGGTCCTACACTGGTGAAACCCACACAGAC
CCTCACACTGACCTGCGTCTTCTCTGGGTTCTCACTCAGCATTACTGGAG
TGCGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT
GCACTCATTTCTTGGGATGATGAAAAGCACTACAGCCCATCTCTGCAGAG
TAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAA
TGACCAACCTGGACCCTGTCGACACAGCCACATATTACTGTGCACGGTCA
ACCGACAGGGGCCACGTCTTACGATATTTTGGCTGGATGTTACCGGGTGA
TGCATTTGATGTCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGC
[0808] TCN-538 (3206_O17) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00492 (SEQ ID NO: 948)
QITLKESGPTLVKPTQTLTLTCVFSGFSLSITGVRVGWIRQPPGKALEWL
ALISWDDEKHYSPSLQSRLTITKDTSKNQVVLTMTNLDPVDTATYYCARS
TDRGHVLRYFGWMLPGDAFDVWGQGTMVTVSS
[0809] TCN-538 (3206_O17) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00493 CDR 1: ITGVRVG (SEQ ID NO: 949) CDR 2:
LISWDDEKHYSPSLQS (SEQ ID NO: 950) CDR 3: STDRGHVLRYFGWMLPGDAFDV
(SEQ ID NO: 951)
[0810] TCN-538 (3206_O17) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00494 CDR 1: GFSLSITG (SEQ ID NO: 952) CDR 2: LISWDDEKH
(SEQ ID NO: 953) CDR 3: STDRGHVLRYFGWMLPGDAFDV (SEQ ID NO: 951)
[0811] TCN-538 (3206_O17) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00495 (SEQ ID NO: 954)
GACATCGTGATGACCCAGTCTCCAGACTTCCTGCCTGTGTCTCTGGGCGA
GAGGGCCACCATCAACTGCAAGTCCAGCCAGAGAGTTTTATACAGCTCCA
ACAATAAAAACTACTTAGCTTGGTACCAGCTGAAACCAGGGCAGCCTCCT
AAGTTGATCATTTATTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG
ATTCAGTGGCAGCGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCC
TGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATAGTCGT
CCGTACACTTTTGGCCAGGGGACCAAGCTCGAGATCAAA
[0812] TCN-538 (3206_O17) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRS Underlined)
TABLE-US-00496 (SEQ ID NO: 955)
DIVMTQSPDFLPVSLGERATINCKSSQRVLYSSNNKNYLAWYQLKPGQPP
KLIIYWASTRESGVPDRFSGSGSGTEFTLTISSLQAEDVAVYYCQQYYSR
PYTFGQGTKLEIK
[0813] TCN-538 (3206_O17) Light Chain Kabat CDRs:
TABLE-US-00497 CDR 1: KSSQRVLYSSNNKNYLA (SEQ ID NO: 956) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYYSRPYT (SEQ ID NO: 958)
[0814] TCN-538 (3206_O17) Light Chain Chothia CDRs:
TABLE-US-00498 CDR 1: KSSQRVLYSSNNKNYLA (SEQ ID NO: 956) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYYSRPYT (SEQ ID NO: 958)
[0815] TCN-539 (5056_A08) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00499 (SEQ ID NO: 959)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGAAATCACCTTCATTACCTATGCTA
TGCACTGGGTCCGCCAGGCCCCAGGCAAGGGGCTGGAGTGGGTGGCACTT
ATATCAGATGATGGAAGCAATAAATTCTACGCAGACTCCGTGAAGGGCCG
ATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGAGAGCTGAGGACACGGCTGCTTATTACTGTGCGAGAGAAGGG
GTTTACTTTGATTCGGGGACTTATAGGGGCTACTTTGACTACTGGGGCCA
GGAAACCCTGGTCACCGTCTCGAGC
[0816] TCN-539 (5056_A08) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00500 (SEQ ID NO: 960)
QVQLVESGGGVVQPGRSLRLSCAASEITFITYAMHWVRQAPGKGLEWVAL
ISDDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAAYYCAREG
VYFDSGTYRGYFDYWGQETLVTVSS
[0817] TCN-539 (5056_A08) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00501 CDR 1: TYAMH (SEQ ID NO: 961) CDR 2:
LISDDGSNKFYADSVKG (SEQ ID NO: 962) CDR 3: EGVYFDSGTYRGYFDY (SEQ ID
NO: 963)
[0818] TCN-539 (5056_A08) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00502 CDR 1: EITFIT (SEQ ID NO: 964) CDR 2: LISDDGSNKF
(SEQ ID NO: 965) CDR 3: EGVYFDSGTYRGYFDY (SEQ ID NO: 963)
[0819] TCN-539 (5056_A08) light chain variable region nucleotide
sequence:
TABLE-US-00503 (SEQ ID NO: 966)
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAG
CCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT
GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTG
CAGTTTATTACTGTCAGCAGCGTAGCCACTGGCCTCCGATCACCTTCGGC
CAAGGGACACGACTGGAGATCAAA
[0820] TCN-539 (5056_A08) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00504 (SEQ ID NO: 967)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSHWPPITFG QGTRLEIK
[0821] TCN-539 (5056_A08) Light Chain Kabat CDRs:
TABLE-US-00505 CDR 1: RASQSVSSYLA (SEQ ID NO: 968) CDR 2: DASNRAT
(SEQ ID NO: 969) CDR 3: QQRSHWPPIT (SEQ ID NO: 970)
[0822] TCN-539 (5056_A08) Light Chain Chothia CDRs:
TABLE-US-00506 CDR 1: RASQSVSSYLA (SEQ ID NO: 968) CDR 2: DASNRAT
(SEQ ID NO: 969) CDR 3: QQRSHWPPIT (SEQ ID NO: 970)
[0823] TCN-540 (5060_F05) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00507 (SEQ ID NO: 971)
CAGGTGCAGCTGGTACAATCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACGCCA
TGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCTATT
ATATCATACGACGGAAATGATCAATACTATACAGACTCCGTGAAGGGCCG
ATTCACCATCTCCAGAGACAGCTCCAAAGTGTATCTCCAAATGCACAGGC
TGAGACCTGAGGACACGGCTGTTTATTACTGTGCGAAAGAATTTGAAACT
AGTGGTTATTTTCATGGGAGTTTTGACTACTGGGGCCAGGGAATCCTGGT
CACCGTCTCGAGC
[0824] TCN-540 (5060_F05) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00508 (SEQ ID NO: 972)
QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAI
ISYDGNDQYYTDSVKGRFTISRDSSKVYLQMHRLRPEDTAVYYCAKEFET
SGYFHGSFDYWGQGILVTVSS
[0825] TCN-540 (5060_F05) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00509 CDR 1: SYAMH (SEQ ID NO: 973) CDR 2:
IISYDGNDQYYTDSVKG (SEQ ID NO: 974) CDR 3: EFETSGYFHGSFDY (SEQ ID
NO: 975)
[0826] TCN-540 (5060_F05) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00510 CDR 1: GFTFSS (SEQ ID NO: 976) CDR 2: IISYDGNDQY
(SEQ ID NO: 977) CDR 3: EFETSGYFHGSFDY (SEQ ID NO: 975)
[0827] TCN-540 (5060_F05) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00511 (SEQ ID NO: 978)
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTC
GATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACT
ATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCTTGATT
TATGAGGTCACTAATTGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGG
ACGAGGCTGACTATTACTGCAGCTCATATGCGGGCAGCAGCACTTGGGTG
TTCGGCGGAGGGACCAGGGTGACCGTTCTA
[0828] TCN-540 (5060_F05) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00512 (SEQ ID NO: 979)
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLLI
YEVTNWPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYAGSSTWV FGGGTRVTVL
[0829] TCN-540 (5060_F05) Light chain Kabat CDRs:
TABLE-US-00513 CDR 1: TGTSSDVGGYNYVS (SEQ ID NO: 980) CDR 2:
EVTNWPS (SEQ ID NO: 981) CDR 3: SSYAGSSTWV (SEQ ID NO: 982)
[0830] TCN-540 (5060_F05) Light Chain Chothia CDRs:
TABLE-US-00514 CDR 1: TGTSSDVGGYNYVS (SEQ ID NO: 980) CDR 2:
EVTNWPS (SEQ ID NO: 981) CDR 3: SSYAGSSTWV (SEQ ID NO: 982)
[0831] TCN-541 (5062_M11) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00515 (SEQ ID NO: 983)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAATAGTTACTACT
GGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGCTAT
ATCTATCACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGT
CACCATTTCGGTAGACACGTCCAAGAACCAGTTCTCCCTGCAGCTGAGCT
CTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGACTCCGGACG
GACTACGGTGACCCCGACTCGGTATACTACTACGGTATGGACGTCTGGGG
CCAAGGGACCACGGTCACCGTCTCGAGC
[0832] TCN-541 (5062_M11) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00516 (SEQ ID NO: 984)
QVQLQESGPGLVKPSETLSLTCTVSGGSINSYYWNWIRQPPGKGLEWIGY
IYHSGSTNYNPSLKSRVTISVDTSKNQFSLQLSSVTAADTAVYYCARLRT
DYGDPDSVYYYGMDVWGQGTTVTVSS
[0833] TCN-541 (5062_M11) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00517 CDR 1: SYYWN (SEQ ID NO: 985) CDR 2:
YIYHSGSTNYNPSLKS (SEQ ID NO: 986) CDR 3: LRTDYGDPDSVYYYGMDV (SEQ ID
NO: 987)
[0834] TCN-541 (5062_M11) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00518 CDR 1: GGSINS (SEQ ID NO: 988) CDR 2: YIYHSGSTN (SEQ
ID NO: 989) CDR 3: LRTDYGDPDSVYYYGMDV (SEQ ID NO: 987)
[0835] TCN-541 (5062_M11) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00519 (SEQ ID NO: 990)
TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGAC
GGCCAGGATCACCTGCTCTGGAGATGCATTGCCAAAGCAAAATGCTTATT
GGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGCTGATATATAAAGAC
AGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGCTCAGG
GACAACAGTCACGTTGACCATCAGTGGAGTCCAGGCAGAGGACGAGGCTG
ACTATTACTGTCAATCAGCAGACAGCAGTGGTACTTCTTGGGTGTTCGGC
GGAGGGACCAAACTGACCGTTCTA
[0836] TCN-541 (5062_M11) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00520 (SEQ ID NO: 991)
SYELTQPPSVSVSPGQTARITCSGDALPKQNAYWYQQKPGQAPVLLIYKD
SERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTSWVFG GGTKLTVL
[0837] TCN-541 (5062_M11) Light Chain Kabat CDRs:
TABLE-US-00521 CDR 1: SGDALPKQNAY (SEQ ID NO: 994) CDR 2: KDSERPS
(SEQ ID NO: 995) CDR 3: QSADSSGTSWV (SEQ ID NO: 996)
[0838] TCN-541 (5062_M11) Light Chain Chothia CDRs:
TABLE-US-00522 CDR 1: SGDALPKQNAY (SEQ ID NO: 994) CDR 2: KDSERPS
(SEQ ID NO: 995) CDR 3: QSADSSGTSWV (SEQ ID NO: 996)
[0839] TCN-542 (5079_A16) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00523 (SEQ ID NO: 992)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTAATT
ACTACTGGAACTGGGTCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT
GGGTACATCTATTACAGAGGGAGCACCTTCTACAACCCGTCCCTCAAGAG
TCGAGTGACCATATCAATAGACACGTCTAAGAACCAGTTCTCCCTGAGGC
TGAGCTCTGTGACGGCCGCGGACACGGCCGTGTATTACTGTGCGAAGGAT
ACAAGGTCGAGCCTAGACAATTACCAGTACGGTATGGACGTCTGGGGCCA
AGGGACCACGGTCACCGTCTCGAGC
[0840] TCN-542 (5079_A16) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00524 (SEQ ID NO: 993)
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGNYYWNWVRQHPGKGLEWI
GYIYYRGSTFYNPSLKSRVTISIDTSKNQFSLRLSSVTAADTAVYYCAKD
TRSSLDNYQYGMDVWGQGTTVTVSS
[0841] TCN-542 (5079_A16) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00525 CDR 1: SGNYYWN (SEQ ID NO: 997) CDR 2:
YIYYRGSTFYNPSLKS (SEQ ID NO: 998) CDR 3: DTRSSLDNYQYGMDV (SEQ ID
NO: 999)
[0842] TCN-542 (5079_A16) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00526 CDR 1: GGSISSGN (SEQ ID NO: 1000) CDR 2: YIYYRGSTF
(SEQ ID NO: 1001) CDR 3: DTRSSLDNYQYGMDV (SEQ ID NO: 999)
[0843] TCN-542 (5079_A16) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00527 (SEQ ID NO: 1002)
CAGACTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGAC
AGTCACTCTCACCTGTGCTTCCAGCACTGGAGCAGTCACCAGTAGTTACT
TTCCAAACTGGTTCCAGCAGAAACCTGGACAAGCGCCCAGGCCACTGATT
TATAGTACAACTATCAGACACTCCTGGACCCCGGCCCGATTCTCAGGCTC
CCTCCTTGGGGGCAAAGCTGCCCTGACACTGTCAGGTGTGCAGCCTGAGG
ACGAGGCTGACTATTACTGCCTGCTCTACTCTGGTGGTGATCCAGTGGCT
TTCGGCGGAGGGACCAAACTGACCGTTCTA
[0844] TCN-542 (5079_A16) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00528 (SEQ ID NO: 1003)
QTVVTQEPSLTVSPGGTVTLTCASSTGAVTSSYFPNWFQQKPGQAPRPLI
YSTTIRHSWTPARFSGSLLGGKAALTLSGVQPEDEADYYCLLYSGGDPVA FGGGTKLTVL
[0845] TCN-542 (5079_A16) Light Chain Kabat CDRs:
TABLE-US-00529 CDR 1: ASSTGAVTSSYFPN (SEQ ID NO: 1004) CDR 2:
STTIRHS (SEQ ID NO: 1005) CDR 3: LLYSGGDPVA (SEQ ID NO: 1006)
[0846] TCN-542 (5079_A16) Light Chain Chothia CDRs:
TABLE-US-00530 CDR 1: ASSTGAVTSSYFPN (SEQ ID NO: 1004) CDR 2:
STTIRHS (SEQ ID NO: 1005) CDR 3: LLYSGGDPVA (SEQ ID NO: 1006)
[0847] TCN-543 (5081_G23) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00531 (SEQ ID NO: 1007)
CAGGTTCATCTGGTGCAGTCTGGAGCTGAGGTGAGGAAGCCTGGGGACTC
AGTGAAGGTCTCCTGTAAGACTTCTGGTTACACCTTTTCCACCTATCCTG
TCGCCTGGGTGCGACAGGTCCCCGGACAAGGGCTTGAGTGGATGGGATGG
ATCAGCACTTACAATGGAAACACAAACTTTGCACAGAACTTCCAGGGCAG
AGTCACCCTGACCACAGACACAACCACGAACACAGCCTACATGGAAGTGA
GGAGCCTGAAATTTGACGACACGGCCGTCTATTACTGTGCGAGAGTGGAA
GGCTCGTACAGGGATTTTTGGAATAATCAAAACAGATTCGACCCCTGGGG
CCAGGGAACCCTGGTCACCGTCTCGAGC
[0848] TCN-543 (5081_G23) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00532 (SEQ ID NO: 1008)
QVHLVQSGAEVRKPGDSVKVSCKTSGYTFSTYPVAWVRQVPGQGLEWMGW
ISTYNGNTNFAQNFQGRVTLTTDTTTNTAYMEVRSLKFDDTAVYYCARVE
GSYRDFWNNQNRFDPWGQGTLVTVSS
[0849] TCN-543 (5081_G23) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00533 CDR 1: TYPVA (SEQ ID NO: 1009) CDR 2:
WISTYNGNTNFAQNFQG (SEQ ID NO: 1010) CDR 3: VEGSYRDFWNNQNRFDP (SEQ
ID NO: 1011)
[0850] TCN-543 (5081_G23) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00534 CDR 1: GYTFST (SEQ ID NO: 1012) CDR 2: WISTYNGNTN
(SEQ ID NO: 1013) CDR 3: VEGSYRDFWNNQNRFDP (SEQ ID NO: 1011)
[0851] TCN-543 (5081_G23) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00535 (SEQ ID NO: 1014)
TCCTATGTACTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGAC
GGCCAGGATTTCCTGTGGGGGAAGCAACATTGGAGGGAAAAGTGTGCACT
GGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGAT
AGCGGCCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGG
GGACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCG
ACTATTTCTGTCAGGTGTGGGATAATTTCGGGGGAGTCTTCGGAACTGGG
ACCAAGGTCACCGTTCTA
[0852] TCN-543 (5081_G23) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00536 (SEQ ID NO: 1015)
SYVLTQPPSVSVAPGQTARISCGGSNIGGKSVHWYQQKPGQAPVLVVYDD
SGRPSGIPERFSGSNSGDTATLTISRVEAGDEADYFCQVWDNFGGVFGTG TKVTVL
[0853] TCN-543 (5081_G23) Light Chain Kabat CDRs:
TABLE-US-00537 CDR 1: GGSNIGGKSVH (SEQ ID NO: 1016) CDR 2: DDSGRPS
(SEQ ID NO: 1017) CDR 3: QVWDNFGGV (SEQ ID NO: 1018)
[0854] TCN-543 (5081_G23) Light Chain Chothia CDRs:
TABLE-US-00538 CDR 1: GGSNIGGKSVH (SEQ ID NO: 1016) CDR 2: DDSGRPS
(SEQ ID NO: 1017) CDR 3: QVWDNFGGV (SEQ ID NO: 1018)
[0855] TCN-544 (5082_A19) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00539 (SEQ ID NO: 1019)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGGCTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTCGTGGCTCCATCGGTCATTACTTCT
GGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGTTAT
ATCTCTTACAGTGGGAGCACCAAGTACAACCCCTCCCTCAGGAGTCGAGT
CACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAATCTGAACT
CTGTCACCGCTACGGACACGGCCCTATATTACTGTGCGAGAGAGGATTAC
GATATTTTGACTGGGGCGGGACCCGGTATGGAGGTCTGGGGCCAAGGGAC
CACGGTCACCGTCTCGAGC
[0856] TCN-544 (5082_A19) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00540 (SEQ ID NO: 1020)
QVQLQESGPGLVKPSETLSLTCTVSRGSIGHYFWSWIRQPPGKGLEWIGY
ISYSGSTKYNPSLRSRVTISVDTSKNQFSLNLNSVTATDTALYYCAREDY
DILTGAGPGMEVWGQGTTVTVSS
[0857] TCN-544 (5082_A19) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00541 CDR 1: HYFWS (SEQ ID NO: 1021) CDR 2:
YISYSGSTKYNPSLRS (SEQ ID NO: 1022) CDR 3: EDYDILTGAGPGMEV (SEQ ID
NO: 1023)
[0858] TCN-544 (5082_A19) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00542 CDR 1: RGSIGH (SEQ ID NO: 1024) CDR 2: YISYSGSTK
(SEQ ID NO: 1025) CDR 3: EDYDILTGAGPGMEV (SEQ ID NO: 1023)
[0859] TCN-544 (5082_A19) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00543 (SEQ ID NO: 1026)
CAGTCTATGTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGTTCTGGGAGCAGCTCCAACATCGGAAGTAATACTG
TCAACTGGTTCAAACATCTCCCAGGAACGGCCCCCAAACTCCTCATCTAC
AGAAATGATCTGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAA
GTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATG
AGGCTGATTATTACTGTGCAACATGGGATGACAGCCTGAATGGTTTTTAT
GTCTTCGGAACTGGGACCAAAGTCACCGTTCTA
[0860] TCN-544 (5082_A19) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00544 (SEQ ID NO: 1027)
QSMLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWFKHLPGTAPKLLIY
RNDLRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSLNGFY VFGTGTKVTVL
[0861] TCN-544 (5082_A19) Light Chain Kabat CDRs:
TABLE-US-00545 CDR 1: SGSSSNIGSNTVN (SEQ ID NO: 1028) CDR 2:
RNDLRPS (SEQ ID NO: 1029) CDR 3: ATWDDSLNGFYV (SEQ ID NO: 1030)
[0862] TCN-544 (5082_A19) Light Chain Chothia CDRs:
TABLE-US-00546 CDR 1: SGSSSNIGSNTVN (SEQ ID NO: 1028) CDR 2:
RNDLRPS (SEQ ID NO: 1029) CDR 3: ATWDDSLNGFYV (SEQ ID NO: 1030)
[0863] TCN-545 (5082_I15) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00547 (SEQ ID NO: 1031)
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGAC
CCTGTCCCTCTCCTGCGCTGTCTTTGGTGGGTCCTTCAGTGATTACTACT
GGACCTGGATACGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGCGAA
ATCAAACATAGTGGAAGAACCAACTACAACCCGTCCCTTGAGAGTCGAGT
CACCATATCAGTGGACACGTCCAAGAACCAGTTTTCCCTGAAACTGAGTT
CTGTGACCGCCGCGGACACGGCTATATATTATTGTGCGAGAGGGACAGAC
CCTGACACGGAGGGATATTGTCGTAGTGGTAGCTGCTCGGCCTTTGACTT
CTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC
[0864] TCN-545 (5082_I15) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in bold, Chothia CDRs underlined)
TABLE-US-00548 (SEQ ID NO: 1032)
QVQLQQWGAGLLKPSETLSLSCAVFGGSFSDYYWTWIRQPPGKGLEWIGE
IKHSGRTNYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAIYYCARGTD
PDTEGYCRSGSCSAFDFWGQGTLVTVSS
[0865] TCN-545 (5082_I15) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00549 CDR 1: DYYWT (SEQ ID NO: 1033) CDR 2:
EIKHSGRTNYNPSLES (SEQ ID NO: 1034) CDR 3: GTDPDTEGYCRSGSCSAFDF (SEQ
ID NO: 1035)
[0866] TCN-545 (5082_I15) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00550 CDR 1: GGSFSD (SEQ ID NO: 1036) CDR 2: EIKHSGRTN
(SEQ ID NO: 1037) CDR 3: GTDPDTEGYCRSGSCSAFDF (SEQ ID NO: 1035)
[0867] TCN-545 (5082_I15) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00551 (SEQ ID NO: 1038)
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCACTTTGTGAACTACAGGTCCT
TAGCCTGGTACCAGCAGACACCTGGCCAGGTTCCCAGGCTCCTCATCTAT
GGTGCGTCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT
TTGCAGTGTATTTCTGTCAGCAGTATGGTGGCTCACCTAGGTACACTTTT
GGCCAGGGGACCAGGCTGGAGATCAAA
[0868] TCN-545 (5082_I15) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00552 (SEQ ID NO: 1039)
EIVLTQSPGTLSLSPGERATLSCRASHFVNYRSLAWYQQTPGQVPRLLIY
GASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYGGSPRYTF GQGTRLEIK
[0869] TCN-545 (5082_I15) Light Chain Kabat CDRs:
TABLE-US-00553 CDR 1: RASHFVNYRSLA (SEQ ID NO: 1040) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QQYGGSPRYT (SEQ ID NO: 1041)
[0870] TCN-545 (5082_I15) Light Chain Chothia CDRs:
TABLE-US-00554 CDR 1: RASHFVNYRSLA (SEQ ID NO: 1040) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QQYGGSPRYT (SEQ ID NO: 1041)
[0871] TCN-546 (5089_L08) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00555 (SEQ ID NO: 1042)
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCGGTGTCTATGGTGGGTCCCTCAGTGATTACTACT
GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGAGAA
ATCAATCATAGTGGAGGCACCAACTACAATCCGTCCCTCAAGAGACGAGT
CACCATATCAGTAGACACGTCAAAGAAGCAATTCTCCCTGAAGATGAACT
CTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGGGACAGAC
CCTGACACGGAAGTATATTGTCGTGCTGGTAACTGCGCGGCCTTTGACTT
CTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC
[0872] TCN-546 (5089_L08) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00556 (SEQ ID NO: 1043)
QVQLQQWGAGLLKPSETLSLTCGVYGGSLSDYYWSWIRQPPGKGLEWIGE
INHSGGTNYNPSLKRRVTISVDTSKKQFSLKMNSVTAADTAVYYCARGTD
PDTEVYCRAGNCAAFDFWGQGTLVTVSS
[0873] TCN-546 (5089_L08) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00557 CDR 1: DYYWS (SEQ ID NO: 1044) CDR 2:
EINHSGGTNYNPSLKR (SEQ ID NO: 1045) CDR 3: GTDPDTEVYCRAGNCAAFDF (SEQ
ID NO: 1046)
[0874] TCN-546 (5089_L08) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00558 CDR 1: GGSLSD (SEQ ID NO: 1047) CDR 2: EINHSGGTN
(SEQ ID NO: 1048) CDR 3: GTDPDTEVYCRAGNCAAFDF (SEQ ID NO: 1046)
[0875] TCN-546 (5089_L08) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00559 (SEQ ID NO: 1049)
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
GAGAGCCACCCTCTCCTGCCGGGCCAGTCACTTTGTTATAGGCAGGGCTG
TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAC
GGTGCATCCAGCAGGGCCACTGGCATCCCGGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGACTGAAGATT
TTGCTGTGTTTTACTGTCAGCACTATGGTAGCTCACCTAGGTACGCTTTT
GGCCAGGGGACCAAGCTGGAGATCAAA
[0876] TCN-546 (5089_L08) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00560 (SEQ ID NO: 1050)
EIVLTQSPGTLSLSPGERATLSCRASHFVIGRAVAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLETEDFAVFYCQHYGSSPRYAF GQGTKLEIK
[0877] TCN-546 (5089_L08) Light Chain Kabat CDRs:
TABLE-US-00561 CDR 1: RASHFVIGRAVA (SEQ ID NO: 1051) CDR 2: GASSRAT
(SEQ ID NO: 768) CDR 3: QHYGSSPRYAF (SEQ ID NO: 1052)
[0878] TCN-546 (5089_L08) Light Chain Chothia CDRs:
TABLE-US-00562 CDR 1: RASHFVIGRAVA (SEQ ID NO: 1051) CDR 2: GASSRAT
(SEQ ID NO: 768) CDR 3: QHYGSSPRYAF (SEQ ID NO: 1052)
[0879] TCN-547 (5092_F11) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00563 (SEQ ID NO: 1053)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATTAGTAGTGTTGATC
ACTACTGGAGCTGGATCCGCCAACACCCAGTGAAGGGCCTGGAGTGGATT
GGGTTCATGTATTACAGTGCGAGCACCTATTACAACCCGTCCCTCAAGAG
TCGAGTTACCATATCAACGGACACGTCTAAGAACCAGTTCTCCCTGAGGC
TGAGTTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGAGAGGC
ACTTGTGCTGGTGACTGCTCCCTTCACTACTACTACTACGGTTTGGACGT
CTGGGGCCAAGGGAGGACGGTCACCGTCTCGAGC
[0880] TCN-547 (5092_F11) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00564 (SEQ ID NO: 1054)
QVQLQESGPGLVKPSQTLSLTCTVSGDSISSVDHYWSWIRQHPVKGLEWI
GFMYYSASTYYNPSLKSRVTISTDTSKNQFSLRLSSVTAADTAVYYCARG
TCAGDCSLHYYYYGLDVWGQGRTVTVSS
[0881] TCN-547 (5092_F11) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00565 CDR 1: SVDHYWS (SEQ ID NO: 1055) CDR 2:
FMYYSASTYYNPSLKS (SEQ ID NO: 1056) CDR 3: GTCAGDCSLHYYYYGLDV (SEQ
ID NO: 1057)
[0882] TCN-547 (5092_F11) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00566 CDR 1: GDSISSVD (SEQ ID NO: 1058) CDR 2: FMYYSASTY
(SEQ ID NO: 1059) CDR 3: GTCAGDCSLHYYYYGLDV (SEQ ID NO: 1057)
[0883] TCN-547 (5092_F11) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00567 (SEQ ID NO: 1060)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA
ATTGGTATCAGCACAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCT
GTATCCATTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGGCAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAGTTCCCCGCTCACTTTCGGCGGA
GGGACCAAGGTGGAGATCAAA
[0884] TCN-547 (5092_F11) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00568 (SEQ ID NO: 1061)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQHKPGKAPKVLMYA
VSILQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQSYSSPLTFGG GTKVEIK
[0885] TCN-547 (5092_F11) Light Chain Kabat CDRs:
TABLE-US-00569 CDR 1: RASQSISSYLN (SEQ ID NO: 1062) CDR 2: AVSILQS
(SEQ ID NO: 1063) CDR 3: QQSYSSPLT (SEQ ID NO: 1064)
[0886] TCN-547 (5092_F11) Light Chain Chothia CDRs:
TABLE-US-00570 CDR 1: RASQSISSYLN (SEQ ID NO: 1062) CDR 2: AVSILQS
(SEQ ID NO: 1063) CDR 3: QQSYSSPLT (SEQ ID NO: 1064)
[0887] TCN-548 (5092_P01) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00571 (SEQ ID NO: 1065)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTAGTGGCCCCATGAGTGATTATTACT
GGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGCAT
GTCTCTGTCTCTCACGGAGGGAGGACCAAATCCAATCCCTCCGTCATGAG
TCGAGTCACCATTTCAGTAGAAACGTCCAAGAACCAATTCTCCCTGAAAC
TGACCTCCGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGATTA
AATTACTATGATAGAAGTGGTTATCATTCGCCTGACGGCCCCTCGAACAA
CTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC
[0888] TCN-548 (5092_P01) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00572 (SEQ ID NO: 1066)
QVQLQESGPGLVKPSETLSLTCTVSSGPMSDYYWSWIRQPPGKGLEWIGH
VSVSHGGRTKSNPSVMSRVTISVETSKNQFSLKLTSVTAADTAVYYCARL
NYYDRSGYHSPDGPSNNWFDPWGQGTLVTVSS
[0889] TCN-548 (5092_P01) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00573 CDR 1: DYYWS (SEQ ID NO: 1044) CDR 2:
HVSVSHGGRTKSNPSVMS (SEQ ID NO: 1067) CDR 3: LNYYDRSGYHSPDGPSNNWFDP
(SEQ ID NO: 1068)
[0890] TCN-548 (5092_P01) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00574 CDR 1: SGPMSD (SEQ ID NO: 1069) CDR 2: HVSVSHGGRTK
(SEQ ID NO: 1070) CDR 3: LNYYDRSGYHSPDGPSNNWFDP (SEQ ID NO:
1068)
[0891] TCN-548 (5092_P01) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00575 (SEQ ID NO: 1071)
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGA
GAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCA
ACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT
AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG
AATCAGCGGCAGCGGGTCTGGGGCAGATTTCACTCTCACCATCAGCAGCC
TGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTATTTTGCTACT
CCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
[0892] TCN-548 (5092_P01) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00576 (SEQ ID NO: 1072)
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRISGSGSGADFTLTISSLQAEDVAVYYCQQYFAT
PRTFGQGTKVEIK
[0893] TCN-548 (5092_P01) Light Chain Kabat CDRs:
TABLE-US-00577 CDR 1: KSSQSVLYSSNNKNYLA (SEQ ID NO: 1073) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYFATPRT (SEQ ID NO: 1074)
[0894] TCN-548 (5092_P01) Light Chain Chothia CDRs:
TABLE-US-00578 CDR 1: KSSQSVLYSSNNKNYLA (SEQ ID NO: 1073) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYFATPRT (SEQ ID NO: 1074)
[0895] TCN-549 (5092_P04) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00579 (SEQ ID NO: 1075)
CAGGTACAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATA
TGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAACCCTAACAGTGGTGACACAAACTATGCACAGAAGTTTCAGGGCAG
GGTCACCATGACCAGGGACACGTCCATCACCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATTCC
CCCTATAGCAGCAGCTGGTCCTTCTTTGACTACTGGGGCCAGGGACCCCT
GGTCACCGTCTCGAGC
[0896] TCN-549 (5092_P04) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00580 (SEQ ID NO: 1076)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGW
INPNSGDTNYAQKFQGRVTMTRDTSITTAYMELSSLRSDDTAVYYCARDS
PYSSSWSFFDYWGQGPLVTVSS
[0897] TCN-549 (5092_P04) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00581 CDR 1: GYYMH (SEQ ID NO: 1077) CDR 2:
WINPNSGDTNYAQKFQG (SEQ ID NO: 1078) CDR 3: DSPYSSSWSFFDY (SEQ ID
NO: 1079)
[0898] TCN-549 (5092_P04) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00582 CDR 1: GYTFTG (SEQ ID NO: 1080) CDR 2: WINPNSGDTN
(SEQ ID NO: 1081) CDR 3: DSPYSSSWSFFDY (SEQ ID NO: 1079)
[0899] TCN-549 (5092_P04) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00583 (SEQ ID NO: 1082)
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGA
GAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCA
ACAATAAGAGCCACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT
AAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG
ATTCAGTGGCAGCGGGTCTGGGACAGATTTCACCCTCATCATCAGCAGCC
TGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAATATTATTTTTCT
CCCCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
[0900] TCN-549 (5092_P04) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00584 (SEQ ID NO: 1083)
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKSHLAWYQQKPGQPP
VKLLIYWASTRESGPDRFSGSGSGTDFTLIISSLQAEDVAVYYCQQYYFS
PLTFGGGTKVEIK
[0901] TCN-549 (5092_P04) Light Chain Kabat CDRs:
TABLE-US-00585 CDR 1: KSSQSVLYSSNNKSHLA (SEQ ID NO: 1084) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYYFSPLT (SEQ ID NO: 1085)
[0902] TCN-549 (5092_P04) Light Chain Chothia CDRs:
TABLE-US-00586 CDR 1: KSSQSVLYSSNNKSHLA (SEQ ID NO: 1084) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYYFSPLT (SEQ ID NO: 1085)
[0903] TCN-550 (5096_F06) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00587 (SEQ ID NO: 1086)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGCCTCCATCAATAGTCACTACT
GGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT
GTCTATTACAGTGGGAGCACCACCTACAACCCCTCCCTCAAGAGTCGAGT
CACCTTATCAGTAGATACGTCCAAGAACCAGTTCTCCCTGAACCTGAGCT
CTGTGACCGCCGCAGACACGGCCTTCTATTACTGTGCGAGACATCCCTAC
GATGTTTTGACTGGTTCCGGGGACTGGTTCGACCCCTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGC
[0904] TCN-550 (5096_F06) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00588 (SEQ ID NO: 1087)
QVQLQESGPGLVKPSETLSLTCTVSGASINSHYWSWIRQPPGKGLEWIGY
VYYSGSTTYNPSLKSRVTLSVDTSKNQFSLNLSSVTAADTAFYYCARHPY
DVLTGSGDWFDPWGQGTLVTVSS
[0905] TCN-550 (5096_F06) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00589 CDR 1: SHYWS (SEQ ID NO: 1088) CDR 2:
YVYYSGSTTYNPSLKS (SEQ ID NO: 1089) CDR 3: HPYDVLTGSGDWFDP (SEQ ID
NO: 1090)
[0906] TCN-550 (5096_F06) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00590 CDR 1: GASINSH (SEQ ID NO: 1091) CDR 2: YVYYSGSTT
(SEQ ID NO: 1092) CDR 3: HPYDVLTGSGDWFDP (SEQ ID NO: 1090)
[0907] TCN-550 (5096_F06) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00591 (SEQ ID NO: 1093)
TCCTATGTTCTGACTCAGGCACCCTCGGTGTCAGTGGCCCCAGGACAGAC
GGCCAGGATTACCTGTGGGGGAAATGCCATTGGAAGTAAAAAAGTTCACT
GGTACCAGCACAAGGCAGGCCAGGCCCCTGTACTCGTCGTCTATGATGAT
ACAGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTTG
GAGCACGGCCACCCTGACCATCAACAGGGTCGAAGCCGGGGATGAGGCCG
ACTATTACTGTCAGGTGTGGGATTTTACCATTGATCATGTGGTCTTCGGC
GGAGGGACCAAGCTGACCGTTCTA
[0908] TCN-550 (5096_F06) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00592 (SEQ ID NO: 1094)
SYVLTQAPSVSVAPGQTARITCGGNAIGSKKVHWYQHKAGQAPVLVVYDD
TDRPSGIPERFSGSNSWSTATLTINRVEAGDEADYYCQVWDFTIDHVVFG GGTKLTVL
[0909] TCN-550 (5096_F06) Light Chain Kabat CDRs:
TABLE-US-00593 CDR 1: GGNAIGSKKVH (SEQ ID NO: 1095) CDR 2: DDTDRPS
(SEQ ID NO: 1096) CDR 3: QVWDFTIDHVV (SEQ ID NO: 1097)
[0910] TCN-550 (5096_F06) Light Chain Chothia CDRs:
TABLE-US-00594 CDR 1: GGNAIGSKKVH (SEQ ID NO: 1095) CDR 2: DDTDRPS
(SEQ ID NO: 1096) CDR 3: QVWDFTIDHVV (SEQ ID NO: 1097)
[0911] TCN-551 (5243_D01) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00595 (SEQ ID NO: 1098)
GAGGTGCAACTGGTTCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTC
TCTGAAGATCTCCTGTAAGGGTTCTGGCTACAGCTTTAGCAACTACTGGA
TCGGCTGGGTGCGCCACATGCCCGGGAAAGGCCTGGAATGGATGGGGATC
ATTTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCA
GGTCACCATGTCAGCCGACAAGTCCAGCAGCACCGTCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATTTATTATTGTGCGAGACGGGGC
GGACATAGTTTTGGATATGGGTCGGGGGGGGACACGCACAGTGAATTCGA
CTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC
[0912] TCN-551 (5243_D01) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00596 (SEQ ID NO: 1099)
EVQLVQSGSEVKKPGESLKISCKGSGYSFSNYWIGWVRHMPGKGLEWMGI
IYPGDSDTRYSPSFQGQVTMSADKSSSTVYLQWSSLKASDTAIYYCARRG
GHSFGYGSGGDTHSEFDSWGQGTLV TVSS
[0913] TCN-551 (5243_D01) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00597 CDR 1: NYWIG (SEQ ID NO: 1100) CDR 2:
IIYPGDSDTRYSPSFQG (SEQ ID NO: 1101) CDR 3: RGGHSFGYGSGGDTHSEFDS
(SEQ ID NO: 1102)
[0914] TCN-551 (5243_D01) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00598 CDR 1: GYSFSN (SEQ ID NO: 1103) CDR 2: IIYPGDSDTR
(SEQ ID NO: 1104) CDR 3: RGGHSFGYGSGGDTHSEFDS (SEQ ID NO: 1102)
[0915] TCN-551 (5243_D01) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00599 (SEQ ID NO: 1105)
CAGTCTGTATTGACGCAGTCGCCCTCAGTGTCTGCGGCCCCAGGACAGAA
GGTCACCATCTCCTGCTCTGGAAGCGACTCCAACATTGGTGATTATTTTG
TATGTTGGTACCAGCACCTCCCAGGAAAACCCCCCCAACTCCTCATCTAT
GAAAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAA
GTCTGGCACGTCAGCCACCCTGGGCATCACCGGAATCCAGACCGGGGACG
AGGCCGATTACTACTGCGCAACTTGGGATGGCAGCCTGAGTGCTTGGGTG
TTCGGCGGAGGGACCAAGCTGACCGTTCTA
[0916] TCN-551 (5243_D01) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00600 (SEQ ID NO: 1106)
QSVLTQSPSVSAAPGQKVTISCSGSDSNIGDYFVCWYQHLPGKPPQLLIY
ENNKRPSGIPDRFSGSKSGTSATLGITGIQTGDEADYYCATWDGSLSAWV FGGGTKLTVL
[0917] TCN-551 (5243_D01) Light Chain Kabat CDRs:
TABLE-US-00601 CDR 1: SGSDSNIGDYFVC (SEQ ID NO: 1107) CDR 2:
ENNKRPS (SEQ ID NO: 1108) CDR 3: ATWDGSLSAWV (SEQ ID NO: 1109)
[0918] TCN-551 (5243_D01) Light Chain Chothia CDRs:
TABLE-US-00602 CDR 1: SGSDSNIGDYFVC (SEQ ID NO: 1107) CDR 2:
ENNKRPS (SEQ ID NO: 1108) CDR 3: ATWDGSLSAWV (SEQ ID NO: 1109)
[0919] TCN-552 (5249.sub.--1123) Heavy Chain Variable Region
Nucleotide Sequence:
TABLE-US-00603 (SEQ ID NO: 1110)
CAGGTCCAAGTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAGGGTCTCCTGCCAGGCTTCTGGAGGCACCTTCATGAATTATGCTA
TCATTTGGGTGCGACGGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG
ATCATCCCTGTCTTTCCTACACCAAACTACGCACAGATGTTCCAGGGCAG
AGTCACGATTTCCACGGACGAATCCAGGAGCACATCCTTCTTGGAACTGA
CCAACCTGAGATATGAGGACACGGCCGTTTATTACTGTGCGAGGCGAATT
TATCACGGTGGTAACTCCGGCTTTGACTTCTGGGGCCAGGGAACCCTGGT
CACCGTCTCGAGC
[0920] TCN-552 (5249_I23) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00604 (SEQ ID NO: 1111)
QVQVVQSGAEVKKPGSSVRVSCQASGGTFMNYAIIWVRRAPGQGLEWMG
GIIPVFPTPNYAQMFQGRVTISTDESRSTSFLELTNLRYEDTAVYYCAR
RIYHGGNSGFDFWGQGTLVTVSS
[0921] TCN-552 (5249_I23) gamma heavy chain Kabat CDRs:
TABLE-US-00605 CDR 1: NYAII (SEQ ID NO: 1112) CDR 2:
GIIPVFPTPNYAQMFQG (SEQ ID NO: 1113) CDR 3: RIYHGGNSGFDF (SEQ ID NO:
1114)
[0922] TCN-552 (5249.sub.--1123) Gamma Heavy Chain Chothia
CDRs:
TABLE-US-00606 CDR 1: GGTFMN (SEQ ID NO: 1115) CDR 2: GIIPVFPTPN
(SEQ ID NO: 1116) CDR 3: RIYHGGNSGFDF (SEQ ID NO: 1114)
[0923] TCN-552 (5249.sub.--1123) Light Chain Variable Region
Nucleotide Sequence:
TABLE-US-00607 (SEQ ID NO: 1117)
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAACTACTTAG
CCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT
TCATCCAACAGGGCCCCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACTCTCACCATCAGCAGCCTCGCGCCTGAAGATTTTG
CAGTTTATTACTGTCAGCAGCGTAGCAAGTGGCCTCCCATGTACAGTTTT
GGCCATGGGACCAAGCTGGAGATCAAA
[0924] TCN-552 (5249_I23) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00608 (SEQ ID NO: 1118)
EIVLTQSPATLSLSPGERATLSCRASQSVGNYLAWYQQKPGQAPRLLIY
DSSNRAPGIPARFSGSGSGTDFTLTISSLAPEDFAVYYCQQRSKWPPMYS FGHGTKLEIK
[0925] TCN-552 (5249.sub.--1123) Light Chain Kabat CDRs:
TABLE-US-00609 CDR 1: RASQSVGNYLA (SEQ ID NO: 1119) CDR 2: DSSNRAP
(SEQ ID NO: 1120) CDR 3: QQRSKWPPMYS (SEQ ID NO: 1121)
[0926] TCN-552 (5249.sub.--1123) Light Chain Chothia CDRs:
TABLE-US-00610 CDR 1: RASQSVGNYLA (SEQ ID NO: 1119) CDR 2: DSSNRAP
(SEQ ID NO: 1120) CDR 3: QQRSKWPPMYS (SEQ ID NO: 1121)
[0927] TCN-553 (5261_C18) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00611 (SEQ ID NO: 1122)
CAGGTCCAGGTGGTGCAGTCTGGGACTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCCAGACTTCTGGAGGCAGGTTCATGAGTTATGCTA
TCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC
ATCGTCCCTGTCTTCGGAACAGCAAACTACGCTCAGAAGTTCCAGGGCAG
AGTCACGATCACCACGGACGATTCCACGCGCACAGCCTATATGGAGTTGA
GCAGCCTGAGAAGTGAGGACACGGCCGTTTATTACTGTGGGTTCCGATAC
GGCTCTGGTTACGGGTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGT CTCGAGC
[0928] TCN-553 (5261_C18) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00612 (SEQ ID NO: 1123)
QVQVVQSGTEVKKPGSSVKVSCQTSGGRFMSYAITWVRQAPGQGLEWMG
GIVPVFGTANYAQKFQGRVTITTDDSTRTAYMELSSLRSEDTAVYYCGF
RYGSGYGFDSWGQGTLVTVSS
[0929] TCN-553 (5261_C18) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00613 CDR 1: SYAIT (SEQ ID NO: 1124) CDR 2:
GIVPVFGTANYAQKFQG (SEQ ID NO: 1125) CDR 3: RYGSGYGFDS (SEQ ID NO:
1126)
[0930] TCN-553 (5261_C18) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00614 CDR 1: GGRFMS (SEQ ID NO: 1127) CDR 2: GIVPVFGTAN
(SEQ ID NO: 1128) CDR 3: RYGSGYGFDS (SEQ ID NO: 1126)
[0931] TCN-553 (5261_C18) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00615 (SEQ ID NO: 1129)
GAAATTGTATTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGTAGCAGCTACT
TAGCCTGGTATCAGAAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCTTCCACTAGGGCCACTGGCATCCCGGACCGGTTCACTGGCAGTGG
GTCTGGGACAGACTTCACTCTCAGCATCAGTAGACTGGAGCCTGAAGATT
TTGCAGTGTATTACTGTCAGCACTTTGGTACCTCAGTCTTCACTTTCGGC
GGAGGGACCAAGGTTGAGATCAAA
[0932] TCN-553 (5261_C18) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00616 (SEQ ID NO: 1130)
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQKKPGQAPRLLIY
GASTRATGIPDRFTGSGSGTDFTLSISRLEPEDFAVYYCQHFGTSVFTFG GGTKVEIK
[0933] TCN-553 (5261_C18) Light Chain Kabat CDRs:
TABLE-US-00617 CDR 1: RASQSVSSSYLA (SEQ ID NO: 944) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QHFGTSVFT (SEQ ID NO: 1131)
[0934] TCN-553 (5261_C18) Light Chain Chothia CDRs:
TABLE-US-00618 CDR 1: RASQSVSSSYLA (SEQ ID NO: 944) CDR 2: GASTRAT
(SEQ ID NO: 755) CDR 3: QHFGTSVFT (SEQ ID NO: 1131)
[0935] TCN-554 (5277_M05) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00619 (SEQ ID NO: 1132)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGATCTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGACTACTATA
TTCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAACCCTGAAAGTGGTGACACAAAGTATGCACAGAAGTTTCAGGGCAG
GGTCACCATGACCAGGGACACGTCCATCACCACAGCCTACATGGAGCTGG
GTAGGCTGAGATCCGACGACACGGCCGTGTATTACTGTGCGAGAGATGTA
AGTACGACCTGGAGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCAC CGTCTCGAGC
[0936] TCN-554 (5277_M05) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00620 (SEQ ID NO: 1133)
QVQLVQSGADLKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMGW
INPESGDTKYAQKFQGRVTMTRDTSITTAYMELGRLRSDDTAVYYCARDV
STTWSWFAPWGQGTLVTVSS
[0937] TCN-554 (5277_M05) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00621 CDR 1: DYYIH (SEQ ID NO: 1134) CDR 2:
WINPESGDTKYAQKFQG (SEQ ID NO: 1135) CDR 3: DVSTTWSWFAP (SEQ ID NO:
1136)
[0938] TCN-554 (5277_M05) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00622 CDR 1: GYTFTD (SEQ ID NO: 1137) CDR 2: WINPESGDTK
(SEQ ID NO: 1138) CDR 3: DVSTTWSWFAP (SEQ ID NO: 1136)
[0939] TCN-554 (5277_M05) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00623 (SEQ ID NO: 1139)
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCAGTGTCTCTGGGCGA
GAGGGCCACCATCAACTGCAGGTCCAGCCAGAGTATTTTCCACAACTCCA
ACAATGAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT
AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG
ATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCC
TGCAGGCTGAAGATGTGGCGGTTTATTTCTGTCAGCAATATTATAATGCT
CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
[0940] TCN-554 (5277_M05) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00624 (SEQ ID NO: 1140)
DIVMTQSPDSLAVSLGERATINCRSSQSIFHNSNNENYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYNA
PLTFGGGTKVEIK
[0941] TCN-554 (5277_M05) Light Chain Kabat CDRs:
TABLE-US-00625 CDR 1: RSSQSIFHNSNNENYLA (SEQ ID NO: 1141) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYYNAPLT (SEQ ID NO: 1142)
[0942] TCN-554 (5277_M05) Light Chain Chothia CDRs:
TABLE-US-00626 CDR 1: RSSQSIFHNSNNENYLA (SEQ ID NO: 1141) CDR 2:
WASTRES (SEQ ID NO: 957) CDR 3: QQYYNAPLT (SEQ ID NO: 1142)
[0943] TCN-555 (5246_L16) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00627 (SEQ ID NO: 1143)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTC
GGTGAAGGTCTCATGCACGGCTTCTGGAGGCATCTTCAGGAAGAATGCAA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAGGG
ATCATCGCAGTCTTTAACACAGCAAATTACGCGCAGAAGTTCCAGAACAG
AGTCAAAATTACCGCAGACGAGTCAGGCAATACGGCCTACATGGAGCTGA
GCAGCCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGTCACCCA
AAATATTTCTATGGTTCGGGGAGTTATCCGGACTTCTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGC
[0944] TCN-555 (5246_L16) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00628 (SEQ ID NO: 1144)
QVQLVQSGAEVKRPGSSVKVSCTASGGIFRKNAISWVRQAPGQGLEWMGG
IIAVFNTANYAQKFQNRVKITADESGNTAYMELSSLTSDDTAVYYCASHP
KYFYGSGSYPDFWGQGTLVTVSS
[0945] TCN-555 (5246_L16) gamma heavy chain Kabat CDRs:
TABLE-US-00629 CDR 1: KNAIS (SEQ ID NO: 796) CDR 2:
GIIAVFNTANYAQKFQN (SEQ ID NO: 797) CDR 3: HPKYFYGSGSYPDF (SEQ ID
NO: 798)
[0946] TCN-555 (5246_L16) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00630 CDR 1: GGIFRK (SEQ ID NO: 799) CDR 2: GIIAVFNTAN
(SEQ ID NO: 800) CDR 3: HPKYFYGSGSYPDF (SEQ ID NO: 798)
[0947] TCN-555 (5246_L16) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00631 (SEQ ID NO: 1145)
CAATCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTC
AATCACCATCTCCTGTACTGGTGGCAGCAGTGATATTGGTGCTTCTAACT
CTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCGTTATT
TTTGATGTCACTGAGCGACCCTCAGGGGTCCCGCATCGGTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGCCTGACG
ACGAGGCTGATTATTTCTGCTGCGCATATGGAGGCAAATATCTTGTGGTC
TTCGGCGGAGGGACCAAGGTGACCGTTCTA
[0948] TCN-555 (5246_L16) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00632 (SEQ ID NO: 1146)
QSALTQPRSVSGSPGQSITISCTGGSSDIGASNSVSWYQQHPGKAPKLVI
FDVTERPSGVPHRFSGSKSGNTASLTVSGLQPDDEADYFCCAYGGKYLVV FGGGTKVTVL
[0949] TCN-555 (5246_L16) Light Chain Kabat CDRs:
TABLE-US-00633 CDR 1: TGGSSDIGASNSVS (SEQ ID NO: 1147) CDR 2:
DVTERPS (SEQ ID NO: 804) CDR 3: CAYGGKYLVV (SEQ ID NO: 805)
[0950] TCN-555 (5246_L16) Light Chain Chothia CDRs:
TABLE-US-00634 CDR 1: TGGSSDIGASNSVS (SEQ ID NO: 1147) CDR 2:
DVTERPS (SEQ ID NO: 804) CDR 3: CAYGGKYLVV (SEQ ID NO: 805)
[0951] TCN-556 (5089_K12) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00635 (SEQ ID NO: 1148)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAACCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCATCGGCTATGATA
TGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAACGCTAAAAGAGGTGGCACAAACTATGCACAAAAGTTTCAGGGCAG
GGTCACCATGACCAGGGACACGTCTATCAGCACAGCCTACATGGAGCTGA
ACAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGGGGTG
GGGTCACGAACTACGATTTTTGGAGTTCTCAACCCGGAATTTGACTACTG
GGGCCAGGGAACCCTGGTCACCGTCTCGAGC
[0952] TCN-556 (5089_K12) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00636 (SEQ ID NO: 1149)
QVQLVQSGAEVKKPGASVKVSCKASGYTFIGYDMHWVRQAPGQGLEWMGW
INAKRGGTNYAQKFQGRVTMTRDTSISTAYMELNSLRSDDTAVYYCARGV
GSRTTIFGVLNPEFDYWGQGTLVTVSS
[0953] TCN-556 (5089_K12) gamma heavy chain Kabat CDRs:
TABLE-US-00637 CDR 1: GYDMH (SEQ ID NO: 1150) CDR 2:
WINAKRGGTNYAQKFQG (SEQ ID NO: 1151) CDR 3: GVGSRTTIFGVLNPEFDY (SEQ
ID NO: 1152)
[0954] TCN-556 (5089_K12) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00638 CDR 1: GYTFIG (SEQ ID NO: 1153) CDR 2: WINAKRGGTN
(SEQ ID NO: 1154) CDR 3: GVGSRTTIFGVLNPEFDY (SEQ ID NO: 1152)
[0955] TCN-556 (5089_K12) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00639 (SEQ ID NO: 1155)
CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTC
AGTCACCATCTCCTGCACTGGATCCAGCAGTGACGTTGGTGGTTATGACT
ATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCCTGATT
TATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGG
ATGAGGCTGATTATTACTGCAGCTCATATGCGGGCAACTACAATCATGTC
TTCGGACCTGGGACCAAGGTCACCGTTCTA
[0956] TCN-556 (5089_K12) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00640 (SEQ ID NO: 1156)
QSALTQPPSASGSPGQSVTISCTGSSSDVGGYDYVSWYQQHPGKAPKLLI
YEVTKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGNYNHV FGPGTKVTVL
[0957] TCN-556 (5089_K12) Light Chain Kabat CDRs:
TABLE-US-00641 CDR 1: TGSSSDVGGYDYVS (SEQ ID NO: 1157) CDR 2:
EVTKRPS (SEQ ID NO: 1158) CDR 3: SSYAGNYNHV (SEQ ID NO: 1159)
[0958] TCN-556 (5089_K12) Light Chain Chothia CDRs:
TABLE-US-00642 CDR 1: TGSSSDVGGYDYVS (SEQ ID NO: 1157) CDR 2:
EVTKRPS (SEQ ID NO: 1158) CDR 3: SSYAGNYNHV (SEQ ID NO: 1159)
[0959] TCN-557 (5081_A04) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00643 (SEQ ID NO: 1160)
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGACACACCTTCACCGGCTACTACA
TACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAACCCTGACAGTGGTGCCACCAGTTCTGCACAGAACTTTCAGGGCAG
GGTCACCATGACCGGGGACACGTCCTCTAGCACAGCCTACATGGAGCTGA
GTAGGCTGAGTTTTGACGACACGGCCGTCTATTACTGTGCGAGAGTACTG
TTTACCAGTCCTTTTGACTTCTGGGGTGAGGGAACCCTGGTCACCGTCTC GAGC
[0960] TCN-557 (5081_A04) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00644 (SEQ ID NO: 1161)
QVQLVQSGAEVKKPGASVKVSCKASGHTFTGYYIHWVRQAPGQGLEWMGW
INPDSGATSSAQNFQGRVTMTGDTSSSTAYMELSRLSFDDTAVYYCARVL
FTSPFDFWGEGTLVTVSS
[0961] TCN-557 (5081_A04) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00645 CDR 1: GYYIH (SEQ ID NO: 1162) CDR 2:
WINPDSGATSSAQNFQG (SEQ ID NO: 1163) CDR 3: VLFTSPFDF (SEQ ID NO:
1164)
[0962] TCN-557 (5081_A04) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00646 CDR 1: GHTFTG (SEQ ID NO: 1165) CDR 2: WINPDSGATS
(SEQ ID NO: 1166) CDR 3: VLFTSPFDF (SEQ ID NO: 1164)
[0963] TCN-557 (5081_A04) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00647 (SEQ ID NO: 1167)
CAGGCTGTGGTGACTCAGGAGCCCTCACTGGCTGTGTCCCCAGGAGGGAC
AGTCACTCTCACCTGTGGCTCCAGCACTGGAGCTGTCACCAGGGGTCATT
ATCCCTATTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGGCACTCATT
TATGATAGTGCAGGCAACAGACACTCCTGGACTCCCGCCCGATTCTCAGG
CTCCCTCCTTGGGGGCAAAGCTGCCCTGACCCTTTCGGGTGCGCAGCCTG
AGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGTCTGGGTG
TTCGGCGGAGGGACGAAGCTGACCGTTCTA
[0964] TCN-557 (5081_A04) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00648 (SEQ ID NO: 1168)
QAVVTQEPSLAVSPGGTVTLTCGSSTGAVTRGHYPYWFQQKPGQAPRALI
YDSAGNRHSWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCLLSYSGVWV FGGGTKLTVL
[0965] TCN-557 (5081_A04) Light Chain Kabat CDRs:
TABLE-US-00649 CDR 1: GSSTGAVTRGHYPY (SEQ ID NO: 1169) CDR 2:
DSAGNRHS (SEQ ID NO: 1170) CDR 3: LLSYSGVWV (SEQ ID NO: 1171)
[0966] TCN-557 (5081_A04) Light Chain Chothia CDRs:
TABLE-US-00650 CDR 1: GSSTGAVTRGHYPY (SEQ ID NO: 1169) CDR 2:
DSAGNRHS (SEQ ID NO: 1170) CDR 3: LLSYSGVWV (SEQ ID NO: 1171)
[0967] TCN-558 (5248_H10b) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00651 (SEQ ID NO: 1172)
CAGGTCCAGCTGGTGCAATCTGGGAGTGAGGTGAAGAAGCCTGGGACCTC
GGTGAAGGTCTCCTGCACGGCCTCTGGAAGTGTCTTCACCAATTATGGAA
TTAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCATCCCTCTCTTTGGCGCAGCCAAGTACGCACAGAAATTCCAGGGCAG
AGTCACCATCACAGCGGACGAATCCACGAAGACAGTCTACATGGAGCTGA
GCAGGCTGACATCTAAAGACACGGCCATATATTTCTGTGCGAAGGCCCCC
CGTGTCTACGAGTACTACTTTGATCAGTGGGGCCAGGGAACCCCAGTCAC CGTCTCCTCA
[0968] TCN-558 (5248_H10b) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00652 (SEQ ID NO: 914)
QVQLVQSGSEVKKPGTSVKVSCTASGSVFTNYGISWVRQAPGQGLEWMGG
IIPLFGAAKYAQKFQGRVTITADESTKTVYMELSRLTSKDTAIYFCAKAP
RVYEYYFDQWGQGTPVTVSS
[0969] TCN-558 (5248_H10b) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00653 CDR 1: NYGIS (SEQ ID NO: 915) CDR 2:
GIIPLFGAAKYAQKFQG (SEQ ID NO: 916) CDR 3: APRVYEYYFDQ (SEQ ID NO:
917)
[0970] TCN-558 (5248_H10b) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00654 CDR 1: GSVFTN (SEQ ID NO: 918) CDR 2: GIIPLFGAAK
(SEQ ID NO: 919) CDR 3: APRVYEYYFDQ (SEQ ID NO: 917)
[0971] TCN-558 (5248_H10b) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00655 (SEQ ID NO: 1173)
GAAATAGTGATGACGCAGTTTCCAGCCACCCTGTCTGTGTCTCCCGGGGA
ACGAGTCACCCTCTCCTGTAGGGCCAGTCAGAGTGTTAGCAACAATTTAG
CCTGGTACCAGCAAAAACCTGGCCAGCCTCCCAGGCTCCTCATCTATGAT
GCATCTACCAGGGCCACGGGTGTCCCAGCCAAGTTCAGTGGCACTGGGTC
TGGCACAGAGTTCACTCTCAGCATCAGCAGCCTGCAGTCCGAAGATTTTG
CAGTTTATTACTGTCAGCAGTATCACAACTGGCCTCCCTCGTACAGTTTT
GGCCTGGGGACCAAGCTGGAGATCAAA
[0972] TCN-558 (5248_H10b) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00656 (SEQ ID NO: 862)
EIVMTQFPATLSVSPGERVTLSCRASOSVSNNLAWYQQKPGQPPRLLIYD
ASTRATGVPAKFSGTGSGTEFTLSISSLQSEDFAVYYCQQYHNWPPSYSF GLGTKLEIK
[0973] TCN-558 (5248_H10b) Light Chain Kabat CDRs:
TABLE-US-00657 CDR 1: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT
(SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865)
[0974] TCN-558 (5248_H10b) Light Chain Chothia CDRs:
TABLE-US-00658 CDR 1: RASQSVSNNLA (SEQ ID NO: 863) CDR 2: DASTRAT
(SEQ ID NO: 864) CDR 3: QQYHNWPPSYS (SEQ ID NO: 865)
[0975] TCN-559 (5097_G08) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00659 (SEQ ID NO: 1174)
CAAGAGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTAGAAAGTCCTTCATTGGCTACTATG
TACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAGCCCTGACAGTGATGCCACAAAGTACGCACAGAAGTTTCAGGGCTC
CGTCATCATGACCAGGGACACGTCCGTCAGCACAGTGTACATGGAGCTGA
GTAGGCTGACATCTGACGACACGGCCCTTTATTACTGTCTCCTTTTTCGA
GGTGGAAACTCCCTCTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAG C
[0976] TCN-559 (5097_G08) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in bold, Chothia CDRs underlined)
TABLE-US-00660 (SEQ ID NO: 1175)
QEQLVQSGAEVKKPGASVKVSCKASRKSFIGYYVHWVRQAPGQGLEWMGW
ISPDSDATKYAQKFQGSVIMTRDTSVSTVYMELSRLTSDDTALYYCLLFR
GGNSLSWGQGTLVTVSS
[0977] TCN-559 (5097_G08) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00661 CDR 1: GYYVH (SEQ ID NO: 1176) CDR 2:
WISPDSDATKYAQKFQG (SEQ ID NO: 1177) CDR 3: FRGGNSLS (SEQ ID NO:
1178)
[0978] TCN-559 (5097_G08) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00662 CDR 1: RKSFIG (SEQ ID NO: 1179) CDR 2: WISPDSDATK
(SEQ ID NO: 1180) CDR 3: FRGGNSLS (SEQ ID NO: 1178)
[0979] TCN-559 (5097_G08) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00663 (SEQ ID NO: 1181)
CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGAC
AGTCACCCTCACCTGTGGCTCCAGCACTGGACCTGTCACCAGTGGTCATT
ATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACATTGATT
TCTGCTACATCCAACACACACTCCTGGACACCTGCCCGCTTCTCAGGCTC
CCTCCTTGGGGGCAGAGCTGCCCTGACCCTTTCGGGTGCGCAGCCTGAGG
ATGAGGCTGACTATTATTGCTTTCTCTCCTACAGTGGTGCTTGGGTGTTC
GGCGGAGGGACCACGCTGACCGTTCTA
[0980] TCN-559 (5097_G08) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00664 (SEQ ID NO: 1182)
QAVVTQEPSLTVSPGGTVTLTCGSSTGPVTSGHYPYWFQQKPGQAPRTLI
SATSNTHSWTPARFSGSLLGGRAALTLSGAQPEDEADYYCFLSYSGAWVF GGGTTLTVL
[0981] TCN-559 (5097_G08) Light Chain Kabat CDRs:
TABLE-US-00665 CDR 1: GSSTGPVTSGHYPY (SEQ ID NO: 1183) CDR 2:
ATSNTHS (SEQ ID NO: 1184) CDR 3: FLSYSGAWV (SEQ ID NO: 1185)
[0982] TCN-559 (5097_G08) Light Chain Chothia CDRs:
TABLE-US-00666 CDR 1: GSSTGPVTSGHYPY (SEQ ID NO: 1183) CDR 2:
ATSNTHS (SEQ ID NO: 1184) CDR 3: FLSYSGAWV (SEQ ID NO: 1185)
[0983] TCN-560 (5084_P10) Heavy Chain Variable Region Nucleotide
Sequence:
TABLE-US-00667 (SEQ ID NO: 1186)
GAGGTGCAGCTGGTGGAATCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTTATCTTTAGAAATTACTGGA
TGAGCTGGGTCCGGCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC
ATAAAACAAGATGGAAGAGAGAAGTACTATGTGGACTCTCTGAGGGGCCG
AGTCAACATCTCCAGAGACAACGCCGAGAACTCATTGTATCTGCACATGA
ACAGCCTGAGAGTCGAGGACACGGCTGTTTATTTCTGTGCGAGAGCTCGG
ATGGTGGTGGTTACTGGCGATGGTTTTGATGTCTGGGGCCATGGGACAAT
GGTCACCGTCTCGAGC
[0984] TCN-560 (5084_P10) Gamma Heavy Chain Variable Region Amino
Acid Sequence: (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00668 (SEQ ID NO: 1187)
EVQLVESGGGLVQPGGSLRLSCAASGFIFRNYWMSWVRQAPGKGLEWVAN
IKQDGREKYYVDSLRGRVNISRDNAENSLYLHMNSLRVEDTAVYFCARAR
MVVVTGDGFDVWGHGTMVTVSS
[0985] TCN-560 (5084_P10) Gamma Heavy Chain Kabat CDRs:
TABLE-US-00669 CDR 1: NYWMS (SEQ ID NO: 1188) CDR 2:
NIKQDGREKYYVDSLRG (SEQ ID NO: 1189) CDR 3: ARMVVVTGDGFDV (SEQ ID
NO: 1190)
[0986] TCN-560 (5084_P10) Gamma Heavy Chain Chothia CDRs:
TABLE-US-00670 CDR 1: GFIFRN (SEQ ID NO: 1191) CDR 2: NIKQDGREKY
(SEQ ID NO: 1192) CDR 3: ARMVVVTGDGFDV (SEQ ID NO: 1190)
[0987] TCN-560 (5084_P10) Light Chain Variable Region Nucleotide
Sequence:
TABLE-US-00671 (SEQ ID NO: 1193)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAATATTAAGAGGTATTTCA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAATTTAGAAAATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTG
CGACTTATTACTGTCAGCAGAGTTTCAGTAAATCGTGGACATTCGGCCAA
GGGACCAACGTGGACATCAAA
[0988] TCN-560 (5084_P10) Light Chain Variable Region Amino Acid
Sequence (Kabat CDRs in Bold, Chothia CDRs Underlined)
TABLE-US-00672 (SEQ ID NO: 1194)
DIQMTQSPSSLSASVGDRVTITCRASQNIKRYFNWYQQKPGKAPKLLIYA
ASNLENGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSKSWTFGQ GTNVDIK
[0989] TCN-560 (5084_P10) Light Chain Kabat CDRs:
TABLE-US-00673 CDR 1: RASQNIKRYFN (SEQ ID NO: 1195) CDR 2: AASNLEN
(SEQ ID NO: 1196) CDR 3: QQSFSKSWT (SEQ ID NO: 1197)
[0990] TCN-560 (5084_P10) Light Chain Chothia CDRs:
TABLE-US-00674 CDR 1: RASQNIKRYFN (SEQ ID NO: 1195) CDR 2: AASNLEN
(SEQ ID NO: 1196) CDR 3: QQSFSKSWT (SEQ ID NO: 1197)
[0991] The invention provides an isolated fully human monoclonal
anti-HA antibody or fragment thereof, wherein said antibody
includes a variable heavy chain (V.sub.H) region comprising CDR1
and CDR2, wherein the V.sub.H region is encoded by a human IGHV1
(or specifically, IGHV1-18, IGHV1-2, IGHV1-69, IGHV1-8), IGHV2 (or
specifically, IGHV2-5), IGHV3 (or specifically, IGHV3-30, IGHV3-33,
IGHV3-49, IGHV3-53, 66, IGHV3-7), IGHV4 (or specifically, IGHV4-31,
IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61), or IGHV5 (or specifically,
IGHV5-51) V.sub.H germline sequence or an allele thereof, or a
nucleic acid sequence that is homologous to the IGHV1, IGHV2,
IGHV3, IGHV4, or IGHV5 V.sub.H germline gene sequence or an allele
thereof. In one aspect, the nucleic acid sequence that is
homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 V.sub.H
germline sequence is at least 75% homologous to the IGHV1, IGHV2,
IGHV3, IGHV4, or IGHV5 V.sub.H germline sequence or an allele
thereof. Exemplary alleles include, but are not limited to,
IGHV1-18*01, IGHV1-2*02, IGHV1-2*04, IGHV1-69*01, IGHV1-69*05,
IGHV1-69*06, IGHV1-69*12, IGHV1-8*01, IGHV2-5*10, IGHV3-30-3*01,
IGHV3-30*03, IGHV3-30*18, IGHV3-33*05, IGHV3-49*04, IGHV3-53*01,
IGHV3-66*03, IGHV3-7*01, IGHV4-31*03, IGHV4-31*06, IGHV4-34*01,
IGHV4-34*02, IGHV4-34*03, IGHV4-34*12, IGHV4-39*01, IGHV4-59*01,
IGHV4-59*03, IGHV4-61*01, IGHV4-61*08, and IGHV5-51*01. Exemplary
sequences for each allele are provided below.
[0992] IGHV1-18*01 Nucleotide Sequence (SEQ ID NO: 1198)
TABLE-US-00675 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAG
AGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGA
GGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA
[0993] IGHV1-2*02 Nucleotide Sequence (SEQ ID NO: 1199)
TABLE-US-00676 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATA
TGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAG
GGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGA
GCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA
[0994] IGHV1-2*04 Nucleotide Sequence (SEQ ID NO: 1200)
TABLE-US-00677 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATA
TGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
ATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCTG
GGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGA
GCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
[0995] IGHV1-69*01 Nucleotide Sequence (SEQ ID NO: 1201)
TABLE-US-00678 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA
[0996] IGHV1-69*05 Nucleotide Sequence (SEQ ID NO: 1202)
TABLE-US-00679 CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCACGGACGAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA
[0997] IGHV1-69*06 Nucleotide Sequence (SEQ ID NO: 1203)
TABLE-US-00680 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA
[0998] IGHV1-69*12 Nucleotide Sequence (SEQ ID NO: 1204)
TABLE-US-00681 CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGA
[0999] IGHV1-8*01 Nucleotide Sequence (SEQ ID NO: 1205)
TABLE-US-00682 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATA
TCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGG
ATGAACCCTAACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAG
AGTCACCATGACCAGGAACACCTCCATAAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGG
[1000] IGHV2-5*10 Nucleotide Sequence (SEQ ID NO: 1206)
TABLE-US-00683 CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGAC
CCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAG
TGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTT
GCACTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAG
CAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAA
TGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACGG
[1001] IGHV3-30-3*01 Nucleotide Sequence (SEQ ID NO: 1207)
TABLE-US-00684 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTA
TGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT
ATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCG
ATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGA
[1002] IGHV3-30*03 Nucleotide Sequence (SEQ ID NO: 1208)
TABLE-US-00685 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCA
TGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT
ATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCG
ATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGA
[1003] IGHV3-30*18 Nucleotide Sequence (SEQ ID NO: 1209)
TABLE-US-00686 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCA
TGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT
ATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCG
ATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGA
[1004] IGHV3-33*05 Nucleotide Sequence (SEQ ID NO: 1210)
TABLE-US-00687 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTC
CCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCA
TGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT
ATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCG
ATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGA
[1005] IGHV3-49*04 Nucleotide Sequence (SEQ ID NO: 1211)
TABLE-US-00688 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTC
CCTGAGACTCTCCTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTA
TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTC
ATTAGAAGCAAAGCTTATGGTGGGACAACAGAATACGCCGCGTCTGTGAA
AGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCTGC
AAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACTAGA GA
[1006] IGHV3-53*01 Nucleotide Sequence (SEQ ID NO: 1212)
TABLE-US-00689 GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGCAACTACA
TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTT
ATTTATAGCGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATT
CACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACA
GCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA
[1007] IGHV3-66*03 Nucleotide Sequence (SEQ ID NO: 1213)
TABLE-US-00690 CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGTTACAGTTTCACCACCTATGGTA
TGAATTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
TTCAACACCTACACTGGGAACCCAACATATGCCCAGGGCTTCACAGGACG
GTTTGTCTTCTCCATGGACACCTCTGCCAGCACAGCATACCTGCAGATCA
GCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATA
[1008] IGHV3-7*01 Nucleotide Sequence (SEQ ID NO: 1214)
TABLE-US-00691 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGA
TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC
ATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCG
ATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGA
[1009] IGHV4-31*03 Nucleotide Sequence (SEQ ID NO: 1215)
TABLE-US-00692 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTT
ACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT
GGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAG
TCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGC
TGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGA
[1010] IGHV4-31*06 Nucleotide Sequence (SEQ ID NO: 1216)
TABLE-US-00693 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTAGTT
ACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATT
GGGTACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAG
TCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGC
TGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTG
[1011] IGHV4-34*01 Nucleotide Sequence (SEQ ID NO: 1217)
TABLE-US-00694 CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTT
ACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATT
GGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAG
TCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGC
TGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACA
[1012] IGHV4-34*02 Nucleotide Sequence (SEQ ID NO: 1218)
TABLE-US-00695 CAGGTGCAGCTACAACAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACT
GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA
ATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGT
CACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCT
CTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGG
[1013] IGHV4-34*03 Nucleotide Sequence (SEQ ID NO: 1219)
TABLE-US-00696 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACT
GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA
ATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGT
CACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCT
CTGTGACCGCCGCGGACACGGCCGTGTATTACTG
[1014] IGHV4-34*12 Nucleotide Sequence (SEQ ID NO: 1220)
TABLE-US-00697 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACT
GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA
ATCATTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGT
CACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCT
CTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGA
[1015] IGHV4-39*01 Nucleotide Sequence (SEQ ID NO: 1221)
TABLE-US-00698 CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTT
ACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATT
GGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAG
TCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGC
TGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACA
[1016] IGHV4-59*01 Nucleotide Sequence (SEQ ID NO: 1222)
TABLE-US-00699 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTAAAGACTGGAGGGGT
CTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTCTGCT
ATGCACTGGGTCCACCAGGCTCCAGGAAAGGGTTTGGAGTGGGTCTCAGT
TATTAGTACAAGTGGTGATACCGTACTCTACACAGACTCTGTGAAGGGCT
GATTCACCATCTCTAGAGACAATGCCCAGAATTCACTGTATCTGCAAATG
AACAGCCTGAGAGCCGACGACATGGCTGTGTATTACTGTGTGAAAGA
[1017] IGHV4-59*03 Nucleotide Sequence (SEQ ID NO: 1223)
TABLE-US-00700 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACT
GGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTAT
ATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGT
CACCATATCAGTAGACACGTCCAAGAACCAATTCTCCCTGAAGCTGAGCT
CTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCG
[1018] IGHV4-61*01 Nucleotide Sequence (SEQ ID NO: 1224)
TABLE-US-00701 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTAGTT
ACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATT
GGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAG
TCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGC
TGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGA
[1019] IGHV4-61*08 Nucleotide Sequence (SEQ ID NO: 1225)
TABLE-US-00702 CAGGTGCAGCTGGTGCAGTCTGGCCATGAGGTGAAGCAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGGCTTCTGGTTACAGTTTCACCACCTATGGTA
TGAATTGGGTGCCACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG
TTCAACACCTACACTGGGAACCCAACATATGCCCAGGGCTTCACAGGACG
GTTTGTCTTCTCCATGGACACCTCTGCCAGCACAGCATACCTGCAGATCA
GCAGCCTAAAGGCTGAGGACATGGCCATGTATTACTGTGCGAGATA
[1020] IGHV5-51*01 Nucleotide Sequence (SEQ ID NO: 1226)
TABLE-US-00703 GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTC
TCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGA
TCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATC
ATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCA
GGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACA
[1021] In certain embodiments of the invention, the antibody
further includes a variable light chain (VL) region encoded by a
human IGKV1 (or specifically, IGKV1-17, IGKV1-27, IGKV1-39,
IGKV1D-39, IGKV1-5), IGKV2 (or specifically, IGKV2-30), IGKV3 (or
specifically, IGKV3-11, IGKV3-15, IGKV3-20), IGKV4 (or
specifically, IGKV4-1, IGKV4-1), IGLV1 (or specifically, IGLV1-40,
IGLV1-44, IGLV1-55), IGLV2 (or specifically, IGLV2-11, IGLV2-14,
IGLV2-8), IGLV3 (or specifically, IGLV3-21 or IGLV3-25), IGLV7 (or
specifically, IGLV7-43 or IGLV7-46), or IGLV9 (or specifically,
IGLV9-49) or an allele thereof. V.sub.L germline gene sequence
IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 or
an allele thereof, or a nucleotide acid sequence that is homologous
to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or
IGLV9 V.sub.L germline gene sequence or an allele thereof.
Furthermore, the nucleic acid sequence that is homologous to the
IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9
V.sub.L germline sequence or an allele thereof is at least 65%
homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3,
IGLV7, or IGLV9 V.sub.L germline sequence or an allele thereof.
[1022] IGKV1-17*01 Nucleotide Sequence (SEQ ID NO: 1227)
TABLE-US-00704 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAG
GCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTG
CAACTTATTACTGTCTACAGCATAATAGTTACCCTCC
[1023] IGKV1-27*01 Nucleotide Sequence (SEQ ID NO: 1228)
TABLE-US-00705 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAG
CCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCT
GCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTG
CAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCC
[1024] IGKV1-39*01 Nucleotide Sequence (SEQ ID NO: 1229)
TABLE-US-00706 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAGTACCCCTCC
[1025] IGKV1D-39*01 Nucleotide Sequence (SEQ ID NO: 1230)
TABLE-US-00707 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGAGTTACAGTACCCCTCC
[1026] IGKV1-5*03 Nucleotide Sequence (SEQ ID NO: 1231)
TABLE-US-00708 GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGG
CCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAG
GCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC
TGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTG
CAACTTATTACTGCCAACAGTATAATAGTTATTCTCC
[1027] IGKV2-30*02 Nucleotide Sequence (SEQ ID NO: 1232)
TABLE-US-00709 GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACA
GCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTGATG
GAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGG
CGCCTAATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATT
CAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCT CC
[1028] IGKV3-11*01 Nucleotide Sequence (SEQ ID NO: 1233)
TABLE-US-00710 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAG
CCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT
GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTG
CAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCC
[1029] IGKV3-15*01 Nucleotide Sequence (SEQ ID NO: 1234)
TABLE-US-00711 GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAG
CCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT
GCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTG
CAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCC
[1030] IGKV3-20*01 Nucleotide Sequence (SEQ ID NO: 1235)
TABLE-US-00712 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT
TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT
TTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCC
[1031] IGKV4-1*01 Nucleotide Sequence (SEQ ID NO: 1236)
TABLE-US-00713 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGA
GAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCA
ACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCT
AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG
ATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCC
TGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACT CCTCC
[1032] IGLV1-40*01 Nucleotide Sequence (SEQ ID NO: 1237)
TABLE-US-00714 CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAG
GGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATG
ATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATC
TATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTC
CAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGG
ATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTTC
[1033] IGLV1-44*01 Nucleotide Sequence (SEQ ID NO: 1238)
TABLE-US-00715 CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTG
TAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT
AGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAA
GTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATG
AGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTCC
[1034] IGLV1-51*02 Nucleotide Sequence (SEQ ID NO: 1239)
TABLE-US-00716 CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAA
GGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATG
TATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTAT
GAAAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAA
GTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACG
AGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGAGTGCTGG
[1035] IGLV2-11*01 Nucleotide Sequence (SEQ ID NO: 1240)
TABLE-US-00717 CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTC
AGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACT
ATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT
TATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGG
ATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTTC
[1036] IGLV2-14*01 Nucleotide Sequence (SEQ ID NO: 1241)
TABLE-US-00718 CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTC
GATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACT
ATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT
TATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGG
ACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACTCTC
[1037] IGLV2-8*01 Nucleotide Sequence (SEQ ID NO: 1242)
TABLE-US-00719 CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTC
AGTCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACT
ATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT
TATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTC
CAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGG
ATGAGGCTGATTATTACTGCAGCTCATATGCAGGCAGCAACAATTTC
[1038] IGLV3-21*02 Nucleotide Sequence (SEQ ID NO: 1243)
TABLE-US-00720 TCCTATGAGCTGACACAGCTACCCTCGGTGTCAGTGTCCCCAGGACAGAC
AGCCAGGATCACCTGCTCTGGAGATGTACTGGGGGAAAATTATGCTGACT
GGTACCAGCAGAAGCCAGGCCAGGCCCCTGAGTTGGTGATATACGAAGAT
AGTGAGCGGTACCCTGGAATCCCTGAACGATTCTCTGGGTCCACCTCAGG
GAACACGACCACCCTGACCATCAGCAGGGTCCTGACCGAAGACGAGGCTG
ACTATTACTGTTTGTCTGGGGATGAGGACAATCC
[1039] IGLV3-25*03 Nucleotide Sequence (SEQ ID NO: 1244)
TABLE-US-00721 TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGAC
GGCCAGGATCACCTGCTCTGGAGATGCATTGCCAAAGCAATATGCTTATT
GGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTGATATATAAAGAC
AGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGCTCAGG
GACAACAGTCACGTTGACCATCAGTGGAGTCCAGGCAGAAGACGAGGCTG
ACTATTACTGTCAATCAGCAGACAGCAGTGGT
[1040] IGLV7-43*01 Nucleotide Sequence (SEQ ID NO: 1245)
TABLE-US-00722 CAGACTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGAC
AGTCACTCTCACCTGTGCTTCCAGCACTGGAGCAGTCACCAGTGGTTACT
ATCCAAACTGGTTCCAGCAGAAACCTGGACAAGCACCCAGGGCACTGATT
TATAGTACAAGCAACAAACACTCCTGGACCCCTGCCCGGTTCTCAGGCTC
CCTCCTTGGGGGCAAAGCTGCCCTGACACTGTCAGGTGTGCAGCCTGAGG
ACGAGGCTGAGTATTACTGCCTGCTCTACTATGGTGGTGCTCAG
[1041] IGLV7-46*01 Nucleotide Sequence (SEQ ID NO: 1246)
TABLE-US-00723 CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGAC
AGTCACTCTCACCTGTGGCTCCAGCACTGGAGCTGTCACCAGTGGTCATT
ATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATT
TATGATACAAGCAACAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTC
CCTCCTTGGGGGCAAAGCTGCCCTGACCCTTTCGGGTGCGCAGCCTGAGG
ATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGCTCGG
[1042] IGLV7-46*02 Nucleotide Sequence (SEQ ID NO: 1247)
TABLE-US-00724 CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGAC
AGTCACTCTCACCTGTGGCTCCAGCACTGGAGCTGTCACCAGTGGTCATT
ATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATT
TATGATACAAGCAACAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTC
CCTCCTTGGGGGCAAAGCTGCCCTGACCCTTTTGGGTGCGCAGCCTGAGG
ATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGGTGCTCGG
[1043] IGLV9-49*01 Nucleotide Sequence (SEQ ID NO: 1248)
TABLE-US-00725 CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTC
GGTCACACTCACCTGCACCCTGAGCAGCGGCTACAGTAATTATAAAGTGG
ACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGGTTTGTGATGCGAGTG
GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTT
CTCAGTCTTGGGCTCAGGCCTGAATCGGTACCTGACCATCAAGAACATCC
AGGAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGG
AGCAACTTCGTGTAACC
[1044] IGLV9-49*03 Nucleotide Sequence (SEQ ID NO: 1249)
TABLE-US-00726 CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTC
GGTCACACTCACCTGCACCCTGAGCAGCGGCTACAGTAATTATAAAGTGG
ACTGGTACCAGCAGAGACCAGGGAAGGGCCCCCGATTTGTGATGCGAGTG
GGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATCCCTGATCGCTT
CTCAGTCTTGGGCTCAGGCCTGAATCGGTACCTGACCATCAAGAACATCC
AGGAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGG
AGCAACTTCGTGTAACC
[1045] The heavy chain of an isolated monoclonal anti-hemagglutinin
(HA) antibody (i.e., anti-hemagglutinin antibody of the invention)
is derived from a germ line V (variable) gene such as, for example,
the IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene or an allele
thereof.
[1046] The HA antibodies of the invention include a variable heavy
chain (V.sub.H) region encoded by a human IGHV1, IGHV2, IGHV3,
IGHV4, or IGHV5 germline gene sequence or an allele thereof. A
IGHV1, IGHV2, IGHV3, IGHV4, or IGHV5 germline gene sequence is
shown, e.g., in SEQ ID NOs: 457 to 485. The HA antibodies of the
invention include a V.sub.H region that is encoded by a nucleic
acid sequence that is at least 75% homologous to the IGHV1, IGHV2,
IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele thereof.
Preferably, the nucleic acid sequence is at least 75%, 80%, 85%,
90%, 95%, 96%, 97% homologous to the IGHV1, IGHV2, IGHV3, IGHV4, or
IGHV5 germline gene sequence or an allele thereof, and more
preferably, at least 98%, 99% homologous to the IGHV1, IGHV2,
IGHV3, IGHV4, or IGHV5 germline gene sequence or an allele thereof.
The V.sub.H region of the HA antibody is at least 75% homologous to
the amino acid sequence of the V.sub.H region encoded by the IGHV1,
IGHV2, IGHV3, IGHV4, or IGHV5 V.sub.H germline gene sequence or an
allele thereof. Preferably, the amino acid sequence of V.sub.H
region of the HA antibody is at least 75%, 80%, 85%, 90%, 95%, 96%,
97% homologous to the amino acid sequence encoded by the 75%, 80%,
85%, 90%, 95%, 96%, 97% germline gene sequence or an allele
thereof, and more preferably, at least 98%, 99% homologous to the
sequence encoded by the 75%, 80%, 85%, 90%, 95%, 96%, 97% germline
gene sequence or an allele thereof.
[1047] The HA antibodies of the invention also include a variable
light chain (V.sub.L) region encoded by a human IGKV1, IGKV2,
IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene
sequence or an allele thereof. A human IGKV1, IGKV2, IGKV3, IGKV4,
IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 V.sub.L germline gene
sequence, or an allele thereof is shown, e.g., at SEQ ID NOs: 486
to 508. Alternatively, the HA antibodies include a IGKV1, IGKV2,
IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 V.sub.L region
that is encoded by a nucleic acid sequence that is at least 65%
homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3,
IGLV7, or IGLV9 germline gene sequence or an allele thereof.
Preferably, the nucleic acid sequence is at least 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97% homologous to the IGKV1, IGKV2, IGKV3,
IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene sequence
or an allele thereof, and more preferably, at least 98%, 99%
homologous to the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3,
IGLV7, or IGLV9 germline gene sequence or an allele thereof. The
V.sub.L region of the HA antibody is at least 65% homologous to the
amino acid sequence of the V.sub.L region encoded the IGKV1, IGKV2,
IGKV3, IGKV4, IGLV1, IGLV2, IGLV3, IGLV7, or IGLV9 germline gene
sequence or an allele thereof. Preferably, the amino acid sequence
of V.sub.L region of the HA antibody is at least 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97% homologous to the amino acid sequence
encoded by the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3,
IGLV7, or IGLV9 germline gene sequence or an allele thereof, and
more preferably, at least 98%, 99% homologous to the sequence
encoded by the IGKV1, IGKV2, IGKV3, IGKV4, IGLV1, IGLV2, IGLV3,
IGLV7, or IGLV9 germline gene sequence or an allele thereof.
HA Antibodies III
[1048] The present invention relates to an immunogen capable of
inducing antibodies against a target peptide of the stem region of
hemagglutinn protein of an influenza virus. The immunogen is a
peptide or a synthetic peptide. In particular, the immunogen of
this invention comprises one or more epitopes or epitope units.
Optionally, the immunogen further comprises a general immune
stimulator. These immunogens of the present invention are capable
of inducing antibodies against influenza A virus to prevent
infection by the virus.
[1049] In one aspect the invention provides an immunogen having an
epitope or epitope unit recognized by a protective monoclonal
antibody having the specificity for the stem region of hemagglutinn
protein of an influenza virus.
[1050] The antibody binds both the HA1 and HA2 peptide. In some
embodiments the epitope is recognized by monoclonal antibody D7,
D8, F10, G17, H40, A66, D80, E88, E90, or H98 or a monoclonal
antibody that competes with the binding of monoclonal antibody D7,
D8, F10, G17, H40, A66, D80, E88, E90, or H98 to the HA protein.
Preferably, the epitope is the F10 epitope.
[1051] In some embodiments the hemagglutinin protein is in the
neutral pH conformation.
[1052] The immunogen is a peptide or a synthetic peptide.
[1053] In some aspects the immunogen is a conjugate having one or
more peptides or peptide fragments that are spatially positioned
relative to each other so that they together form a non-linear
sequence which mimics the tertiary structure of an F10 epitope.
Optionally, the one or more peptides or peptide fragments are
linked to a backbone. The conjugate competes with the binding of
monoclonal antibody F10 to the HA protein.
[1054] The e conformation of the epitope is defined by amino acid
residues 18, 38, 39, 40 and 291 of HA1 and 18, 19, 20, 21, 38, 41,
42, 45, 49, 52, 53, and 56 of HA2 when the hemagglutinin in the
neutral pH conformation.
[1055] In some embodiments the immunogen is a peptide having one or
more of the following amino acid sequences.
[1056] [Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-[Xaa.sub.0].sub.p,
wherein, preferably, Xaa.sub.1 is S, T, F H or Y and Xaa 2 is H, Y,
M, L or Q. Most preferably, Xaa.sub.1 is Y. Most preferably,
Xaa.sub.2 is H.
[1057] [Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-[Xaa.sub.0].sub.p,
wherein, preferably, Xaa.sub.1 is H, Q, Y, S, D, N or T and
Xaa.sub.2 of is Q, E, K, I, V, M, E, R or T. Most preferably,
Xaa.sub.1 is H. Most preferably, Xaa.sub.2 is Q.
[1058]
[Xaa.sub.0].sub.m-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-[Xaa.sub.-
0].sub.p, wherein, preferably, Xaa.sub.1 is I, V, M, or L;
Xaa.sub.2 is D, N, H, Y, D, A, S or E, Xaa.sub.3 is G or A, and
Xaa.sub.4 is W, R, or G. Most preferably, Xaa.sub.1 is V; Xaa.sub.2
is D, Xaa.sub.3 is G, and Xaa.sub.4 is W.
[1059] [Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q
Xaa.sub.4-[Xaa.sub.0].sub.r-Xaa.sub.5-[Xaa.sub.0].sub.q-Xaa.sub.6-Xaa.sub-
.7-[Xaa.sub.0].sub.q-Xaa.sub.8-[Xaa.sub.0].sub.p, and
[Xaa.sub.0].sub.m-Xaa.sub.1-[Xaa.sub.0].sub.q
Xaa.sub.2-Xaa.sub.3-[Xaa.sub.0].sub.q-Xaa.sub.4-[Xaa.sub.0].sub.r-Xaa.sub-
.5-[Xaa.sub.0].sub.q-Xaa.sub.6
Xaa.sub.7-[Xaa.sub.0].sub.s-[Xaa.sub.8].sub.t-[Xaa.sub.0].sub.p,
wherein, preferably Xaa.sub.1 is K, Q, R, N, L, G, F, H or Y;
Xaa.sub.2 is S or T, Xaa.sub.3 is Q or P; Xaa.sub.4 is F, V, I, M,
L, or T; Xaa.sub.5 is I, T, S, N, Q, D, or A; Xaa.sub.6 is I, V, M,
or L; Xaa.sub.7 is N, S, T, or D and Xaa.sub.8 is I, F, V, A, or T.
Most preferably, Xaa.sub.1 is K; Xaa.sub.2 is T, Xaa.sub.3 is Q;
Xaa.sub.4 is I; Xaa.sub.5 is T; Xaa.sub.6 is V; Xaa.sub.7 is N, and
Xaa.sub.8 is I.
[1060] For all of the preceding sequences, m, and p are
independently 0 or 1-100, preferably about 1-90, 1-80, 1-70, 1-60,
1-50, 1-40, 1-30, 1-20 or 1-10; q is 2, r is 3, s is 0 or 2, and t
is 0 or 1, and Xaa.sub.0, is independently any amino acid.
Preferably s is 2 and t is 1.
[1061] In some aspects of the inventions, one or more amino acids
are D-amino acids.
[1062] Optionally, the immunogen further comprises an adjuvant or
is conjugated to a carrier.
[1063] In various aspects the invention includes a composition
containing the immunogen together with one or more pharmaceutically
acceptable excipients, diluents, and/or adjuvants. In some
embodiments the composition further comprises an anti-influenza
antibody of antigen binding fragment thereof. Preferably, the
antibody is monoclonal antibody D7, D8, F10, G17, H40, A66, D80,
E88, E90, or H98 or a monoclonal antibody that competes with the
binding of monoclonal antibody D7, D8, F10, G17, H40, A66, D80,
E88, E90, or H98 to the HA protein. Also provided by the invention
are nucleic acids encoding the immunogens of the invention and
composition comprising the nucleic acids.
[1064] The invention further comprises a method preventing a
disease or disorder caused by an influenza virus by administering
to person at risk of suffering from said disease or disorder an
immunogen composition described herein. Optionally, the method
includes further administering an anti-viral drug, a viral entry
inhibitor or a viral attachment inhibitor. The anti-viral drug is a
neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or
an M2 ion channel. The M2 ion channel inhibitor is amantadine or,
rimantadine. The neuraminidase inhibitor zanamivir, or oseltamivir
phosphate.
[1065] In another aspect the method includes further administering
one or more antibodies specific to a Group I influenza virus and or
a Group II influenza virus. The antibody is administered at a dose
sufficient to neutralize the influenza virus.
[1066] Administration is prior to or after exposure to influenza
virus.
[1067] Also disclosed are methods of treating subjects and methods
of screening and producing antibodies. For example, disclosed is a
method of treating a subject suffering or at risk of influenza
infection, the method comprising administering to the subject one
or more of the disclosed antibodies, such as the disclosed HA stem
antibodies. For example, disclosed is a method of treating a
subject, the method comprising administering to the subject the
stem region of influenza hemagglutinin in the neutral pH
conformation in isolation from other components of influenza virus,
wherein the subject produces an immune response to the stem region.
For example, disclosed is a method of treating a subject, the
method comprising administering to the subject the stem region of
influenza hemagglutinin in the neutral pH conformation in isolation
from the head region of hemagglutinin, wherein the subject produces
an immune response to the stem region. For example, disclosed is a
method of treating a subject, the method comprising administering
to the subject influenza hemagglutinin in the neutral pH
conformation in isolation from other components of influenza virus,
wherein the head region of the hemagglutinin is modified to reduce
the antigenicity of the head region, wherein the subject produces
an immune response to the stem region. For example, disclosed is a
method, the method comprising screening antibodies reactive to
hemagglutinin for binding to hemagglutinin immobilized on a
surface, thereby identifying antibodies of interest. For example,
disclosed is a method comprising screening antibodies reactive to
hemagglutinin for binding to the stem region of influenza
hemagglutinin in the neutral pH conformation in isolation from the
head region of hemagglutinin, thereby identifying antibodies of
interest. For example, disclosed is a method comprising screening
antibodies reactive to hemagglutinin for binding to influenza
hemagglutinin in the neutral pH conformation in isolation from
other components of influenza virus, wherein the head region of the
hemagglutinin is modified to reduce the antigenicity of the head
region, thereby identifying antibodies of interest.
[1068] In some forms, the head region of the hemagglutinin can be
modified by removing or replacing glycosylation sites. In some
forms, the head region of the hemagglutinin can be modified by
adding glycosylation sites. In some forms, the head region of the
hemagglutinin can be modified by removing all or a portion of the
head region.
[1069] In some forms, the disclosed antibodies, disclosed
hemagglutinins, and disclosed methods can produce an immune
reaction in a subject. For example, in some forms, the subject can
produce an immune response that prevents or reduces the severity of
an influenza infection. In some forms, the immune response can be
reactive to influenza viruses within a subtype. In some forms, the
immune response can be reactive to influenza viruses in each
subtype within a cluster. In some forms, the immune response can be
reactive to influenza viruses in each cluster within a group. In
some forms, the immune response can be reactive to all influenza
viruses in each subtype within a group. In some forms, the immune
response can be reactive to influenza viruses within group 1.
[1070] In some forms, the disclosed methods can further comprise
screening the antibodies of interest for competing with antibody
F10 for binding to hemagglutinin, thereby identifying F10-competing
antibodies. In some forms, the hemagglutinin can be hemagglutinin
from a group 2 influenza virus. In some forms, the hemagglutinin
can be hemagglutinin from a group 1 influenza virus. In some forms,
the disclosed methods can further comprising producing the
identified antibodies. Also disclosed are antibodies produced by
the disclosed methods. Also disclosed are antibodies identified by
the disclosed methods.
[1071] The disclosed compositions and methods are based upon the
discovery of monoclonal antibodies which neutralize the influenza
virus, e.g. influenza A virus. The influenza A virus is a Group I
influenza A virus such as a H1 cluster influenza virus. The H1
cluster influenza virus is an H1a cluster or an H1b cluster. The
monoclonal antibody is fully human. In some forms, the monoclonal
antibody can be a bivalent antibody, a monovalent antibody, a
single chain antibody or fragment thereof. Specifically, such
monoclonal can bind to an epitope on the stem region of the
hemagglutinin protein (HA), such as HA1 or HA2 polypeptide. The
epitope can be non-linear.
[1072] 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.
[1073] The disclosed compositions and methods are further based
upon the discovery of a protocol for generating broadly
neutralizing human antibodies that target a highly conserved
epitope in the stem region of HA. Using the trimeric H5 ectodomain
expressed in baculovirus which produces shorter N-glycans and
uncharged mannoses absorbed on a plastic surface, allowed for the
dominant presentation of the stem epitope while masking the
normally immunodominat globular head. Accordingly, also disclosed
is a method of producing an isolated antibody that specifically
binds a pathogenic enveloped virus by exposing a single chain or
Fab expression library to a membrane fusion protein of the virus,
identifying an antibody in the library that specifically binds said
protein; and isolating the antibody from the library. The fusion
protein can be immobilized on a solid surface, e.g. plastic. In
some forms the fusion protein can have modified glycosylations
compared to a wild type fusion protein. For example, the fusion can
be produced in a non-mammalian cell, such as an insect cell. The
fusion protein can be, for example, a trimeric hemagglutinin (HA)
protein.
[1074] Also disclosed is a method of vaccinating a subject against
pathogenic enveloped virus such as an influenza virus by
administering to the subject, for example, a membrane fusion
protein (e.g., a trimeric hemagglutinin (HA) protein coated) or
embedded in a biologically compatible matrix. In some forms the
fusion protein can have modified glycosylations compared to a wild
type fusion protein.
[1075] Also disclosed is a composition comprising a monoclonal
antibody as described herein and kits containing the composition in
one or more containers and instructions for use. The invention
further provides a method of screening a compound for binding to an
F10 antibody by contacting said F10 antibody with a compound of
interest and detecting a compound-antibody complex. Also included
in the invention are the compound identified by the method and
their use as immunogens.
[1076] High affinity, cross-subtype, broadly-neutralizing human
anti-HA mAbs have been identified. Specifically, a human Ab phage
display library and H5 hemagglutinin (HA) ectodomain was used to
select ten neutralizing mAbs (nAbs) with a remarkably broad range
among Group 1 influenza viruses, including the H5N1 "bird flu" and
the H1N1 "Spanish flu" and "Swine flu" strains. These nAbs inhibit
the post-attachment fusion process by recognizing a novel and
highly conserved neutralizing epitope within the stem region at a
point where key elements of the conformational change--the fusion
peptide and the exposed surface of helix aA--are brought into close
apposition. The crystal structure of one mAb (mAbF10) bound to H5N1
HA reveals that only the heavy chain inserts into a highly
conserved pocket in the HA stem region, inhibiting the
conformational changes required for membrane fusion. It has been
discovered that nAbs targeting this pocket can provide broad
protection against both seasonal and pandemic influenza A
infections. The crystal structure further revealed that the epitope
to which the F10 mAb is defined by amino acid residues 18, 38, 39,
40 and 291 of HA1 and 18, 19, 20, 21, 38, 41, 42, 45, 49, 52, 53,
and 56 of HA2. This epitope is referred to herein as the F10
epitope. Structural and sequence analysis of all 16 HA subtypes
points to the existence of only two variants of this epitope,
corresponding to the two phylogenetic groupings of HA (Groups 1 and
2). This discovery indicates that a small cocktail of nAbs derived
from a subset of each group can provide broad protection against
both seasonal and pandemic influenza.
[1077] Remarkably, nAbs were isolated that utilize the same VH
germline gene, IGHV1-69*01, and encode a CDR3 loop containing a
tyrosine at an equivalent position to Y102, from a non-immune
library. This indicates that broad anti-HA cross-immunity
pre-exists in the H5-naive population, possibly due to previous
exposure to H1, and, for library donors born before 1968, H2
subtypes. The recurrent use of this germline VH segment, the
commonality of the CDR3 tyrosine introduced through N insertion
and/or germline D gene assembly, and the promiscuous use of VL
genes by the discovered nAbs discovered indicate that the precursor
frequency of rearranged VH segments that could recognize this
epitope is significant. This indicates that with suitable exposure
to the F10 epitope identified here, these broad-spectrum nAbs can
be readily induced in vivo. These discoveries led to the disclosed
simple solution to provide universal protection against virus
subtypes in both groups.
[1078] Three unique anti-HA-1 scFvs were identified by sequencing
analysis of the 58 HA-1 positive clones. These scFvs were
designated as 38B and 1C. The VH and VL amino acid sequence of 2A
is shown herein. Ten unique anti-HAO scFvs were identified by
sequencing analysis of the 97 HAO positive clones. These scFvs were
designated as 7, 8, 10, 17, 40, 66, 80, 88, 90, and 98. Six
different VH and 10 different VL genes were revealed. Some scFvs
shared the same VH gene. Five out of the six different VH genes
belonged to the IGHV1-69 gene family. Three out often VL genes were
kappa chain. 2A scFv is a moderate neutralizing antibody, 38B and
1C are non-neutralizing antibodies. Ten scFvs, 7, 8, 10, 17, 40,
66, 80, 88, 90, and 98 are potent neutralizing antibodies. The
nucleic acid and amino acid sequence of the neutralizing influenza
antibodies are provided below. Methods of making these antibodies
are disclosed in PCT/US2009/054950 (Publication No. WO
2010/027818), the entire contents of which are incorporated herein
by reference.
[1079] Antibody 2A: Variable Region Nucleic Acid Sequences
[1080] VH Chain of 2A (SEQ ID NO: 1305)
TABLE-US-00727 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGTGACAATGCTA
TCAGCTGGGTGCGACAGGCCCCAGGACAAGGGCTTGAGTGGATGGGGGGC
ATCATTCCTATCTTTGGAAAACCAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACTGCGGACGAATCCACGAGCACAGCCTACATGGACCTGA
GGAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAGATTCA
GACGCGTATTACTATGGTTCGGGGGGTATGGACGTCTGGGGCCAAGGCAC
CCTGGTCACCGTCTCCTCA
[1081] VL Chain of 2A (SEQ ID NO: 1306)
TABLE-US-00728 CTGCCTGTGCTGACTCAATCATCCTCTGCCTCTGCTTCCCTGGGATCCTC
GGTCAAGCTCACCTGCACTCTGAGCAGTGGGCATAGTAACTACATCATCG
CATGGCATCAACAGCAGCCAGGGAAGGCCCCTCGGTACTTGATGAAGGTT
AATAGTGATGGCAGCCACACCAAGGGGGACGGGATCCCTGATCGCTTCTC
AGGCTCCAGCTCTGGGGCTGACCGCTACCTCACCATCTCCAACCTCCAGT
CTGAGGATGAGGCTAGTTATTTCTGTGAGACCTGGGACACTAAGATTCAT
GTCTTCGGAACTGGGACCAAGGTCTCCGTCCTCAG
[1082] Antibody 2A: Variable Region Amino Acid Sequences
[1083] VH Chain of 2A (SEQ ID NO: 1307)
TABLE-US-00729 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNAISWVRQAPGQGLEWMGG
IIPIFGKPNYAQKFQGRVTITADESTSTAYMDLRSLRSEDTAVYYCARDS
DAYYYGSGGMDVWGQGTLVTVSS
[1084] VL Chain of 2A (SEQ ID NO: 1308)
TABLE-US-00730 LPVLTQSSSASASLGSSVKLTCTLSSGHSNYIIAWHQQQPGKAPRYLMKV
NSDGSHTKGDGIPDRFSGSSSGADRYLTISNLQSEDEASYFCETWDTKIH VFGTGTKVSVL
[1085] Antibody D7: Variable Region Nucleic Acid Sequences
[1086] VH Chain of D7 (SEQ ID NO: 1309)
TABLE-US-00731 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTCCTGGAGGTATCTTCAACACCAATGCTT
TCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGG
GTCATCCCTTTGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAG
AGTCACCATTACCGCGGACGAATCCACGAACACAGCCTACATGGAGCTTA
CCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGT
GGTTACCATTTTAGGAGTCACTTTGACTCCTGGGGCCTGGGAACCCTGGT
CACCGTCTCCTCA
[1087] VL Chain of D7 (SEQ ID NO: 1310)
TABLE-US-00732 AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGCGTCTCCGGGGAAGAC
GGTGACCATCTCCTGCACCGGCAGCAGTGGCAACATTGCCGCCAACTATG
TGCAGTGGTACCAACAACGTCCGGGCAGTGCCCCCACTACTGTGATCTAT
GAGGATGACCGAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCAT
CGACAGGTCCTCCAACTCTGCCTCCCTCACCATCTCAGGACTGAAGACTG
AGGACGAGGCTGACTACTACTGTCAGACTTATGATACCAACAATCATGCT
GTGTTCGGAGGAGGCACCCACCTGACCGTCCTC
[1088] Antibody H98: Variable Region Nucleic Acid Sequences
[1089] VH Chain of H98 (SEQ ID NO: 1311)
TABLE-US-00733 CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTCCTGGAGGTATCTTCAACACCAATGCTT
TCAGCTGGGTCCGACAGGCCCCTGGACAAGGTCTTGAGTGGGTGGGAGGG
GTCATCCCTTTGTTTCGAACAGCAAGCTACGCACAGAACGTCCAGGGCAG
AGTCACCATTACCGCGGACGAATCCACGAACACAGCCTACATGGAGCTTA
CCAGCCTGAGATCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGTAGT
GGTTACCATTTTAGGAGTCACTTTGACTCCTGGGGCCTGGGAACCCTGGT
CACCGTCTCCTCA
[1090] VL Chain of H98 (SEQ ID NO: 1312)
TABLE-US-00734 TCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGAAACACGGGCAGAG
GGTCACCATCTCTTGTTCTGGAGGCACCTCCAACATCGGACGTAATCATG
TTAACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTAT
AGTAATGAACAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAA
ATCTGGCACCTCCGCCTCCCTGGCCGTGAGTGGGCTCCAGTCTGAGGATG
AGGCTGATTATTACTGTGCATCATGGGATGACAACTTGAGTGGTTGGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTAT
[1091] Antibody D7 and H98: Variable Region Chain Amino Acid
Sequences
[1092] VH Chain of D7 and H98 (SEQ ID NO: 1313)
TABLE-US-00735 QVQLVQSGAEVKKPGSSVKVSCKAPGGIFNTNAFSWVRQAPGQGLEWVGG
VIPLFRTASYAQNVQGRVT I TADESTNTAYMELTSLRSADTAVYYCAR
SSGYHFRSHFDSWGLGTLVTVSS
[1093] VL Chain of D7 (SEQ ID NO: 1314)
TABLE-US-00736 NFMLTQPHSVSASPGKTVTISCTGSSGNIAANYVQWYQQRPGSAPTTVIY
EDDRRPSGVPDRFSGSIDRSSNSASLTISGLKTEDEADYYCQTYDTNNHA VFGGGTHLTVL
[1094] VL Chain of H98 (SEQ ID NO: 1315)
TABLE-US-00737 SYELTQPPSASGKHGQRVTISCSGGTSNIGRNHVNWYQQLPGTAPKLLIY
SNEQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEADYYCASWDDNLSGWV FGGGTKLTVL
[1095] Antibody D8: Variable Region Nucleic Acid Sequences
[1096] VH Chain of D8 (SEQ ID NO: 1316)
TABLE-US-00738 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCGCTTATGCTT
TCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC
ATCACCGGAATGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCGCGGACGAACTCACGAGCACAGCCTACATGGAGTTGA
GCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTG
TATTACTATGAGAGTAGTCTTGACTATTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAG
[1097] VL Chain of D8 (SEQ ID NO: 1317)
TABLE-US-00739 CAGTCTGTGCTGACTCAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTC
AGTCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACT
CTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATT
TATGAGGTCACTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGCCTC
CAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGG
ATGAGGCTGATTATTTCTGCTGCTCATATGCAGGCCACAGTGCTTATGTC
TTCGGAACTGGGACCAAGGTCACCGTCCTG
[1098] Antibody D80: Variable Region Nucleic Acid Sequences
[1099] VH Chain of D80 (SEQ ID NO: 1318)
TABLE-US-00740 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCAGGGCTTCTGGAGGCACCTTCAGCGCTTATGCTT
TCACCTGGGTGCGGCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGC
ATCACCGGAATGTTTGGCACAGCAAACTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCGCGGACGAACTCACGAGCACAGCCTACATGGAGTTGA
GCTCCCTGACATCTGAAGACACGGCCCTTTATTATTGTGCGAGAGGATTG
TATTACTATGAGAGTAGTCTTGACTATTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAG
[1100] VK Chain of D80 (SEQ ID NO: 1319)
TABLE-US-00741 GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTCTTAGCAGCAAGTACT
TAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG
GTCTGGGACAGACTTCACCCTCACCATCAGTAGACTGGAGCCTGAAGATT
TTGCAGTGTATTCCTGTCAGCAGTATGATGGCGTACCTCGGACGTTCGGC
CAAGGGACCACGGTGGAAATCAAA
[1101] Antibody D8 and D80: Variable Region Chain Amino Acid
Sequences
[1102] VH Chain of D8 and D80 (SEQ ID NO: 1320)
TABLE-US-00742 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYAFTWVRQAPGQGLEWMGG
ITGMFGTANYAQKFQGRVTITADELTSTAYMELSSLTSEDTALYYCARGL
YYYESSLDYWGQGTLVTVSS
[1103] VL Chain of D8 (SEQ ID NO: 1321)
TABLE-US-00743 QSVLTQPPSASGSPGQSVTISCTGTSSDVGGYNSVSWYQQHPGKAPKLMI
YEVTKRPSGVPDRFSASKSGNTASLTVSGLQAEDEADYFCCSYAGHSAYV FGTGTKVTVL
[1104] VK Chain of D80 (SEQ ID NO: 1322)
TABLE-US-00744 EIVLTQSPGTLSLSPGERATLSCRASQSLSSKYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYSCQQYDGVPRTFG QGTTVEIK
[1105] Antibody F10: Variable Region Nucleic Acid Sequences
[1106] VH Chain of F10 (SEQ ID NO: 1323)
TABLE-US-00745 CAGGTGCAGCTGGTGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCACGTCCTCTGAAGTCACCTTCAGTAGTTTTGCTA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGG
ATCAGCCCTATGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAG
AGTCACCATTACCGCGGACCAGTCCACGAGGACAGCCTACATGGACCTGA
GGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCT
TCTTACATTTGTTCTGGTGGAACCTGCGTCTTTGACCATTGGGGCCAGGG
AACCCTGGTCACCGTCTCCTCA
[1107] VL Chain of F10 (SEQ ID NO: 1324)
TABLE-US-00746 CAGCCTGGGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGAC
CGCCACACTCACCTGCACTGGGAACAGCAACAATGTTGGCAACCAAGGAG
CAGCTTGGCTGCAGCAGCACCAGGGCCACCCTCCCAAACTCCTATCCTAC
AGGAATAATGACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAG
GTCAGGAAACACAGCCTCCCTGACCATTACTGGACTCCAGCCTGAGGACG
AGGCTGACTATTACTGCTCAACATGGGACAGCAGCCTCAGTGCTGTGGTA
TTCGGCGGAGGGACCAAGCTGACCGTCCTA
[1108] Antibody E90: Variable Region Nucleic Acid Sequences
[1109] VH Chain of E90 (SEQ ID NO: 1325)
TABLE-US-00747 CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTC
GGTGAAGGTCTCCTGCACGTCCTCTGAAGTCACCTTCAGTAGTTTTGCTA
TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGG
ATCAGCCCTATGTTTGGAACACCTAATTACGCGCAGAAGTTCCAAGGCAG
AGTCACCATTACCGCGGACCAGTCCACGAGGACAGCCTACATGGACCTGA
GGAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGATCTCCT
TCTTACATTTGTTCTGGTGGAACCTGCGTCTTTGACCATTGGGGCCAGGG
AACCCTGGTCACCGTCTCCTCA
[1110] VL Chain of E90 (SEQ ID NO: 1326)
TABLE-US-00748 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCAAAGAGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGACTTCACTCTCACCATTAGCAGCCTGCAGCCTGAAGATTTTG
CAGTGTATTACTGTCAGCAGTATGATAGTTCACCGTACACTTTTGGCCAG
GGGACCAAGGTAGAGATCAAA
[1111] Antibody F10 and E90 Variable Region Amino Acid
Sequences
[1112] VH Chain of F10 and E90 (SEQ ID NO: 1327)
TABLE-US-00749 QVQLVQSGAEVKKPGSSVKVSCTSSEVTFSSFAISWVRQAPGQGLEWLGG
ISPMFGTPNYAQKFQGRVTITADQSTRTAYMDLRSLRSEDTAVYYCARSP
SYICSGGTCVFDHWGQGTLVTVSS
[1113] VL Chain of F10 (SEQ ID NO: 1328)
TABLE-US-00750 QPGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSY
RNNDRPSGISERFSASRSGNTASLTITGLQPEDEADYYCSTWDSSLSAVV FGGGTKLTVL
[1114] VL Chain of E90 (SEQ ID NO: 1329)
TABLE-US-00751 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQRGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYDSSPYTFGQ GTKVEIK
[1115] Antibody G17: Variable Region Nucleic Acid Sequences
[1116] VH Chain of G17 (SEQ ID NO: 1330)
TABLE-US-00752 CAGGTGCAGCTGGTGCAATCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTC
AGTGAAGGTCTCCTGCAAGACTTCTGGAGTCACCTTCAGCAGCTATGCTA
TCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG
ATCATCGGTGTCTTTGGTGTACCAAAGTACGCGCAGAACTTCCAGGGCAG
AGTCACAATTACCGCGGACAAACCGACGAGTACAGTCTACATGGAGCTGA
ACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGCCC
GGGTACTACGTAGGAAAGAATGGTTTTGATGTCTGGGGCCAAGGGACAAT
GGTCACCGTCTCTTCA
[1117] VL Chain of G17 (SEQ ID NO: 1331)
TABLE-US-00753 TCCTATGAGCTGACTCAGCCACCCTCGGTGTCCAAGGGCTTGAGACAGAC
CGCCATACTCACCTGCACTGGAGACAGCAACAATGTTGGCCACCAAGGTA
CAGCTTGGCTGCAACAACACCAGGGCCACCCTCCCAAACTCCTATCCTAC
AGGAATGGCAACCGGCCCTCAGGGATCTCAGAGAGATTCTCTGCATCCAG
GTCAGGAAATACAGCCTCCCTGACCATTATTGGACTCCAGCCTGAGGACG
AGGCTGACTACTACTGCTCAGTATGGGACAGCAGCCTCAGTGCCTGGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA
[1118] Antibody G17 Variable Region Amino Acid Sequences
[1119] VH Chain of G17 (SEQ ID NO: 1332)
TABLE-US-00754 QVQLVQSGAEVKKPGASVKVSCKTSGVTFSSYAISWVRQAPGQGLEWMGG
IIGVFGVPKYAQNFQGRVTITADKPTSTVYMELNSLRAEDTAVYYCAREP
GYYVGKNGFDVWGQGTMVTVSS
[1120] VL Chain of G17 (SEQ ID NO: 1333)
TABLE-US-00755 SYELTQPPSVSKGLRQTAILTCTGDSNNVGHQGTAWLQQHQGHPPKLLSY
RNGNRPSGISERFSASRSGNTASLTIIGLQPEDEADYYCSVWDSSLSAWV FGGGTKLTVL
[1121] Antibody H40: Variable Region Nucleic Acid Sequences
[1122] VH Chain of H40 (SEQ ID NO: 1334)
TABLE-US-00756 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGGCCTC
AGTGAAGGTCTCATGTAAGGCTTCTGGATACACCTTCACCGGTTATTATA
TTCACTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGTTGG
ATCAACCCTATGACTGGTGGCACAAACTATGCACAGAAGTTTCAGGTCTG
GGTCACCATGACCCGGGACACGTCCATCAACACAGCCTACATGGAGGTGA
GCAGGCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGGGGGGCT
TCCGTATTACGATATTTTGACTGGCAGCCCGAGGCTCTTGATATCTGGGG
CCTCGGGACCACGGTCACCGTCTCCTCA
[1123] VL Chain of H40 (SEQ ID NO: 1335)
TABLE-US-00757 CAGCCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGAC
GGCCAGCATTCCCTGTGGGGGGAACAACATTGGAGGCTACAGTGTACACT
GGTACCAACAAAAGCCGGGCCAGGCCCCCCTCTTGGTCATTTATGACGAT
AAAGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCGCCAACTCTGG
GAGCACGGCCACCCTGACAATCAGCAGGGTCGAAGCCGGGGATGAGGGCG
ACTACTACTGTCAGGTGTGGGATAGTGGTAATGATCGTCCGCTGTTCGGC
GGAGGGACCAAGCTGACCGTCCTA
[1124] Antibody H40: Variable Region Amino Acid Sequences
[1125] VH Chain of H40 (SEQ ID NO: 1336)
TABLE-US-00758 QVQLVQSGAEVRKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGW
INPMTGGTNYAQKFQVWVTMTRDTSINTAYMEVSRLTSDDTAVYYCARGA
SVLRYFDWQPEALDIWGLGTTVTVSS
[1126] VL Chain of H40 (SEQ ID NO: 1337)
TABLE-US-00759 QPVLTQPPSVSVAPGQTASIPCGGNNIGGYSVHWYQQKPGQAPLLVIYDD
KDRPSGIPERFSGANSGSTATLTISRVEAGDEGDYYCQVWDSGNDRPLFG GGTKLTVL
[1127] Antibody A66 Variable Region Nucleic Acid Sequences
[1128] VH Chain of A66 (SEQ ID NO: 1338)
TABLE-US-00760 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTC
GGTGAAGGTTTCCTGCAAGGCTTCTGGAGGCCCCTTCAGCATGACTGCTT
TCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGG
ATCAGCCCTATCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCGCGGACGAATCCACGAACACAGCCAACATGGAGCTGA
CCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTT
TCCTCCTACCAACCGAATAATGATGCTTTTGCTATCTGGGGCCAAGGGAC
AATGGTCACCGTCTCTTCA
[1129] VK Chain of A66 (SEQ ID NO: 1339)
TABLE-US-00761 GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAG
CCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT
GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC
TGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTG
CAGTCTATTTCTGTCAGCAGTATGGTAGCTCACCTCAATTCGGCCAAGGG
ACACGACTGGAGATTAAA
[1130] Antibody A66 Variable Region Amino Acid Sequences
[1131] VH Chain of A66 (SEQ ID NO: 1340)
TABLE-US-00762 QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGG
ISPIFRTPKYAQKFQGRVTITADESTNTANMELTSLKSEDTAVYYCARTL
SSYQPNNDAFAIWGQGTMVTVSS
[1132] VK Chain of A66 (SEQ ID NO: 1341)
TABLE-US-00763 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLT I SRLEPEDFAVYFCQQYGSSPQFG QGTRLEIK
[1133] Antibody E88 Variable Region Nucleic Acid Sequences
[1134] VH Chain of E88 (SEQ ID NO: 1342)
TABLE-US-00764 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGCTCCTC
GGTGAAGGTTTCCTGCAAGGCTTCTGGAGGCCCCTTCAGCATGACTGCTT
TCACCTGGCTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGG
ATCAGCCCTATCTTTCGTACACCGAAGTACGCACAGAAGTTCCAGGGCAG
AGTCACGATTACCGCGGACGAATCCACGAACACAGCCAACATGGAGCTGA
CCAGCCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACCCTT
TCCTCCTACCAACCGAATAATGATGCTTTTGCTATCTGGGGCCAAGGGAC
AATGGTCACCGTCTCTTCA
[1135] VL Chain of E88 (SEQ ID NO: 1343)
TABLE-US-00765 CTGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAG
GGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTG
TAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTAT
AGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAG
GTCAGGCACCTCAGCCTCCCTGGCCATCATTGGACTCCGGCCTGAGGATG
AAGCTGATTATTACTGTCAGTCGTATGACAGCAGGCTCAGTGCTTCTCTC
TTCGGAACTGGGACCACGGTCACCGTCCTC
[1136] Antibody E88 Variable Region Amino Acid Sequences
[1137] VH Chain of E88 (SEQ ID NO: 1344)
TABLE-US-00766 QVQLVQSGAEVKKPGSSVKVSCKASGGPFSMTAFTWLRQAPGQGLEWMGG
ISPIFRTPKYAQKFQGRVTITADESTNTANMELTSLKSEDTAVYYCARTL
SSYQPNNDAFAIWGQGTMVTVSS
[1138] VL Chain of E88 (SEQ ID NO: 1345)
TABLE-US-00767 LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIY
SNNQRPSGVPDRFSGSRSGTSASLAIIGLRPEDEADYYCQSYDSRLSASL FGTGTTVTVL
[1139] The amino acid sequences of the heavy and light chain
complementary determining regions of the neutralizing influenza
antibodies are shown below in Table 18.
TABLE-US-00768 TABLE 18 SEQ SEQ SEQ ID ID ANTIBODY CHAIN CDR1 ID
NO: CDR2 NO: CDR3 NO: CONSENSUS HEAVY SYAFS 299 GIIPMFGTPNYAQK 1263
SSGYYYGGGFDV 1284 FQG D7/H98 HEAVY TNAFS 302 GVIPLFRTASYAQN 1264
SSGYHFGRSHFDS 1285 VQN D8/D80 HEAVY AYAFT 305 GITGMFGTANYAQ 1265
GLYYYESSLDY 1286 KFQG F10/E90 HEAVY SFAIS 600 GISPMFGTPNYAQK 1266
SPSYICSGGTCVF 1287 FQG DH G17 HEAVY SYAIS 606 GIIGVFGVPKYAQK 1267
EPGYYGKNGFDV 1288 FQG H40 HEAVY GYYIH 630 WINPMTGGTNYAQ 1268
GASVLRYFDWQP 1289 KFQV EALDI A66 HEAVY MTAFT 641 GISPIFRTPKYAQKF
1269 TLSSYQPNNDAF 1290 QG AI E88 HEAVY MTAFT 647 GISPIFRTPKYAQKF
1270 TLSSYQPNNDAF 1291 QG AI 2A HEAVY DNAIS 668 GIIPIFGKPNYAQKF
1271 DSDAYYYGSGG 1292 QG MDV CONSENSUS LIGHT TGSSSMN 757 SNSDRPS
1272 QSYDSLSAYV 1293 IGNYVA D7 LIGHT TGSSSNIA 1252 EDDRRPS 1273
QTYDTNNHAV 1294 ANYVQ D8 LIGHT TGTSSDV 1253 EVTKRPS 1274 CSYAGHSAYV
1295 GGYNSVS F10 LIGHT TGNSNNV 1254 RNNDRPS 1275 STWDSSLSAVV 1296
GNQGAA G17 LIGHT TGNSNNV 1255 RNGNRPS 1276 SVWDSSLSAWV 1297 GHQGTA
H40 LIGHT GGNNIGG 1256 DDKDRPS 1277 QVWDSGNDRPL 1298 YSVH A66 LIGHT
RASQSVS 1257 DASNRAT 1278 QQYGSSPQF 1299 SYLA D80 LIGHT RASQSLS
1258 GASSRAT 1279 QQYDGVPRT 1300 SKYLA E88 LIGHT SGSSSNIG 1259
SNNQRPS 1280 QSYDSRLSASL 1301 SNTVN E90 LIGHT RASQSISS 1260 AASSLQR
1281 QQYDSSPYT 1302 YLN H98 LIGHT SGGTSNI 1261 SNEQRPS 1282
ASWDDNLSGWV 1303 GRNHVN 2A LIGHT TLSSGHS 1262 VNSDGSHTKGD 1283
ETWDTKIHV 1304 NYIIA
Antibodies
[1140] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
practice of the present invention will employ, unless indicated
specifically to the contrary, conventional methods of virology,
immunology, microbiology, molecular biology and recombinant DNA
techniques within the skill of the art, many of which are described
below for the purpose of illustration. Such techniques are
explained fully in the literature. See, e.g., Sambrook, et al.
Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);
Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA
Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);
Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., 1985);
Transcription and Translation (B. Hames & S. Higgins, eds.,
1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A
Practical Guide to Molecular Cloning (1984).
[1141] The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[1142] The following definitions are useful in understanding the
present invention:
[1143] The term "antibody" (Ab) as used herein includes monoclonal
antibodies, polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies), and antibody fragments, so long as they
exhibit the desired biological activity. The term "immunoglobulin"
(Ig) is used interchangeably with "antibody" herein.
[1144] An "isolated antibody" is one that has been separated and/or
recovered from a component of its natural environment. Contaminant
components of its natural environment are materials that would
interfere with diagnostic or therapeutic uses for the antibody, and
may include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In preferred embodiments, the antibody is
purified: (1) to greater than 95% by weight of antibody as
determined by the Lowry method, and most preferably more than 99%
by weight; (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[1145] The basic four-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain. In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
g and s isotypes. Each L chain has at the N-terminus, a variable
domain (V.sub.L) followed by a constant domain (C.sub.L) at its
other end. The V.sub.L is aligned with the V.sub.H and the C.sub.L
is aligned with the first constant domain of the heavy chain
(C.sub.H1). Particular amino acid residues are believed to form an
interface between the light chain and heavy chain variable domains.
The pairing of a V.sub.H and V.sub.L together forms a single
antigen-binding site. For the structure and properties of the
different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71, and Chapter 6.
[1146] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa (.kappa.) and
lambda (.lamda.), based on the amino acid sequences of their
constant domains (C.sub.L). Depending on the amino acid sequence of
the constant domain of their heavy chains (C.sub.H),
immunoglobulins can be assigned to different classes or isotypes.
There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, having heavy chains designated alpha (.alpha.), delta
(.delta.), epsilon (.epsilon.), gamma (.gamma.) and mu (.mu.),
respectively. The .gamma. and .alpha. classes are further divided
into subclasses on the basis of relatively minor differences in
C.sub.H sequence and function, e.g., humans express the following
subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
[1147] The term "variable" refers to the fact that certain segments
of the V domains differ extensively in sequence among antibodies.
The V domain mediates antigen binding and defines specificity of a
particular antibody for its particular antigen. However, the
variability is not evenly distributed across the 110-amino acid
span of the variable domains. Instead, the V regions consist of
relatively invariant stretches called framework regions (FRs) of
15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody dependent
cellular cytotoxicity (ADCC).
[1148] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H when numbered in accordance with the Kabat
numbering system; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)); and/or those residues
from a "hypervariable loop" (e.g., residues 24-34 (L), 50-56 (L2)
and 89-97 (L3) in the V.sub.L, and 26-32 (H1), 52-56 (H2) and
95-101 (H3) in the V.sub.H 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 (L), 56-65 (L2) and 105-120 (L3) in
the V.sub.L, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the
V.sub.H when numbered in accordance with the IMGT numbering system;
Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M.
e al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody
has symmetrical insertions at one or more of the following points
28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the V.sub.L, and 28, 36
(H1), 63, 74-75 (H2) and 123 (H3) in the V.sub.H when numbered in
accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol.
309:657-670 (2001)).
[1149] By "germline nucleic acid residue" is meant the nucleic acid
residue that naturally occurs in a germline gene encoding a
constant or variable region. "Germline gene" is the DNA found in a
germ cell (i.e., a cell destined to become an egg or in the sperm).
A "germline mutation" refers to a heritable change in a particular
DNA that has occurred in a germ cell or the zygote at the
single-cell stage, and when transmitted to offspring, such a
mutation is incorporated in every cell of the body. A germline
mutation is in contrast to a somatic mutation which is acquired in
a single body cell. In some cases, nucleotides in a germline DNA
sequence encoding for a variable region are mutated (i.e., a
somatic mutation) and replaced with a different nucleotide.
[1150] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations that include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies useful in the present invention may be
prepared by the hybridoma methodology first described by Kohler et
al., Nature, 256:495 (1975), or may be made using recombinant DNA
methods in bacterial, eukaryotic animal or plant cells (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[1151] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 (1984)). The present invention
provides variable domain antigen-binding sequences derived from
human antibodies. Accordingly, chimeric antibodies of primary
interest herein include antibodies having one or more human antigen
binding sequences (e.g., CDRs) and containing one or more sequences
derived from a non-human antibody, e.g., an FR or C region
sequence. In addition, chimeric antibodies of primary interest
herein include those comprising a human variable domain antigen
binding sequence of one antibody class or subclass and another
sequence, e.g., FR or C region sequence, derived from another
antibody class or subclass. Chimeric antibodies of interest herein
also include those containing variable domain antigen-binding
sequences related to those described herein or derived from a
different species, such as a non-human primate (e.g., Old World
Monkey, Ape, etc). Chimeric antibodies also include primatized and
humanized antibodies.
[1152] Furthermore, chimeric antibodies may comprise residues that
are not found in the recipient antibody or in the donor antibody.
These modifications are made to further refine antibody
performance. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[1153] A "humanized antibody" is generally considered to be a human
antibody that has one or more amino acid residues introduced into
it from a source that is non-human. These non-human amino acid
residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization is
traditionally performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting import hypervariable region
sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567) wherein substantially less than an intact
human variable domain has been substituted by the corresponding
sequence from a non-human species.
[1154] A "human antibody" is an antibody containing only sequences
present in an antibody naturally produced by a human. However, as
used herein, human antibodies may comprise residues or
modifications not found in a naturally occurring human antibody,
including those modifications and variant sequences described
herein. These are typically made to further refine or enhance
antibody performance.
[1155] An "intact" antibody is one that comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H 1, C.sub.H 2 and C.sub.H 3. The constant
domains may be native sequence constant domains (e.g., human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[1156] 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').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8 (10):
1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[1157] The phrase "functional fragment or analog" of an antibody is
a compound having qualitative biological activity in common with a
full-length antibody. For example, a functional fragment or analog
of an anti-IgE antibody is one that can bind to an IgE
immunoglobulin in such a manner so as to prevent or substantially
reduce the ability of such molecule from having the ability to bind
to the high affinity receptor, Fc.sub..epsilon.RI.
[1158] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H 1). Each Fab fragment
is monovalent with respect to antigen binding, i.e., it has a
single antigen-binding site. Pepsin treatment of an antibody yields
a single large F(ab').sub.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 C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments that
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[1159] The "Fc" fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[1160] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (three loops
each from the H and L chain) that contribute the amino acid
residues for antigen binding and confer antigen binding specificity
to the antibody. However, even a single variable domain (or half of
an Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[1161] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains that enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Borrebaeck 1995, infra.
[1162] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[1163] As used herein, an antibody that "internalizes" is one that
is taken up by (i.e., enters) the cell upon binding to an antigen
on a mammalian cell (e.g., a cell surface polypeptide or receptor).
The internalizing antibody will of course include antibody
fragments, human or chimeric antibody, and antibody conjugates. For
certain therapeutic applications, internalization in vivo is
contemplated. The number of antibody molecules internalized will be
sufficient or adequate to kill a cell or inhibit its growth,
especially an infected cell. Depending on the potency of the
antibody or antibody conjugate, in some instances, the uptake of a
single antibody molecule into the cell is sufficient to kill the
target cell to which the antibody binds. For example, certain
toxins are highly potent in killing such that internalization of
one molecule of the toxin conjugated to the antibody is sufficient
to kill the infected cell.
[1164] As used herein, an antibody is said to be "immunospecific,"
"specific for" or to "specifically bind" an antigen if it reacts at
a detectable level with the antigen, preferably with an affinity
constant, K.sub.a, of greater than or equal to about 10.sup.4
M.sup.-1, or greater than or equal to about 10.sup.5 M.sup.-1,
greater than or equal to about 10.sup.6 M.sup.-1, greater than or
equal to about 10.sup.7 M.sup.-1, or greater than or equal to
10.sup.8 M.sup.-1. Affinity of an antibody for its cognate antigen
is also commonly expressed as a dissociation constant K.sub.D, and
in certain embodiments, HuM2e antibody specifically binds to M2e if
it binds with a K.sub.D of less than or equal to 10.sup.-4 M, less
than or equal to about 10.sup.-5 M, less than or equal to about
10.sup.-6 M, less than or equal to 10.sup.-7 M, or less than or
equal to 10.sup.-8 M. Affinities of antibodies can be readily
determined using conventional techniques, for example, those
described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660
(1949)).
[1165] Binding properties of an antibody to antigens, cells or
tissues thereof may generally be determined and assessed using
immunodetection methods including, for example,
immunofluorescence-based assays, such as immuno-histochemistry
(IHC) and/or fluorescence-activated cell sorting (FACS).
[1166] An antibody having a "biological characteristic" of a
designated antibody is one that possesses one or more of the
biological characteristics of that antibody which distinguish it
from other antibodies. For example, in certain embodiments, an
antibody with a biological characteristic of a designated antibody
will bind the same epitope as that bound by the designated antibody
and/or have a common effector function as the designated
antibody.
[1167] The term "antagonist" antibody is used in the broadest
sense, and includes an antibody that partially or fully blocks,
inhibits, or neutralizes a biological activity of an epitope,
polypeptide, or cell that it specifically binds. Methods for
identifying antagonist antibodies may comprise contacting a
polypeptide or cell specifically bound by a candidate antagonist
antibody with the candidate antagonist antibody and measuring a
detectable change in one or more biological activities normally
associated with the polypeptide or cell.
[1168] An "antibody that inhibits the growth of infected cells" or
a "growth inhibitory" antibody is one that binds to and results in
measurable growth inhibition of infected cells expressing or
capable of expressing an M2e epitope bound by an antibody.
Preferred growth inhibitory antibodies inhibit growth of infected
cells by greater than 20%, preferably from about 20% to about 50%,
and even more preferably, by greater than 50% (e.g., from about 50%
to about 100%) as compared to the appropriate control, the control
typically being infected cells not treated with the antibody being
tested. Growth inhibition can be measured at an antibody
concentration of about 0.1 to 30 .mu.g/ml or about 0.5 nM to 200 nM
in cell culture, where the growth inhibition is determined 1-10
days after exposure of the infected cells to the antibody. Growth
inhibition of infected cells in vivo can be determined in various
ways known in the art. The antibody is growth inhibitory in vivo if
administration of the antibody at about 1 .mu.g/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.
[1169] An antibody that "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). Preferably the cell is an
infected cell. Various methods are available for evaluating the
cellular events associated with apoptosis. For example,
phosphatidyl serine (PS) translocation can be measured by annexin
binding; DNA fragmentation can be evaluated through DNA laddering;
and nuclear/chromatin condensation along with DNA fragmentation can
be evaluated by any increase in hypodiploid cells. Preferably, the
antibody that induces apoptosis is one that results in about 2 to
50 fold, preferably about 5 to 50 fold, and most preferably about
10 to 50 fold, induction of annexin binding relative to untreated
cell in an annexin binding assay.
[1170] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[1171] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound to Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are required for such
killing. The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may
be performed. Useful effector cells for such assays include
peripheral blood mononuclear cells (PBMC) and Natural Killer (NK)
cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al., PNAS (USA)
95:652-656 (1998).
[1172] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In certain embodiments, the FcR is a
native sequence human FcR. Moreover, a preferred FcR is one that
binds an IgG antibody (a gamma receptor) and includes receptors of
the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses,
including allelic variants and alternatively spliced forms of these
receptors. FC.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[1173] "Human effector cells" are leukocytes that express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes that mediate ADCC include PBMC, NK
cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and
NK cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[1174] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) that are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[1175] The terms "influenza A" and "Influenzavirus A" refer to a
genus of the Orthomyxoviridae family of viruses. Influenzavirus A
includes only one species: influenza A virus which cause influenza
in birds, humans, pigs, and horses. Strains of all subtypes of
influenza A virus have been isolated from wild birds, although
disease is uncommon. Some isolates of influenza A virus cause
severe disease both in domestic poultry and, rarely, in humans.
[1176] A "mammal" for purposes of treating n infection, refers to
any mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, cats, cattle, horses, sheep,
pigs, goats, rabbits, etc. Preferably, the mammal is human.
[1177] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures;
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. Those in need of treatment
include those already with the disorder as well as those prone to
have the disorder or those in whom the disorder is to be prevented.
A subject or mammal is successfully "treated" for an infection if,
after receiving a therapeutic amount of an antibody according to
the methods of the present invention, the patient shows observable
and/or measurable reduction in or absence of one or more of the
following: reduction in the number of infected cells or absence of
the infected cells; reduction in the percent of total cells that
are infected; and/or relief to some extent, one or more of the
symptoms associated with the specific infection; reduced morbidity
and mortality, and improvement in quality of life issues. The above
parameters for assessing successful treatment and improvement in
the disease are readily measurable by routine procedures familiar
to a physician.
[1178] The term "therapeutically effective amount" refers to an
amount of an antibody or a drug effective to "treat" a disease or
disorder in a subject or mammal. See preceding definition of
"treating."
[1179] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[1180] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[1181] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM. polyethylene glycol (PEG), and PLURONICS.TM..
[1182] 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.sup.211, I.sup.113, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.,
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described
below.
[1183] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, either in
vitro or in vivo. Examples of growth inhibitory agents include
agents that block cell cycle progression, such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vinca alkaloids (vincristine, vinorelbine and
vinblastine), taxanes, and topoisomerase II inhibitors such as
doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogenes, and antineoplastic
drugs" by Murakami et al. (W B Saunders: Philadelphia, 1995),
especially p. 13. The taxanes (paclitaxel and docetaxel) are
anticancer drugs both derived from the yew tree. Docetaxel
(TAXOTERE.TM., Rhone-Poulenc Rorer), derived from the European yew,
is a semisynthetic analogue of paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb). Paclitaxel and docetaxel promote the
assembly of microtubules from tubulin dimers and stabilize
microtubules by preventing depolymerization, which results in the
inhibition of mitosis in cells.
[1184] "Label" as used herein refers to a detectable compound or
composition that is conjugated directly or indirectly to the
antibody so as to generate a "labeled" antibody. The label may be
detectable by itself (e.g., radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition that is
detectable.
[1185] The term "epitope tagged" as used herein refers to a
chimeric polypeptide comprising a polypeptide fused to a "tag
polypeptide." The tag polypeptide has enough residues to provide an
epitope against which an antibody can be made, yet is short enough
such that it does not interfere with activity of the polypeptide to
which it is fused. The tag polypeptide is also preferably fairly
unique so that the antibody does not substantially cross-react with
other epitopes. Suitable tag polypeptides generally have at least
six amino acid residues and usually between about 8 and 50 amino
acid residues (preferably, between about 10 and 20 amino acid
residues).
[1186] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[1187] The terms "nucleic acid" and "polynucleotide" are used
interchangeably herein to refer to single- or double-stranded RNA,
DNA, or mixed polymers. Polynucleotides may include genomic
sequences, extra-genomic and plasmid sequences, and smaller
engineered gene segments that express, or may be adapted to express
polypeptides.
[1188] An "isolated nucleic acid" is a nucleic acid that is
substantially separated from other genome DNA sequences as well as
proteins or complexes such as ribosomes and polymerases, which
naturally accompany a native sequence. The term embraces a nucleic
acid sequence that has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates and
chemically synthesized analogues or analogues biologically
synthesized by heterologous systems. A substantially pure nucleic
acid includes isolated forms of the nucleic acid. Of course, this
refers to the nucleic acid as originally isolated and does not
exclude genes or sequences later added to the isolated nucleic acid
by the hand of man.
[1189] The term "polypeptide" is used in its conventional meaning,
i.e., as a sequence of amino acids. The polypeptides are not
limited to a specific length of the product. Peptides,
oligopeptides, and proteins are included within the definition of
polypeptide, and such terms may be used interchangeably herein
unless specifically indicated otherwise. This term also does not
refer to or exclude post-expression modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like, as well as other modifications known
in the art, both naturally occurring and non-naturally occurring. A
polypeptide may be an entire protein, or a subsequence thereof.
Particular polypeptides of interest in the context of this
invention are amino acid subsequences comprising CDRs and being
capable of binding an antigen or Influenza A-infected cell.
[1190] An "isolated polypeptide" is one that has been identified
and separated and/or recovered from a component of its natural
environment. In preferred embodiments, the isolated polypeptide
will be purified (1) to greater than 95% by weight of polypeptide
as determined by the Lowry method, and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or non-reducing conditions using Coomassie blue or,
preferably, silver stain. Isolated polypeptide includes the
polypeptide in situ within recombinant cells since at least one
component of the polypeptide's natural environment will not be
present. Ordinarily, however, isolated polypeptide will be prepared
by at least one purification step.
[1191] A "native sequence" polynucleotide is one that has the same
nucleotide sequence as a polynucleotide derived from nature. A
"native sequence" polypeptide is one that has the same amino acid
sequence as a polypeptide (e.g., antibody) derived from nature
(e.g., from any species). Such native sequence polynucleotides and
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means.
[1192] A polynucleotide "variant," as the term is used herein, is a
polynucleotide that typically differs from a polynucleotide
specifically disclosed herein in one or more substitutions,
deletions, additions and/or insertions. Such variants may be
naturally occurring or may be synthetically generated, for example,
by modifying one or more of the polynucleotide sequences of the
invention and evaluating one or more biological activities of the
encoded polypeptide as described herein and/or using any of a
number of techniques well known in the art.
[1193] A polypeptide "variant," as the term is used herein, is a
polypeptide that typically differs from a polypeptide specifically
disclosed herein in one or more substitutions, deletions, additions
and/or insertions. Such variants may be naturally occurring or may
be synthetically generated, for example, by modifying one or more
of the above polypeptide sequences of the invention and evaluating
one or more biological activities of the polypeptide as described
herein and/or using any of a number of techniques well known in the
art.
[1194] Modifications may be made in the structure of the
polynucleotides and polypeptides of the present invention and still
obtain a functional molecule that encodes a variant or derivative
polypeptide with desirable characteristics. When it is desired to
alter the amino acid sequence of a polypeptide to create an
equivalent, or even an improved, variant or portion of a
polypeptide of the invention, one skilled in the art will typically
change one or more of the codons of the encoding DNA sequence.
[1195] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of its ability to bind other polypeptides (e.g., antigens) or
cells. Since it is the binding capacity and nature of a protein
that defines that protein's biological functional activity, certain
amino acid sequence substitutions can be made in a protein
sequence, and, of course, its underlying DNA coding sequence, and
nevertheless obtain a protein with like properties. It is thus
contemplated that various changes may be made in the peptide
sequences of the disclosed compositions, or corresponding DNA
sequences that encode said peptides without appreciable loss of
their biological utility or activity.
[1196] In many instances, a polypeptide variant will contain one or
more conservative substitutions. A "conservative substitution" is
one in which an amino acid is substituted for another amino acid
that has similar properties, such that one skilled in the art of
peptide chemistry would expect the secondary structure and
hydropathic nature of the polypeptide to be substantially
unchanged.
[1197] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a protein is
generally understood in the art (Kyte and Doolittle, 1982). It is
accepted that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like. Each amino acid has been
assigned a hydropathic index on the basis of its hydrophobicity and
charge characteristics (Kyte and Doolittle, 1982). These values
are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);
alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine
(-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);
asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[1198] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e. still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred. It is also understood in the
art that the substitution of like amino acids can be made
effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101
states that the greatest local average hydrophilicity of a protein,
as governed by the hydrophilicity of its adjacent amino acids,
correlates with a biological property of the protein.
[1199] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent protein. In such
changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[1200] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
that take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine.
[1201] Amino acid substitutions may further be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and
valine; glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain nonconservative
changes. In a preferred embodiment, variant polypeptides differ
from a native sequence by substitution, deletion or addition of
five amino acids or fewer. Variants may also (or alternatively) be
modified by, for example, the deletion or addition of amino acids
that have minimal influence on the immunogenicity, secondary
structure and hydropathic nature of the polypeptide.
[1202] Polypeptides may comprise a signal (or leader) sequence at
the N-terminal end of the protein, which co-translationally or
post-translationally directs transfer of the protein. The
polypeptide may also be conjugated to a linker or other sequence
for ease of synthesis, purification or identification of the
polypeptide (e.g., poly-His), or to enhance binding of the
polypeptide to a solid support. For example, a polypeptide may be
conjugated to an immunoglobulin Fc region.
[1203] When comparing polynucleotide and polypeptide sequences, two
sequences are said to be "identical" if the sequence of nucleotides
or amino acids in the two sequences is the same when aligned for
maximum correspondence, as described below. Comparisons between two
sequences are typically performed by comparing the sequences over a
comparison window to identify and compare local regions of sequence
similarity. A "comparison window" as used herein, refers to a
segment of at least about 20 contiguous positions, usually 30 to
about 75, 40 to about 50, in which a sequence may be compared to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned.
[1204] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins-Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E.
W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971)
Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.
[1205] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[1206] One preferred example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0
can be used, for example with the parameters described herein, to
determine percent sequence identity for the polynucleotides and
polypeptides of the invention. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information.
[1207] In one illustrative example, cumulative scores can be
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, and expectation (E) of 10,
and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50,
expectation (E) of 10, M=5, N=-4 and a comparison of both
strands.
[1208] For amino acid sequences, a scoring matrix can be used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment.
[1209] In one approach, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residues
occur in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e., the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[1210] "Homology" refers to the percentage of residues in the
polynucleotide or polypeptide sequence variant that are identical
to the non-variant sequence after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology. In particular embodiments, polynucleotide and polypeptide
variants have at least 70%, at least 75%, at least 80%, at least
90%, at least 95%, at least 98%, or at least 99% polynucleotide or
polypeptide homology with a polynucleotide or polypeptide described
herein.
[1211] "Vector" includes shuttle and expression vectors. Typically,
the plasmid construct will also include an origin of replication
(e.g., the ColE1 origin of replication) and a selectable marker
(e.g., ampicillin or tetracycline resistance), for replication and
selection, respectively, of the plasmids in bacteria. An
"expression vector" refers to a vector that contains the necessary
control sequences or regulatory elements for expression of the
antibodies including antibody fragment of the invention, in
bacterial or eukaryotic cells. Suitable vectors are disclosed
below.
[1212] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
[1213] The present invention includes human monoclonal
anti-influenza antibodies comprising a polypeptide of the present
invention, as well as fragments and variants thereof. In one
embodiment, the antibody is an antibody designated herein as
TCN-032 (8I10), 21B15, TCN-031 (23K12), 3241_G23, 3244_I10,
3243_J07, 3259_J21, 3245_O19, 3244_H04, 3136_G05, 3252_C13,
3255_J06, 3420_I23, 3139_P23, 3248_P18, 3253_P10, 3260_D19,
3362_B11, 3242_P05, TCN-522 (3212_I12), TCN-521 (3280_D18), TCN-523
(5248_A17), TCN-563 (5237_B21), TCN-526 (5084_C17), TCN-527
(5086_C06), TCN-528 (5087_P17), TCN-529 (5297_H01), TCN-530
(5248_H10), TCN-531 (5091_H13), TCN-532 (5262_H18), TCN-533
(5256_A17), TCN-534 (5249_B02), TCN-535 (5246_P19), TCN-536
(5095_N01), TCN-537 (3194_D21), TCN-538 (3206_O17), TCN-539
(5056_A08), TCN-540 (5060_F05), TCN-541 (5062_M11), TCN-542
(5079_A16), TCN-543 (5081_G23), TCN-544 (5082_A19), TCN-545
(5082_I15), TCN-546 (5089_L08), TCN-547 (5092_F11), TCN-548
(5092_P01), TCN-549 (5092_P04), TCN-550 (5096_F06), TCN-551
(5243_D01), TCN-552 (5249_I23), TCN-553 (5261_C18), TCN-554
(5277_M05), TCN-555 (5246_L16), TCN-556 (5089_K12), TCN-557
(5081_A04), TCN 558 (5248_H10b), TCN-559 (5097_G08), TCN-560
(5084_P10), TCN-504 (3251_K17), SC06-141, SC06-255, SC06-257,
SC06-260, SC06-261, SC06-262, SC06-268, SC06-272, SC06-296,
SC06-301, SC06-307, SC06-310, SC06-314, SC06-323, SC06-325,
SC06-327, SC06-328, SC06-329, SC06-331, SC06-332, SC06-334,
SC06-336, SC06-339, SC06-342, SC06-343, SC06-344, CR6141, CR6255,
CR6257, CR6260, CR6261, CR6262, CR6268, CR6272, CR6296, CR6301,
CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329,
CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, or CR6344.
These antibodies preferentially bind to or specifically bind to
influenza A infected cells as compared to uninfected control cells
of the same cell type.
[1214] In particular embodiments, the antibodies of the present
invention bind to the M2 or HA protein. In certain embodiments, the
present invention provides human anti-influenza antibodies that
bind to epitopes within M2e or HA that are only present in the
native conformation, i.e., as expressed in cells. In particular
embodiments, these antibodies fail to specifically bind to an
isolated M2e polypeptide, e.g., the 23 amino acid residue M2e
fragment or an isolated HA polypeptide. It is understood that these
antibodies recognize non-linear (i.e. conformational) epitope(s) of
the M2 or HA peptide or protein.
[1215] These specific conformational epitopes within the M2 or HA
protein, and particularly within M2e, may be used as vaccines to
prevent the development of influenza infection within a
subject.
[1216] As will be understood by the skilled artisan, general
description of antibodies herein and methods of preparing and using
the same also apply to individual antibody polypeptide constituents
and antibody fragments.
[1217] The antibodies of the present invention may be polyclonal or
monoclonal antibodies. However, in preferred embodiments, they are
monoclonal. In particular embodiments, antibodies of the present
invention are fully human antibodies. Methods of producing
polyclonal and monoclonal antibodies are known in the art and
described generally, e.g., in U.S. Pat. No. 6,824,780. Typically,
the antibodies of the present invention are produced recombinantly,
using vectors and methods available in the art, as described
further below. Human antibodies may also be generated by in vitro
activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
[1218] Human antibodies may also be produced in transgenic animals
(e.g., mice) that are capable of producing a full repertoire of
human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array into such germ-line mutant mice
results in the production of human antibodies upon antigen
challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos.
5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No.
5,545,807; and WO 97/17852. Such animals may be genetically
engineered to produce human antibodies comprising a polypeptide of
the present invention.
[1219] 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 Primatized.TM.. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[1220] 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.
[1221] The choice of non-human sequences, both light and heavy, to
be used in making the chimeric antibodies is important to reduce
antigenicity and human anti-non-human antibody responses when the
antibody is intended for human therapeutic use. It is further
important that chimeric antibodies retain high binding affinity for
the antigen and other favorable biological properties. To achieve
this goal, according to a preferred method, chimeric antibodies are
prepared by a process of analysis of the parental sequences and
various conceptual chimeric products using three-dimensional models
of the parental human and non-human sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the recipient and
import sequences so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the hypervariable region residues are directly and most
substantially involved in influencing antigen binding.
[1222] As noted above, antibodies (or immunoglobulins) can be
divided into five different classes, based on differences in the
amino acid sequences in the constant region of the heavy chains.
All immunoglobulins within a given class have very similar heavy
chain constant regions. These differences can be detected by
sequence studies or more commonly by serological means (i.e. by the
use of antibodies directed to these differences). Antibodies, or
fragments thereof, of the present invention may be any class, and
may, therefore, have a gamma, mu, alpha, delta, or epsilon heavy
chain. A gamma chain may be gamma 1, gamma 2, gamma 3, or gamma 4;
and an alpha chain may be alpha 1 or alpha 2.
[1223] In a preferred embodiment, an antibody of the present
invention, or fragment thereof, is an IgG. IgG is considered the
most versatile immunoglobulin, because it is capable of carrying
out all of the functions of immunoglobulin molecules. IgG is the
major Ig in serum, and the only class of Ig that crosses the
placenta. IgG also fixes complement, although the IgG4 subclass
does not. Macrophages, monocytes, PMN's and some lymphocytes have
Fc receptors for the Fc region of IgG. Not all subclasses bind
equally well; IgG2 and IgG4 do not bind to Fc receptors. A
consequence of binding to the Fc receptors on PMN's, monocytes and
macrophages is that the cell can now internalize the antigen
better. IgG is an opsonin that enhances phagocytosis. Binding of
IgG to Fc receptors on other types of cells results in the
activation of other functions. Antibodies of the present invention
may be of any IgG subclass.
[1224] In another preferred embodiment, an antibody, or fragment
thereof, of the present invention is an IgE. IgE is the least
common serum Ig since it binds very tightly to Fc receptors on
basophils and mast cells even before interacting with antigen. As a
consequence of its binding to basophils an mast cells, IgE is
involved in allergic reactions. Binding of the allergen to the IgE
on the cells results in the release of various pharmacological
mediators that result in allergic symptoms. IgE also plays a role
in parasitic helminth diseases. Eosinophils have Fc receptors for
IgE and binding of eosinophils to IgE-coated helminths results in
killing of the parasite. IgE does not fix complement.
[1225] In various embodiments, antibodies of the present invention,
and fragments thereof, comprise a variable light chain that is
either kappa or lambda. The lamba chain may be any of subtype,
including, e.g., lambda 1, lambda 2, lambda 3, and lambda 4.
[1226] As noted above, the present invention further provides
antibody fragments comprising a polypeptide of the present
invention. In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. For example, the
smaller size of the fragments allows for rapid clearance, and may
lead to improved access to certain tissues, such as solid tumors.
Examples of antibody fragments include: Fab, Fab', F(ab').sub.2 and
Fv fragments; diabodies; linear antibodies; single-chain
antibodies; and multispecific antibodies formed from antibody
fragments.
[1227] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Fab'-SH fragments can be directly
recovered from E. coli and chemically coupled to form F(ab').sub.2
fragments (Carter et al., Bio/Technology 10:163-167 (1992)).
According to another approach, F(ab').sub.2 fragments can be
isolated directly from recombinant host cell culture. Fab and
F(ab').sub.2 fragment with increased in vivo half-life comprising a
salvage receptor binding epitope residues are described in U.S.
Pat. No. 5,869,046. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner.
[1228] In other embodiments, the antibody of choice is a single
chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894;
and U.S. Pat. No. 5,587,458. Fv and sFv are the only species with
intact combining sites that are devoid of constant regions. Thus,
they are suitable for reduced nonspecific binding during in vivo
use. sFv fusion proteins may be constructed to yield fusion of an
effector protein at either the amino or the carboxy terminus of an
sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody
fragment may also be a "linear antibody", e.g., as described in
U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments
may be monospecific or bispecific.
[1229] In certain embodiments, antibodies of the present invention
are bispecific or multi-specific. Bispecific antibodies are
antibodies that have binding specificities for at least two
different epitopes. Exemplary bispecific antibodies may bind to two
different epitopes of a single antigen. Other such antibodies may
combine a first antigen binding site with a binding site for a
second antigen. Alternatively, an anti-M2e arm may be combined with
an arm that binds to a triggering molecule on a leukocyte, such as
a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16), so as to focus and localize cellular defense
mechanisms to the infected cell. Bispecific antibodies may also be
used to localize cytotoxic agents to infected cells. These
antibodies possess an M2e-binding arm and an arm that binds the
cytotoxic agent (e.g., saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific
antibodies). WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[1230] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of ten different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[1231] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the
construction provide the optimum yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant affect on the yield of the desired chain
combination.
[1232] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[1233] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers that are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H 3 domain. In this method,
one or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[1234] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[1235] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent, sodium arsenite, to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[1236] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.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.
[1237] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol., 148
(5):1547-1553 (1992). The leucine zipper peptides from the Fos and
Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
V.sub.H connected to a V.sub.L by a linker that is too short to
allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
[1238] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147: 60 (1991). A multivalent antibody may be
internalized (and/or catabolized) faster than a bivalent antibody
by a cell expressing an antigen to which the antibodies bind. The
antibodies of the present invention can be multivalent antibodies
with three or more antigen binding sites (e.g., tetravalent
antibodies), which can be readily produced by recombinant
expression of nucleic acid encoding the polypeptide chains of the
antibody. The multivalent antibody can comprise a dimerization
domain and three or more antigen binding sites. The preferred
dimerization domain comprises (or consists of) an Fc region or a
hinge region. In this scenario, the antibody will comprise an Fc
region and three or more antigen binding sites amino-terminal to
the Fc region. The preferred multivalent antibody herein comprises
(or consists of) three to about eight, but preferably four, antigen
binding sites. The multivalent antibody comprises at least one
polypeptide chain (and preferably two polypeptide chains), wherein
the polypeptide chain(s) comprise two or more variable domains. For
instance, the polypeptide chain(s) may comprise
VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a first variable
domain, VD2 is a second variable domain, Fc is one polypeptide
chain of an Fc region, X1 and X2 represent an amino acid or
polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a C.sub.L
domain.
[1239] Antibodies of the present invention further include single
chain antibodies.
[1240] In particular embodiments, antibodies of the present
invention are internalizing antibodies.
[1241] Amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the antibody. Amino acid sequence variants of the antibody may
be prepared by introducing appropriate nucleotide changes into a
polynucleotide that encodes the antibody, or a chain thereof, or by
peptide synthesis. Such modifications include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequences of the antibody. Any
combination of deletion, insertion, and substitution may be made to
arrive at the final antibody, provided that the final construct
possesses the desired characteristics. The amino acid changes also
may alter post-translational processes of the antibody, such as
changing the number or position of glycosylation sites. Any of the
variations and modifications described above for polypeptides of
the present invention may be included in antibodies of the present
invention.
[1242] A useful method for identification of certain residues or
regions of an antibody that are preferred locations for mutagenesis
is called "alanine scanning mutagenesis" as described by Cunningham
and Wells in Science, 244:1081-1085 (1989). Here, a residue or
group of target residues are identified (e.g., charged residues
such as arg, asp, his, lys, and glu) and replaced by a neutral or
negatively charged amino acid (most preferably alanine or
polyalanine) to affect the interaction of the amino acids with PSCA
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
anti-antibody variants are screened for the desired activity.
[1243] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of an antibody include the
fusion to the N- or C-terminus of the antibody to an enzyme (e.g.,
for ADEPT) or a polypeptide that increases the serum half-life of
the antibody.
[1244] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative and non-conservative substitutions are
contemplated.
[1245] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain.
[1246] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability (particularly where
the antibody is an antibody fragment such as an Fv fragment).
[1247] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody.
Generally, the resulting variant(s) selected for further
development will have improved biological properties relative to
the parent antibody from which they are generated. A convenient way
for generating such substitutional variants involves affinity
maturation using phage display. Briefly, several hypervariable
region sites (e.g., 6-7 sites) are mutated to generate all possible
amino substitutions at each site. The antibody variants thus
generated are displayed in a monovalent fashion from filamentous
phage particles as fusions to the gene III product of M13 packaged
within each particle. The phage-displayed variants are then
screened for their biological activity (e.g., binding affinity) as
herein disclosed. In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and an antigen or infected cell. Such contact residues and
neighboring residues are candidates for substitution according to
the techniques elaborated herein. Once such variants are generated,
the panel of variants is subjected to screening as described herein
and antibodies with superior properties in one or more relevant
assays may be selected for further development.
[1248] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[1249] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[1250] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[1251] The antibody of the invention is modified with respect to
effector function, e.g., so as to enhance antigen-dependent
cell-mediated cyotoxicity (ADCC) and/or complement dependent
cytotoxicity (CDC) of the antibody. This may be achieved by
introducing one or more amino acid substitutions in an Fc region of
the antibody. Alternatively or additionally, cysteine residue(s)
may be introduced in the Fc region, thereby allowing interchain
disulfide bond formation in this region. The homodimeric antibody
thus generated may have improved internalization capability and/or
increased complement-mediated cell killing and antibody-dependent
cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.
176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922
(1992). Homodimeric antibodies with enhanced anti-infection
activity may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al., Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement
lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug
Design 3:219-230 (1989).
[1252] To increase the serum half-life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
[1253] Antibodies of the present invention may also be modified to
include an epitope tag or label, e.g., for use in purification or
diagnostic applications. The invention also pertains to therapy
with immunoconjugates comprising an antibody conjugated to an
anti-cancer agent such as a cytotoxic agent or a growth inhibitory
agent. Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above.
[1254] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
[1255] In one preferred embodiment, an antibody (full length or
fragments) of the invention is conjugated to one or more
maytansinoid molecules. Maytansinoids are mitototic inhibitors that
act by inhibiting tubulin polymerization. Maytansine was first
isolated from the east African shrub Maytenus serrata (U.S. Pat.
No. 3,896,111). Subsequently, it was discovered that certain
microbes also produce maytansinoids, such as maytansinol and C-3
maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol
and derivatives and analogues thereof are disclosed, for example,
in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[1256] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)
described immunoconjugates comprising a maytansinoid designated DM1
linked to the monoclonal antibody C242 directed against human
colorectal cancer. The conjugate was found to be highly cytotoxic
towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor growth assay.
[1257] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[1258] There are many linking groups known in the art for making
antibody conjugates, including, for example, those disclosed in
U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et
al., Cancer Research 52: 127-131 (1992). The linking groups include
disulfide groups, thioether groups, acid labile groups, photolabile
groups, peptidase labile groups, or esterase labile groups, as
disclosed in the above-identified patents, disulfide and thioether
groups being preferred.
[1259] Immunoconjugates may be made using a variety of bifunctional
protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage. For example, a ricin immunotoxin can be prepared
as described in Vitetta et al., Science 238: 1098 (1987).
Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent
for conjugation of radionucleotide to the antibody. See WO94/11026.
The linker may be a "cleavable linker" facilitating release of the
cytotoxic drug in the cell. For example, an acid-labile linker,
Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) may be
used.
[1260] Another immunoconjugate of interest comprises an antibody
conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Another drug that the antibody can be conjugated is QFA which is an
antifolate. Both calicheamicin and QFA have intracellular sites of
action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
[1261] Examples of other agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[1262] Enzymatically active toxins and fragments thereof that can
be used include, e.g., diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin
and the tricothecenes. See, for example, WO 93/21232.
[1263] The present invention further includes an immunoconjugate
formed between an antibody and a compound with nucleolytic activity
(e.g., a ribonuclease or a DNA endonuclease such as a
deoxyribonuclease; DNase).
[1264] For selective destruction of infected cells, the antibody
includes a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-PSCA antibodies. Examples include At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Rc.sup.88, Sm.sup.153, Bi.sup.212,
P.sup.32, Pb.sup.212 and radioactive isotopes of Lu. When the
conjugate is used for diagnosis, it may comprise a radioactive atom
for scintigraphic studies, for example tc.sup.99m or I.sup.123, or
a spin label for nuclear magnetic resonance (NMR) imaging (also
known as magnetic resonance imaging, mri), such as iodine-123,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[1265] The radio- or other label is incorporated in the conjugate
in known ways. For example, the peptide may be biosynthesized or
may be synthesized by chemical amino acid synthesis using suitable
amino acid precursors involving, for example, fluorine-19 in place
of hydrogen. Labels such as tc.sup.99m or I.sup.123, Re.sup.186,
Re.sup.188 and In.sup.111 can be attached via a cysteine residue in
the peptide. Yttrium-90 can be attached via a lysine residue. The
IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun.
80: 49-57 can be used to incorporate iodine-123. "Monoclonal
Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989)
describes other methods in detail.
[1266] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent is made, e.g., by recombinant techniques or peptide
synthesis. The length of DNA may comprise respective regions
encoding the two portions of the conjugate either adjacent one
another or separated by a region encoding a linker peptide which
does not destroy the desired properties of the conjugate.
[1267] The antibodies of the present invention are also used in
antibody dependent enzyme mediated prodrug therapy (ADET) by
conjugating the antibody to a prodrug-activating enzyme which
converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see
WO81/01145) to an active anti-cancer drug (see, e.g., WO 88/07378
and U.S. Pat. No. 4,975,278).
[1268] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form. Enzymes that
are useful in the method of this invention include, but are not
limited to, alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs;
cytosine deaminase useful for converting non-toxic 5-fluorocytosine
into the anti-cancer drug, 5-fluorouracil; proteases, such as
serratia protease, thermolysin, subtilisin, carboxypeptidases and
cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a infected cell population.
[1269] The enzymes of this invention can be covalently bound to the
antibodies by techniques well known in the art such as the use of
the heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the antigen
binding region of an antibody of the invention linked to at least a
functionally active portion of an enzyme of the invention can be
constructed using recombinant DNA techniques well known in the art
(see, e.g., Neuberger et al., Nature, 312: 604-608 (1984).
[1270] Other modifications of the antibody are contemplated herein.
For example, the antibody may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol. The antibody also may be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization (for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively), in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules), or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
16th edition, Oslo, A., Ed., (1980).
[1271] The antibodies disclosed herein are also formulated as
immunoliposomes. A "liposome" is a small vesicle composed of
various types of lipids, phospholipids and/or surfactant that is
useful for delivery of a drug to a mammal. The components of the
liposome are commonly arranged in a bilayer formation, similar to
the lipid arrangement of biological membranes. Liposomes containing
the antibody are prepared by methods known in the art, such as
described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688
(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980);
U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published
Oct. 23, 1997. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[1272] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired a
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19)
1484 (1989).
[1273] Antibodies of the present invention, or fragments thereof,
may possess any of a variety of biological or functional
characteristics. In certain embodiments, these antibodies are
Influenza A specific or M2 protein specific antibodies, indicating
that they specifically bind to or preferentially bind to Influenza
A or the M2 protein thereof, respectively, as compared to a normal
control cell. In particular embodiments, the antibodies are HuM2e
antibodies, indicating that they specifically bind to a M2e
protein, preferably to an epitope of the M2e domain that is only
present when the M2 protein is expressed in cells or present on a
virus, as compared to a normal control cell.
[1274] In particular embodiments, an antibody of the present
invention is an antagonist antibody, which partially or fully
blocks or inhibits a biological activity of a polypeptide or cell
to which it specifically or preferentially binds. In other
embodiments, an antibody of the present invention is a growth
inhibitory antibody, which partially or fully blocks or inhibits
the growth of an infected cell to which it binds. In another
embodiment, an antibody of the present invention induces apoptosis.
In yet another embodiment, an antibody of the present invention
induces or promotes antibody-dependent cell-mediated cytotoxicity
or complement dependent cytotoxicity.
Methods of Identifying and Producing Antibodies Specific for
Influenza Virus
[1275] The present invention provides novel methods for the
identification of human anti-influenza antibodies raised against
the M2e protein, as exemplified in Example 4, and for the
identification of human anti-influenza antibodies raised against
the HA protein, as exemplified in Example 13. These methods may be
readily adapted to identify antibodies specific for other
polypeptides expressed on the cell surface by infectious agents, or
even polypeptides expressed on the surface of an infectious agent
itself.
[1276] In general, the methods include obtaining serum samples from
patients that have been infected with or vaccinated against an
infectious agent. These serum samples are then screened to identify
those that contain antibodies specific for a particular polypeptide
associated with the infectious agent, such as, e.g., a polypeptide
or protein specifically expressed on the surface of cells infected
with the infectious agent, but not uninfected cells. In particular
embodiments, the serum samples are screened by contacting the
samples with a cell that has been transfected with an expression
vector that expresses the polypeptide expressed on the surface of
infected cells.
[1277] Once a patient is identified as having serum containing an
antibody specific for the infectious agent polypeptide of interest
is identified, mononuclear and/or B cells obtained from the same
patient are used to identify a cell or clone thereof that produces
the antibody, using any of the methods described herein or
available in the art. Once a B cell that produces the antibody is
identified, cDNAs encoding the variable regions or fragments
thereof of the antibody may be cloned using standard RT-PCR vectors
and primers specific for conserved antibody sequences, and
subcloned in to expression vectors used for the recombinant
production of monoclonal antibodies specific for the infectious
agent polypeptide of interest.
[1278] In one embodiment, the present invention provides a method
of identifying an antibody that specifically binds influenza
A-infected cells, comprising: contacting an Influenza A virus or a
cell expressing the M2 protein with a biological sample obtained
from a patient having been infected by Influenza A; determining an
amount of antibody in the biological sample that binds to the cell;
and comparing the amount determined with a control value, wherein
if the value determined is at least two-fold greater than the
control value, an antibody that specifically binds influenza
A-infected cells is indicated.
[1279] In various embodiments, the cells expressing an M2 or HA
protein are cells infected with an Influenza A virus or cells that
have been transfected with a polynucleotide that expressed the M2
or HA protein. Alternatively, the cells may express a portion of
the M2 protein that includes the M2e domain and enough additional
M2 sequence that the protein remains associated with the cell and
the M2e domain is presented on the cell surface in the same manner
as when present within full length M2 protein. Methods of preparing
an M2 or HA expression vector and transfecting an appropriate cell,
including those described herein, may be readily accomplished, in
view of the M2 and HA sequences being publicly available. See, for
example, the Influenza Sequence Database (ISD) (flu.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., Osterhaus & Hampson (Eds.),
Elsevier Science, Amsterdam, pp. 103-106) and the Microbial
Sequencing Center (MSC) at The Institute for Genomic Research
(TIGR) (tigr.org/msc/infl_a_virus.shtml on the World Wide Web).
[1280] The M2e- or HA-expressing cells or virus described above are
used to screen the biological sample obtained from a patient
infected with influenza A for the presence of antibodies that
preferentially bind to the cell expressing the M2 or HA polypeptide
using standard biological techniques. For example, in certain
embodiments, the antibodies may be labeled, and the presence of
label associated with the cell detected, e.g., using FMAT or FACs
analysis. In particular embodiments, the biological sample is
blood, serum, plasma, bronchial lavage, or saliva. Methods of the
present invention may be practiced using high throughput
techniques.
[1281] Identified human antibodies may then be characterized
further. For example the particular conformational epitopes with in
the M2e or HA protein that are necessary or sufficient for binding
of the antibody may be determined, e.g., using site-directed
mutagenesis of expressed M2e or HA polypeptides. These methods may
be readily adapted to identify human antibodies that bind any
protein expressed on a cell surface. Furthermore, these methods may
be adapted to determine binding of the antibody to the virus
itself, as opposed to a cell expressing recombinant M2e or HA, or
infected with the virus.
[1282] Polynucleotide sequences encoding the antibodies, variable
regions thereof, or antigen-binding fragments thereof may be
subcloned into expression vectors for the recombinant production of
human monoclonal anti-M2e or anti-HA antibodies. In one embodiment,
this is accomplished by obtaining mononuclear cells from the
patient from the serum containing the identified human monoclonal
anti-M2e or anti-HA antibody was obtained; producing B cell clones
from the mononuclear cells; inducing the B cells to become
antibody-producing plasma cells; and screening the supernatants
produced by the plasma cells to determine if it contains the human
monoclonal anti-M2e or anti-HA antibody. Once a B cell clone that
produces a human monoclonal anti-M2e or anti-HA antibody is
identified, reverse-transcription polymerase chain reaction
(RT-PCR) is performed to clone the DNAs encoding the variable
regions or portions thereof of the human monoclonal anti-M2e or
anti-HA antibody. These sequences are then subcloned into
expression vectors suitable for the recombinant production of human
monoclonal anti-M2e or anti-HA antibodies. The binding specificity
may be confirmed by determining the recombinant antibody's ability
to bind cells expressing M2e or HA polypeptide or protein.
[1283] In particular embodiments of the methods described herein, B
cells isolated from peripheral blood or lymph nodes are sorted,
e.g., based on their being CD19 positive, and plated, e.g., as low
as a single cell specificity per well, e.g., in 96, 384, or 1536
well configurations. The cells are induced to differentiate into
antibody-producing cells, e.g., plasma cells, and the culture
supernatants are harvested and tested for binding to cells
expressing the infectious agent polypeptide on their surface using,
e.g., FMAT or FACS analysis. Positive wells are then subjected to
whole well RT-PCR to amplify heavy and light chain variable regions
of the IgG molecule expressed by the clonal daughter plasma cells.
The resulting PCR products encoding the heavy and light chain
variable regions, or portions thereof, are subcloned into human
antibody expression vectors for recombinant expression. The
resulting recombinant antibodies are then tested to confirm their
original binding specificity and may be further tested for
pan-specificity across various strains of isolates of the
infectious agent.
[1284] Thus, in one embodiment, a method of identifying human
monoclonal anti-M2e or anti-HA antibodies is practiced as follows.
First, full length or approximately full length M2 or HA cDNAs are
transfected into a cell line for expression of M2 or HA protein.
Secondly, individual human plasma or sera samples are tested for
antibodies that bind the cell-expressed M2 or HA. And lastly, MAbs
derived from plasma- or serum-positive individuals are
characterized for binding to the same cell-expressed M2 or HA.
Further definition of the fine specificities of the MAbs can be
performed at this point.
[1285] These methods may be practiced to identify a variety of
different HuM2e antibodies, including antibodies specific for (a)
epitopes in a linear M2e peptide, (b) common epitopes in multiple
variants of M2e, (c) conformational determinants of an M2
homotetramer, and (d) common conformational determinants of
multiple variants of the M2 homotetramer. The last category is
particularly desirable, as this specificity is perhaps specific for
all A strains of influenza.
[1286] These methods may be practiced to identify a variety of
different human monoclonal anti-HA antibodies, including antibodies
specific for (a) epitopes in a linear HA peptide, (b) common
epitopes in multiple variants of HA, (c) conformational
determinants of an HA protein or homotrimer, and (d) common
conformational determinants of multiple variants of the HA protein
or homotrimer. The last category is particularly desirable, as this
specificity is perhaps specific for all A strains of influenza.
[1287] Polynucleotides that encode the human monoclonal anti-M2e or
anti-HA antibodies or portions thereof of the present invention may
be isolated from cells expressing human monoclonal anti-M2e or
anti-HA antibodies, according to methods available in the art and
described herein, including amplification by polymerase chain
reaction using primers specific for conserved regions of human
antibody polypeptides. For example, light chain and heavy chain
variable regions may be cloned from the B cell according to
molecular biology techniques described in WO 92/02551; U.S. Pat.
No. 5,627,052; or Babcook et al., Proc. Natl. Acad. Sci. USA
93:7843-48 (1996). In certain embodiments, polynucleotides encoding
all or a region of both the heavy and light chain variable regions
of the IgG molecule expressed by the clonal daughter plasma cells
expressing the human monoclonal anti-M2e or anti-HA antibody are
subcloned and sequenced. The sequence of the encoded polypeptide
may be readily determined from the polynucleotide sequence.
[1288] Isolated polynucleotides encoding a polypeptide of the
present invention may be subcloned into an expression vector to
recombinantly produce antibodies and polypeptides of the present
invention, using procedures known in the art and described
herein.
[1289] 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
(1HC) and/or fluorescence-activated cell sorting (FACS).
Immunoassay methods may include controls and procedures to
determine whether antibodies bind specifically to M2e from one or
more specific strains of Influenza A, and do not recognize or
cross-react with normal control cells.
[1290] Following pre-screening of serum to identify patients that
produce antibodies to an infectious agent or polypeptide thereof,
e.g., M2 or HA, the methods of the present invention typically
include the isolation or purification of B cells from a biological
sample previously obtained from a patient or subject. The patient
or subject may be currently or previously diagnosed with or suspect
or having a particular disease or infection, or the patient or
subject may be considered free or a particular disease or
infection. Typically, the patient or subject is a mammal and, in
particular embodiments, a human. The biological sample may be any
sample that contains B cells, including but not limited to, lymph
node or lymph node tissue, pleural effusions, peripheral blood,
ascites, tumor tissue, or cerebrospinal fluid (CSF). In various
embodiments, B cells are isolated from different types of
biological samples, such as a biological sample affected by a
particular disease or infection. However, it is understood that any
biological sample comprising B cells may be used for any of the
embodiments of the present invention.
[1291] Once isolated, the B cells are induced to produce
antibodies, e.g., by culturing the B cells under conditions that
support B cell proliferation or development into a plasmacyte,
plasmablast, or plasma cell. The antibodies are then screened,
typically using high throughput techniques, to identify an antibody
that specifically binds to a target antigen, e.g., a particular
tissue, cell, infectious agent, or polypeptide. In certain
embodiments, the specific antigen, e.g., cell surface polypeptide
bound by the antibody is not known, while in other embodiments, the
antigen specifically bound by the antibody is known.
[1292] According to the present invention, B cells may be isolated
from a biological sample, e.g., a tumor, tissue, peripheral blood
or lymph node sample, by any means known and available in the art.
B cells are typically sorted by FACS based on the presence on their
surface of a B cell-specific marker, e.g., CD19, CD138, and/or
surface IgG. However, other methods known in the art may be
employed, such as, e.g., column purification using CD19 magnetic
beads or IgG-specific magnetic beads, followed by elution from the
column. However, magnetic isolation of B cells utilizing any marker
may result in loss of certain B cells. Therefore, in certain
embodiments, the isolated cells are not sorted but, instead,
phicol-purified mononuclear cells isolated from tumor are directly
plated to the appropriate or desired number of specificities per
well.
[1293] In order to identify B cells that produce an infectious
agent-specific antibody, the B cells are typically plated at low
density (e.g., a single cell specificity per well, 1-10 cells per
well, 10-100 cells per well, 1-100 cells per well, less than 10
cells per well, or less than 100 cells per well) in multi-well or
microtitre plates, e.g., in 96, 384, or 1536 well configurations.
When the B cells are initially plated at a density greater than one
cell per well, then the methods of the present invention may
include the step of subsequently diluting cells in a well
identified as producing an antigen-specific antibody, until a
single cell specificity per well is achieved, thereby facilitating
the identification of the B cell that produces the antigen-specific
antibody. Cell supernatants or a portion thereof and/or cells may
be frozen and stored for future testing and later recovery of
antibody polynucleotides.
[1294] In certain embodiments, the B cells are cultured under
conditions that favor the production of antibodies by the B cells.
For example, the B cells may be cultured under conditions favorable
for B cell proliferation and differentiation to yield
antibody-producing plasmablast, plasmacytes, or plasma cells. In
particular embodiments, the B cells are cultured in the presence of
a B cell mitogen, such as lipopolysaccharide (LPS) or CD40 ligand.
In one specific embodiment, B cells are differentiated to
antibody-producing cells by culturing them with feed cells and/or
other B cell activators, such as CD40 ligand.
[1295] Cell culture supernatants or antibodies obtained therefrom
may be tested for their ability to bind to a target antigen, using
routine methods available in the art, including those described
herein. In particular embodiments, culture supernatants are tested
for the presence of antibodies that bind to a target antigen using
high-throughput methods. For example, B cells may be cultured in
multi-well microtitre dishes, such that robotic plate handlers may
be used to simultaneously sample multiple cell supernatants and
test for the presence of antibodies that bind to a target antigen.
In particular embodiments, antigens are bound to beads, e.g.,
paramagnetic or latex beads) to facilitate the capture of
antibody/antigen complexes. In other embodiments, antigens and
antibodies are fluorescently labeled (with different labels) and
FACS analysis is performed to identify the presence of antibodies
that bind to target antigen. In one embodiment, antibody binding is
determined using FMAT.TM. analysis and instrumentation (Applied
Biosystems, Foster City, Calif.). FMAT.TM. is a fluorescence
macro-confocal platform for high-throughput screening, which
mix-and-read, non-radioactive assays using live cells or beads.
[1296] In the context of comparing the binding of an antibody to a
particular target antigen (e.g., a biological sample such as
infected tissue or cells, or infectious agents) as compared to a
control sample (e.g., a biological sample such as uninfected cells,
or a different infectious agent), in various embodiments, the
antibody is considered to preferentially bind a particular target
antigen if at least two-fold, at least three-fold, at least
five-fold, or at least ten-fold more antibody binds to the
particular target antigen as compared to the amount that binds a
control sample.
[1297] Polynucleotides encoding antibody chains, variable regions
thereof, or fragments thereof, may be isolated from cells utilizing
any means available in the art. In one embodiment, polynucleotides
are isolated using polymerase chain reaction (PCR), e.g., reverse
transcription-PCR(RT-PCR) using oligonucleotide primers that
specifically bind to heavy or light chain encoding polynucleotide
sequences or complements thereof using routine procedures available
in the art. In one embodiment, positive wells are subjected to
whole well RT-PCR to amplify the heavy and light chain variable
regions of the IgG molecule expressed by the clonal daughter plasma
cells. These PCR products may be sequenced.
[1298] The resulting PCR products encoding the heavy and light
chain variable regions or portions thereof are then subcloned into
human antibody expression vectors and recombinantly expressed
according to routine procedures in the art (see, e.g., U.S. Pat.
No. 7,112,439). The nucleic acid molecules encoding a
tumor-specific antibody or fragment thereof, as described herein,
may be propagated and expressed according to any of a variety of
well-known procedures for nucleic acid excision, ligation,
transformation, and transfection. Thus, in certain embodiments
expression of an antibody fragment may be preferred in a
prokaryotic host cell, such as Escherichia coli (see, e.g.,
Pluckthun et al., Methods Enzymol. 178:497-515 (1989)). In certain
other embodiments, expression of the antibody or an antigen-binding
fragment thereof may be preferred in a eukaryotic host cell,
including yeast (e.g., Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Pichia pastoris); animal cells
(including mammalian cells); or plant cells. Examples of suitable
animal cells include, but are not limited to, myeloma, COS, CHO, or
hybridoma cells. Examples of plant cells include tobacco, corn,
soybean, and rice cells. By methods known to those having ordinary
skill in the art and based on the present disclosure, a nucleic
acid vector may be designed for expressing foreign sequences in a
particular host system, and then polynucleotide sequences encoding
the tumor-specific antibody (or fragment thereof) may be inserted.
The regulatory elements will vary according to the particular
host.
[1299] One or more replicable expression vectors containing a
polynucleotide encoding a variable and/or constant region may be
prepared and used to transform an appropriate cell line, for
example, a non-producing myeloma cell line, such as a mouse NSO
line or a bacterium, such as E. coli, in which production of the
antibody will occur. In order to obtain efficient transcription and
translation, the polynucleotide sequence in each vector should
include appropriate regulatory sequences, particularly a promoter
and leader sequence operatively linked to the variable domain
sequence. Particular methods for producing antibodies in this way
are generally well known and routinely used. For example, molecular
biology procedures are described by Sambrook et al. (Molecular
Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory, New York, 1989; see also Sambrook et al., 3rd ed., Cold
Spring Harbor Laboratory, New York, (2001)). While not required, in
certain embodiments, regions of polynucleotides encoding the
recombinant antibodies may be sequenced. DNA sequencing can be
performed as described in Sanger et al. (Proc. Natl. Acad. Sci. USA
74:5463 (1977)) and the Amersham International plc sequencing
handbook and including improvements thereto.
[1300] In particular embodiments, the resulting recombinant
antibodies or fragments thereof are then tested to confirm their
original specificity and may be further tested for pan-specificity,
e.g., with related infectious agents. In particular embodiments, an
antibody identified or produced according to methods described
herein is tested for cell killing via antibody dependent cellular
cytotoxicity (ADCC) or apoptosis, and/or well as its ability to
internalize.
Polynucleotides
[1301] The present invention, in other aspects, provides
polynucleotide compositions. In preferred embodiments, these
polynucleotides encode a polypeptide of the invention, e.g., a
region of a variable chain of an antibody that binds to Influenza
A, M2, M2e, or HA (soluble or recombinant). Polynucleotides of the
invention are single-stranded (coding or antisense) or
double-stranded DNA (genomic, cDNA or synthetic) or RNA molecules.
RNA molecules include, but are not limited to, HnRNA molecules,
which contain introns and correspond to a DNA molecule in a
one-to-one manner, and mRNA molecules, which do not contain
introns. Alternatively, or in addition, coding or non-coding
sequences are present within a polynucleotide of the present
invention. Also alternatively, or in addition, a polynucleotide is
linked to other molecules and/or support materials of the
invention. Polynucleotides of the invention are used, e.g., in
hybridization assays to detect the presence of an Influenza A
antibody in a biological sample, and in the recombinant production
of polypeptides of the invention.
[1302] Therefore, according to another aspect of the present
invention, polynucleotide compositions are provided that include
some or all of a polynucleotide sequences set forth herein,
complements of these polynucleotide sequences, and degenerate
variants of these polynucleotide sequences. In certain preferred
embodiments, the polynucleotide sequences set forth herein encode
polypeptides capable of preferentially binding a Influenza
A-infected cell as compared to a normal control uninfected cell,
including a polypeptide having a sequence set forth herein.
Furthermore, the invention includes all polynucleotides that encode
any polypeptide of the present invention.
[1303] In other related embodiments, the invention provides
polynucleotide variants having substantial identity to the
sequences set forth herein, for example those comprising at least
70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% or higher, sequence identity compared to a
polynucleotide sequence of this invention, as determined using the
methods described herein, (e.g., BLAST analysis using standard
parameters). One skilled in this art will recognize that these
values can be appropriately adjusted to determine corresponding
identity of proteins encoded by two nucleotide sequences by taking
into account codon degeneracy, amino acid similarity, reading frame
positioning, and the like.
[1304] Typically, polynucleotide variants contain one or more
substitutions, additions, deletions and/or insertions, preferably
such that the immunogenic binding properties of the polypeptide
encoded by the variant polynucleotide is not substantially
diminished relative to a polypeptide encoded by a polynucleotide
sequence specifically set forth herein.
[1305] In additional embodiments, the present invention provides
polynucleotide fragments comprising various lengths of contiguous
stretches of sequence identical to or complementary to one or more
of the sequences disclosed herein. For example, polynucleotides are
provided by this invention that comprise at least about 10, 15, 20,
30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more
contiguous nucleotides of one or more of the sequences disclosed
herein as well as all intermediate lengths there between. As used
herein, the term "intermediate lengths" is meant to describe any
length between the quoted values, such as 16, 17, 18, 19, etc.; 21,
22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101,
102, 103, etc.; 150, 151, 152, 153, etc.; including all integers
through 200-500; 500-1,000, and the like.
[1306] In another embodiment of the invention, polynucleotide
compositions are provided that are capable of hybridizing under
moderate to high stringency conditions to a polynucleotide sequence
provided herein, or a fragment thereof, or a complementary sequence
thereof. Hybridization techniques are well known in the art of
molecular biology. For purposes of illustration, suitable
moderately stringent conditions for testing the hybridization of a
polynucleotide of this invention with other polynucleotides include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-60.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC
containing 0.1% SDS. One skilled in the art will understand that
the stringency of hybridization can be readily manipulated, such as
by altering the salt content of the hybridization solution and/or
the temperature at which the hybridization is performed. For
example, in another embodiment, suitable highly stringent
hybridization conditions include those described above, with the
exception that the temperature of hybridization is increased, e.g.,
to 60-65.degree. C. or 65-70.degree. C.
[1307] In preferred embodiments, the polypeptide encoded by the
polynucleotide variant or fragment has the same binding specificity
(i.e., specifically or preferentially binds to the same epitope or
Influenza A strain) as the polypeptide encoded by the native
polynucleotide. In certain preferred embodiments, the
polynucleotides described above, e.g., polynucleotide variants,
fragments and hybridizing sequences, encode polypeptides that have
a level of binding activity of at least about 50%, preferably at
least about 70%, and more preferably at least about 90% of that for
a polypeptide sequence specifically set forth herein.
[1308] The polynucleotides of the present invention, or fragments
thereof, regardless of the length of the coding sequence itself,
may be combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. A nucleic acid
fragment of almost any length is employed, with the total length
preferably being limited by the ease of preparation and use in the
intended recombinant DNA protocol. For example, illustrative
polynucleotide segments with total lengths of about 10,000, about
5000, about 3000, about 2,000, about 1,000, about 500, about 200,
about 100, about 50 base pairs in length, and the like, (including
all intermediate lengths) are included in many implementations of
this invention.
[1309] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
multiple nucleotide sequences that encode a polypeptide as
described herein. Some of these polynucleotides bear minimal
homology to the nucleotide sequence of any native gene.
Nonetheless, polynucleotides that encode a polypeptide of the
present invention but which vary due to differences in codon usage
are specifically contemplated by the invention. Further, alleles of
the genes including the polynucleotide sequences provided herein
are within the scope of the invention. Alleles are endogenous genes
that are altered as a result of one or more mutations, such as
deletions, additions and/or substitutions of nucleotides. The
resulting mRNA and protein may, but need not, have an altered
structure or function. Alleles may be identified using standard
techniques (such as hybridization, amplification and/or database
sequence comparison).
[1310] In certain embodiments of the present invention, mutagenesis
of the disclosed polynucleotide sequences is performed in order to
alter one or more properties of the encoded polypeptide, such as
its binding specificity or binding strength. Techniques for
mutagenesis are well-known in the art, and are widely used to
create variants of both polypeptides and polynucleotides. A
mutagenesis approach, such as site-specific mutagenesis, is
employed for the preparation of variants and/or derivatives of the
polypeptides described herein. By this approach, specific
modifications in a polypeptide sequence are made through
mutagenesis of the underlying polynucleotides that encode them.
These techniques provides a straightforward approach to prepare and
test sequence variants, for example, incorporating one or more of
the foregoing considerations, by introducing one or more nucleotide
sequence changes into the polynucleotide.
[1311] Site-specific mutagenesis allows the production of mutants
through the use of specific oligonucleotide sequences include the
nucleotide sequence of the desired mutation, as well as a
sufficient number of adjacent nucleotides, to provide a primer
sequence of sufficient size and sequence complexity to form a
stable duplex on both sides of the deletion junction being
traversed. Mutations are employed in a selected polynucleotide
sequence to improve, alter, decrease, modify, or otherwise change
the properties of the polynucleotide itself, and/or alter the
properties, activity, composition, stability, or primary sequence
of the encoded polypeptide.
[1312] In other embodiments of the present invention, the
polynucleotide sequences provided herein are used as probes or
primers for nucleic acid hybridization, e.g., as PCR primers. The
ability of such nucleic acid probes to specifically hybridize to a
sequence of interest enable them to detect the presence of
complementary sequences in a given sample. However, other uses are
also encompassed by the invention, such as the use of the sequence
information for the preparation of mutant species primers, or
primers for use in preparing other genetic constructions. As such,
nucleic acid segments of the invention that include a sequence
region of at least about 15 nucleotides long contiguous sequence
that has the same sequence as, or is complementary to, a 15
nucleotide long contiguous sequence disclosed herein is
particularly useful. Longer contiguous identical or complementary
sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000
(including all intermediate lengths) including full length
sequences, and all lengths in between, are also used in certain
embodiments.
[1313] Polynucleotide molecules having sequence regions consisting
of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even
of 100-200 nucleotides or so (including intermediate lengths as
well), identical or complementary to a polynucleotide sequence
disclosed herein, are particularly contemplated as hybridization
probes for use in, e.g., Southern and Northern blotting, and/or
primers for use in, e.g., polymerase chain reaction (PCR). The
total size of fragment, as well as the size of the complementary
stretch(es), ultimately depends on the intended use or application
of the particular nucleic acid segment. Smaller fragments are
generally used in hybridization embodiments, wherein the length of
the contiguous complementary region may be varied, such as between
about 15 and about 100 nucleotides, but larger contiguous
complementarity stretches may be used, according to the length
complementary sequences one wishes to detect.
[1314] The use of a hybridization probe of about 15-25 nucleotides
in length allows the formation of a duplex molecule that is both
stable and selective. Molecules having contiguous complementary
sequences over stretches greater than 12 bases in length are
generally preferred, though, in order to increase stability and
selectivity of the hybrid, and thereby improve the quality and
degree of specific hybrid molecules obtained. Nucleic acid
molecules having gene-complementary stretches of 15 to 25
contiguous nucleotides, or even longer where desired, are generally
preferred.
[1315] Hybridization probes are selected from any portion of any of
the sequences disclosed herein. All that is required is to review
the sequences set forth herein, or to any continuous portion of the
sequences, from about 15-25 nucleotides in length up to and
including the full length sequence, that one wishes to utilize as a
probe or primer. The choice of probe and primer sequences is
governed by various factors. For example, one may wish to employ
primers from towards the termini of the total sequence.
[1316] Polynucleotide of the present invention, or fragments or
variants thereof, are readily prepared by, for example, directly
synthesizing the fragment by chemical means, as is commonly
practiced using an automated oligonucleotide synthesizer. Also,
fragments are obtained by application of nucleic acid reproduction
technology, such as the PCR.TM. technology of U.S. Pat. No.
4,683,202, by introducing selected sequences into recombinant
vectors for recombinant production, and by other recombinant DNA
techniques generally known to those of skill in the art of
molecular biology.
Vectors, Host Cells and Recombinant Methods
[1317] The invention provides vectors and host cells comprising a
nucleic acid of the present invention, as well as recombinant
techniques for the production of a polypeptide of the present
invention. Vectors of the invention include those capable of
replication in any type of cell or organism, including, e.g.,
plasmids, phage, cosmids, and mini chromosomes. In various
embodiments, vectors comprising a polynucleotide of the present
invention are vectors suitable for propagation or replication of
the polynucleotide, or vectors suitable for expressing a
polypeptide of the present invention. Such vectors are known in the
art and commercially available.
[1318] Polynucleotides of the present invention are synthesized,
whole or in parts that are then combined, and inserted into a
vector using routine molecular and cell biology techniques,
including, e.g., subcloning the polynucleotide into a linearized
vector using appropriate restriction sites and restriction enzymes.
Polynucleotides of the present invention are amplified by
polymerase chain reaction using oligonucleotide primers
complementary to each strand of the polynucleotide. These primers
also include restriction enzyme cleavage sites to facilitate
subcloning into a vector. The replicable vector components
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, and one or
more marker or selectable genes.
[1319] In order to express a polypeptide of the present invention,
the nucleotide sequences encoding the polypeptide, or functional
equivalents, are inserted into an appropriate expression vector,
i.e., a vector that contains the necessary elements for the
transcription and translation of the inserted coding sequence.
Methods well known to those skilled in the art are used to
construct expression vectors containing sequences encoding a
polypeptide of interest and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Such techniques are described, for example,
in Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.
M. et al. (1989) Current Protocols in Molecular Biology, John Wiley
& Sons, New York. N.Y.
[1320] A variety of expression vector/host systems are utilized to
contain and express polynucleotide sequences. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems.
[1321] Within one embodiment, the variable regions of a gene
expressing a monoclonal antibody of interest are amplified from a
hybridoma cell using nucleotide primers. These primers are
synthesized by one of ordinary skill in the art, or may be
purchased from commercially available sources (see, e.g.,
Stratagene (La Jolla, Calif.), which sells primers for amplifying
mouse and human variable regions. The primers are used to amplify
heavy or light chain variable regions, which are then inserted into
vectors such as ImmunoZAP.TM. H or ImmunoZAP.TM. L (Stratagene),
respectively. These vectors are then introduced into E. coli,
yeast, or mammalian-based systems for expression. Large amounts of
a single-chain protein containing a fusion of the V.sub.H and
V.sub.L domains are produced using these methods (see Bird et al.,
Science 242:423-426 (1988)).
[1322] The "control elements" or "regulatory sequences" present in
an expression vector are those non-translated regions of the
vector, e.g., enhancers, promoters, 5' and 3' untranslated regions,
that interact with host cellular proteins to carry out
transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, is
used.
[1323] Examples of promoters suitable for use with prokaryotic
hosts include the phoa promoter, p-lactamase and lactose promoter
systems, alkaline phosphatase promoter, a tryptophan (trp) promoter
system, and hybrid promoters such as the tac promoter. However,
other known bacterial promoters are suitable. Promoters for use in
bacterial systems also usually contain a Shine-Dalgarno sequence
operably linked to the DNA encoding the polypeptide. Inducible
promoters such as the hybrid lacZ promoter of the PBLUESCRIPT
phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco
BRL, Gaithersburg, Md.) and the like are used.
[1324] A variety of promoter sequences are known for eukaryotes and
any are used according to the present invention. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[1325] In mammalian cell systems, promoters from mammalian genes or
from mammalian viruses are generally preferred. Polypeptide
expression from vectors in mammalian host cells aer controlled, for
example, by promoters obtained from the genomes of viruses such as
polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2),
bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV),
a retrovirus, hepatitis-B virus and most preferably Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin promoter, and from heat-shock
promoters, provided such promoters are compatible with the host
cell systems. If it is necessary to generate a cell line that
contains multiple copies of the sequence encoding a polypeptide,
vectors based on SV40 or EBV may be advantageously used with an
appropriate selectable marker. One example of a suitable expression
vector is pcDNA-3.1 (Invitrogen, Carlsbad, Calif.), which includes
a CMV promoter.
[1326] A number of viral-based expression systems are available for
mammalian expression of polypeptides. For example, in cases where
an adenovirus is used as an expression vector, sequences encoding a
polypeptide of interest may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter
and tripartite leader sequence. Insertion in a non-essential E1 or
E3 region of the viral genome may be used to obtain a viable virus
that is capable of expressing the polypeptide in infected host
cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci.
81:3655-3659). In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells.
[1327] In bacterial systems, any of a number of expression vectors
are selected depending upon the use intended for the expressed
polypeptide. For example, when large quantities are desired,
vectors that direct high level expression of fusion proteins that
are readily purified are used. Such vectors include, but are not
limited to, the multifunctional E. coli cloning and expression
vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding the polypeptide of interest may be ligated into the vector
in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase, so that a hybrid
protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster
(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors
(Promega, Madison, Wis.) are also used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione.
Proteins made in such systems are designed to include heparin,
thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at
will.
[1328] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH are used. Examples of other
suitable promoter sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase or other glycolytic
enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogcnase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. For reviews, see Ausubel et al. (supra) and Grant et
al. (1987) Methods Enzymol. 153:516-544. Other yeast promoters that
are inducible promoters having the additional advantage of
transcription controlled by growth conditions include the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, metallothionein, glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Suitable vectors and promoters for use in yeast
expression are further described in EP 73,657. Yeast enhancers also
are advantageously used with yeast promoters.
[1329] In cases where plant expression vectors are used, the
expression of sequences encoding polypeptides are driven by any of
a number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV are used alone or in combination with the
omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311. Alternatively, plant promoters such as the small subunit
of RUBISCO or heat shock promoters are used (Coruzzi, G. et al.
(1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science
224:838-843; and Winter, J., et al. (1991) Results Probl. Cell
Differ. 17:85-105). These constructs can be introduced into plant
cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, e.g., Hobbs, S. or Murry, L. E.
in McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New York, N.Y.; pp. 191-196).
[1330] An insect system is also used to express a polypeptide of
interest. For example, in one such system, Autographa californica
nuclear polyhedrosis virus (AcNPV) is used as a vector to express
foreign genes in Spodoptera frugiperda cells or in Trichoplusia
larvae. The sequences encoding the polypeptide are cloned into a
non-essential region of the virus, such as the polyhedrin gene, and
placed under control of the polyhedrin promoter. Successful
insertion of the polypeptide-encoding sequence renders the
polyhedrin gene inactive and produce recombinant virus lacking coat
protein. The recombinant viruses are then used to infect, for
example, S. frugiperda cells or Trichoplusia larvae, in which the
polypeptide of interest is expressed (Engelhard, E. K. et al.
(1994) Proc. Natl. Acad. Sci. 91:3224-3227).
[1331] Specific initiation signals are also used to achieve more
efficient translation of sequences encoding a polypeptide of
interest. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding the
polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous translational control signals including the
ATG initiation codon are provided. Furthermore, the initiation
codon is in the correct reading frame to ensure correct translation
of the inserted polynucleotide. Exogenous translational elements
and initiation codons are of various origins, both natural and
synthetic.
[1332] Transcription of a DNA encoding a polypeptide of the
invention is often increased by inserting an enhancer sequence into
the vector. Many enhancer sequences are known, including, e.g.,
those identified in genes encoding globin, elastase, albumin,
.alpha.-fetoprotein, and insulin. Typically, however, an enhancer
from a eukaryotic cell virus is used. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer is
spliced into the vector at a position 5' or 3' to the
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[1333] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) typically also contain sequences necessary
for the termination of transcription and for stabilizing the mRNA.
Such sequences are commonly available from the 5' and, occasionally
3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA
encoding anti-PSCA antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO94/11026 and the expression vector disclosed therein.
[1334] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, plant or higher eukaryote
cells described above. Examples of suitable prokaryotes for this
purpose include eubacteria, such as Gram-negative or Gram-positive
organisms, for example, Enterobacteriaceae such as Escherichia,
e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia
marcescans, and Shigella, as well as Bacilli such as B. subtilis
and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD
266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa,
and Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[1335] Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species, and strains are
commonly available and used herein, such as Schizosaccharomyces
pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis
(ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC
24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),
K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia
pastoris. (EP 183,070); Candida; Trichoderma reesia (EP 244,234);
Neurospora crassa; Schwanniomyces such as Schwanniomyces
occidentalis; and filamentous fungi such as, e.g., Neurospora,
Penicillium, Tolypocladium, and Aspergillus hosts such as A.
nidulans and A. niger.
[1336] In certain embodiments, a host cell strain is chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation. glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing that
cleaves a "prepro" form of the protein is also used to facilitate
correct insertion, folding and/or function. Different host cells
such as CHO, COS, HeLa, MDCK, HEK293, and WI38, which have specific
cellular machinery and characteristic mechanisms for such
post-translational activities, are chosen to ensure the correct
modification and processing of the foreign protein.
[1337] Methods and reagents specifically adapted for the expression
of antibodies or fragments thereof are also known and available in
the art, including those described, e.g., in U.S. Pat. Nos.
4,816,567 and 6,331,415. In various embodiments, antibody heavy and
light chains, or fragments thereof, are expressed from the same or
separate expression vectors. In one embodiment, both chains are
expressed in the same cell, thereby facilitating the formation of a
functional antibody or fragment thereof.
[1338] Full length antibody, antibody fragments, and antibody
fusion proteins are produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
infected cell destruction. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523, which describes translation initiation
region (TIR) and signal sequences for optimizing expression and
secretion. After expression, the antibody is isolated from the E.
coli cell paste in a soluble fraction and can be purified through,
e.g., a protein A or G column depending on the isotype. Final
purification can be carried out using a process similar to that
used for purifying antibody expressed e.g., in CHO cells.
[1339] Suitable host cells for the expression of glycosylated
polypeptides and antibodies are derived from multicellular
organisms. Examples of invertebrate cells include plant and insect
cells. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopicius (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses are used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells. Plant cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco are also utilized as
hosts.
[1340] Methods of propagation of antibody polypeptides and
fragments thereof in vertebrate cells in culture (tissue culture)
are encompassed by the invention. Examples of mammalian host cell
lines used in the methods of the invention are monkey kidney CV1
line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic
kidney line (293 or 293 cells subcloned for growth in suspension
culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster
kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary
cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216
(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251
(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells
(Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2).
[1341] Host cells are transformed with the above-described
expression or cloning vectors for polypeptide production and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[1342] For long-term, high-yield production of recombinant
proteins, stable expression is generally preferred. For example,
cell lines that stably express a polynucleotide of interest are
transformed using expression vectors that contain viral origins of
replication and/or endogenous expression elements and a selectable
marker gene on the same or on a separate vector. Following the
introduction of the vector, cells are allowed to grow for 1-2 days
in an enriched media before they are switched to selective media.
The purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
that successfully express the introduced sequences. Resistant
clones of stably transformed cells are proliferated using tissue
culture techniques appropriate to the cell type.
[1343] A plurality of selection systems are used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1990) Cell 22:817-23) genes that are employed in tk.sup.- or
aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance is used as the basis for selection; for
example, dhfr, which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which
confers resistance to the aminoglycosides, neomycin and G-418
(Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14); and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described. For example, trpB
allows cells to utilize indole in place of tryptophan, and hisD
allows cells to utilize histinol in place of histidine (Hartman, S.
C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).
The use of visible markers has gained popularity with such markers
as anthocyanins, beta-glucuronidase and its substrate GUS, and
luciferase and its substrate luciferin, being widely used not only
to identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.
55:121-131).
[1344] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression is confirmed. For example, if the sequence encoding
a polypeptide is inserted within a marker gene sequence,
recombinant cells containing sequences are identified by the
absence of marker gene function. Alternatively, a marker gene is
placed in tandem with a polypeptide-encoding sequence under the
control of a single promoter. Expression of the marker gene in
response to induction or selection usually indicates expression of
the tandem gene as well.
[1345] Alternatively, host cells that contain and express a desired
polynucleotide sequence are identified by a variety of procedures
known to those of skill in the art. These procedures include, but
are not limited to, DNA-DNA or DNA-RNA hybridizations and protein
bioassay or immunoassay techniques which include, for example,
membrane, solution, or chip based technologies for the detection
and/or quantification of nucleic acid or protein.
[1346] A variety of protocols for detecting and measuring the
expression of polynucleotide-encoded products, using either
polyclonal or monoclonal antibodies specific for the product are
known in the art. Nonlimiting examples include enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), and
fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on a given polypeptide is
preferred for some applications, but a competitive binding assay
may also be employed. These and other assays are described, among
other places, in Hampton, R. et al. (1990; Serological Methods, a
Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et
al. (1983; J. Exp. Med. 158:1211-1216).
[1347] Various labels and conjugation techniques are known by those
skilled in the art and are used in various nucleic acid and amino
acid assays. Means for producing labeled hybridization or PCR
probes for detecting sequences related to polynucleotides include
oligolabeling, nick translation, end-labeling or PCR amplification
using a labeled nucleotide. Alternatively, the sequences, or any
portions thereof are cloned into a vector for the production of an
mRNA probe. Such vectors are known in the art, are commercially
available, and are used to synthesize RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures are conducted using a
variety of commercially available kits. Suitable reporter molecules
or labels, which are used include, but are not limited to,
radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[1348] The polypeptide produced by a recombinant cell is secreted
or contained intracellularly depending on the sequence and/or the
vector used. Expression vectors containing polynucleotides of the
invention are designed to contain signal sequences that direct
secretion of the encoded polypeptide through a prokaryotic or
eukaryotic cell membrane.
[1349] In certain embodiments, a polypeptide of the invention is
produced as a fusion polypeptide further including a polypeptide
domain that facilitates purification of soluble proteins. Such
purification-facilitating domains include, but are not limited to,
metal chelating peptides such as histidine-tryptophan modules that
allow purification on immobilized metals, protein A domains that
allow purification on immobilized immunoglobulin, and the domain
utilized in the FLAGS extension/affinity purification system
(Amgen, Seattle, Wash.). The inclusion of cleavable linker
sequences such as those specific for Factor XA or enterokinase
(Invitrogen. San Diego, Calif.) between the purification domain and
the encoded polypeptide are used to facilitate purification. An
exemplary expression vector provides for expression of a fusion
protein containing a polypeptide of interest and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography) as described in Porath, J. et al. (1992, Prot. Exp.
Purif. 3:263-281) while the enterokinase cleavage site provides a
means for purifying the desired polypeptide from the fusion
protein. A discussion of vectors used for producing fusion proteins
is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.
12:441-453).
[1350] In certain embodiments, a polypeptide of the present
invention is fused with a heterologous polypeptide, which may be a
signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells, the signal sequence is
selected, for example, from the group of the alkaline phosphatase,
penicillinase, 1 pp, or heat-stable enterotoxin II leaders. For
yeast secretion, the signal sequence is selected from, e.g., the
yeast invertase leader, a factor leader (including Saccharomyces
and Kluyveromyces a factor leaders), or acid phosphatase leader,
the C. albicans glucoamylase leader, or the signal described in WO
90/13646. In mammalian cell expression, mammalian signal sequences
as well as viral secretory leaders, for example, the herpes simplex
gD signal, are available.
[1351] When using recombinant techniques, the polypeptide or
antibody is produced intracellularly, in the periplasmic space, or
directly secreted into the medium. If the polypeptide or antibody
is produced intracellularly, as a first step, the particulate
debris, either host cells or lysed fragments, are removed, for
example, by centrifugation or ultrafiltration. Carter et al.,
Bio/Technology 10:163-167 (1992) describe a procedure for isolating
antibodies that are secreted to the periplasmic space of E. coli.
Briefly, cell paste is thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
min. Cell debris is removed by centrifugation. Where the
polypeptide or antibody is secreted into the medium, supernatants
from such expression systems are generally first concentrated using
a commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. Optionally, a
protease inhibitor such as PMSF is included in any of the foregoing
steps to inhibit proteolysis and antibiotics are included to
prevent the growth of adventitious contaminants.
[1352] The polypeptide or antibody composition prepared from the
cells are purified using, for example, hydroxylapatite
chromatography, gel electrophoresis, dialysis, and affinity
chromatography, with affinity chromatography being the preferred
purification technique. The suitability of protein A as an affinity
ligand depends on the species and isotype of any immunoglobulin Fc
domain that is present in the polypeptide or antibody. Protein A is
used to purify antibodies or fragments thereof that are based on
human .gamma..sub.1, .gamma..sub.2, or .gamma..sub.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma..sub.3
(Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the
affinity ligand is attached is most often agarose, but other
matrices are available. Mechanically stable matrices such as
controlled pore glass or poly(styrenedivinyl)benzene allow for
faster flow rates and shorter processing times than can be achieved
with agarose. Where the polypeptide or antibody comprises a C.sub.H
3 domain, the Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg,
N.J.) is useful for purification. Other techniques for protein
purification such as fractionation on an ion-exchange column,
ethanol precipitation, Reverse Phase HPLC, chromatography on
silica, chromatography on heparin SEPHAROSE.TM. chromatography on
an anion or cation exchange resin (such as a polyaspartic acid
column), chromatofocusing, SDS-PAGE, and ammonium sulfate
precipitation are also available depending on the polypeptide or
antibody to be recovered.
[1353] Following any preliminary purification step(s), the mixture
comprising the polypeptide or antibody of interest and contaminants
are subjected to low pH hydrophobic interaction chromatography
using an elution buffer at a pH between about 2.5-4.5, preferably
performed at low salt concentrations (e.g., from about 0-0.25M
salt).
Pharmaceutical Compositions
[1354] The invention further includes pharmaceutical formulations
including a polypeptide, antibody, or modulator of the present
invention, at a desired degree of purity, and a pharmaceutically
acceptable carrier, excipient, or stabilizer (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). In
certain embodiments, pharmaceutical formulations are prepared to
enhance the stability of the polypeptide or antibody during
storage, e.g., in the form of lyophilized formulations or aqueous
solutions.
[1355] Acceptable carriers, excipients, or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and
include, e.g., buffers such as acetate, Tris, phosphate, citrate,
and other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; tonicifiers such as
trehalose and sodium chloride; sugars such as sucrose, mannitol,
trehalose or sorbitol; surfactant such as polysorbate; salt-forming
counter-ions such as sodium; metal complexes (e.g. Zn-protein
complexes); and/or non-ionic surfactants such as TWEEN.TM.,
PLURONICS.TM. or polyethylene glycol (PEG). In certain embodiments,
the therapeutic formulation preferably comprises the polypeptide or
antibody at a concentration of between 5-200 mg/ml, preferably
between 10-100 mg/ml.
[1356] The formulations herein also contain one or more additional
therapeutic agents suitable for the treatment of the particular
indication, e.g., infection being treated, or to prevent undesired
side-effects. Preferably, the additional therapeutic agent has an
activity complementary to the polypeptide or antibody of the resent
invention, and the two do not adversely affect each other. For
example, in addition to the polypeptide or antibody of the
invention, an additional or second antibody, anti-viral agent,
anti-infective agent and/or cardioprotectant is added to the
formulation. Such molecules are suitably present in the
pharmaceutical formulation in amounts that are effective for the
purpose intended.
[1357] The active ingredients, e.g., polypeptides and antibodies of
the invention and other therapeutic agents, are also entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and polymethylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[1358] Sustained-release preparations are prepared. Suitable
examples of sustained-release preparations include, but are not
limited to, semi-permeable matrices of solid hydrophobic polymers
containing the antibody, which matrices are in the form of shaped
articles, e.g., films, or microcapsules. Nonlimiting examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxyburyric acid.
[1359] Formulations to be used for in vivo administration are
preferably sterile. This is readily accomplished by filtration
through sterile filtration membranes.
Diagnostic Uses
[1360] Antibodies and fragments thereof, and therapeutic
compositions, of the invention specifically bind or preferentially
bind to infected cells or tissue, as compared to normal control
cells and tissue. Thus, these influenza A antibodies are used to
detect infected cells or tissues in a patient, biological sample,
or cell population, using any of a variety of diagnostic and
prognostic methods, including those described herein. The ability
of an anti-M2e or anti-HA specific antibody to detect infected
cells depends upon its binding specificity, which is readily
determined by testing its ability to bind to infected cells or
tissues obtained from different patients, and/or from patients
infected with different strains of Influenza A.
[1361] Diagnostic methods generally involve contacting a biological
sample obtained from a patient, such as, e.g., blood, serum,
saliva, urine, sputum, a cell swab sample, or a tissue biopsy, with
an Influenza A, e.g., human monoclonal anti-M2e or anti-HA
antibody, and determining whether the antibody preferentially binds
to the sample as compared to a control sample or predetermined
cut-off value, thereby indicating the presence of infected cells.
In particular embodiments, at least two-fold, three-fold, or
five-fold more human monoclonal anti-M2e or anti-HA antibody binds
to an infected cell as compared to an appropriate control normal
cell or tissue sample. A pre-determined cut-off value is
determined, e.g., by averaging the amount of human monoclonal
anti-M2e or anti-HA antibody that binds to several different
appropriate control samples under the same conditions used to
perform the diagnostic assay of the biological sample being tested.
Alternatively, or in addition, a hemagglutinin (HA) protein is
substituted for an Influenza virus in the above method. The HA
protein is presented on the surface of a virus, host cell (e.g. any
mammalian cell), or in a recombinant and soluble form. In the HA
version of this diagnostic method, the control protein is a
denatured HA protein, a linear HA peptide, an unrelated protein of
similar size and shape, but dissimilar sequence, or a
pre-determined cut-off value.
[1362] Bound antibody is detected using procedures described herein
and known in the art. In certain embodiments, diagnostic methods of
the invention are practiced using human monoclonal anti-M2e or
anti-HA antibodies that are conjugated to a detectable label, e.g.,
a fluorophore, to facilitate detection of bound antibody. However,
they are also practiced using methods of secondary detection of the
human monoclonal anti-M2e or anti-HA antibody. These include, for
example, RIA, ELISA, precipitation, agglutination, complement
fixation and immuno-fluorescence.
[1363] In certain procedures, the human monoclonal anti-M2e or
anti-HA antibodies are labeled. The label is detected directly.
Exemplary labels that are detected directly include, but are not
limited to, radiolabels and fluorochromes. Alternatively, or in
addition, labels are moieties, such as enzymes, that must be
reacted or derivatized to be detected. Nonlimiting examples of
isotope labels are .sup.99Tc, .sup.14C, .sup.131I, .sup.125I,
.sup.3H, .sup.32P and .sup.35S. Fluorescent materials that are used
include, but are not limited to, for example, fluorescein and its
derivatives, rhodamine and its derivatives, auramine, dansyl,
umbelliferone, luciferia, 2,3-dihydrophthalazinediones, horseradish
peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate
dehydrogenase.
[1364] An enzyme label is detected by any of the currently utilized
colorimetric, spectrophotometric, fluorospectro-photometric or
gasometric techniques. Many enzymes which are used in these
procedures are known and utilized by the methods of the invention.
Nonlimiting examples are peroxidase, alkaline phosphatase,
.beta.-glucuronidase, .beta.-D-glucosidase, .beta.-D-galactosidase,
urease, glucose oxidase plus peroxidase, galactose oxidase plus
peroxidase and acid phosphatase.
[1365] The antibodies are tagged with such labels by known methods.
For instance, coupling agents such as aldehydes, carbodiimides,
dimaleimide, imidates, succinimides, bid-diazotized benzadine and
the like are used to tag the antibodies with the above-described
fluorescent, chemiluminescent, and enzyme labels. An enzyme is
typically combined with an antibody using bridging molecules such
as carbodiimides, periodate, diisocyanates, glutaraldehyde and the
like. Various labeling techniques are described in Morrison,
Methods in Enzymology 32b, 103 (1974), Syvanen et al., J. Biol.
Chem. 284, 3762 (1973) and Bolton and Hunter, Biochem J. 133, 529
(1973).
[1366] Human monoclonal anti-M2e or anti-HA antibodies of the
present invention are capable of differentiating between patients
with and patients without an Influenza A infection, and determining
whether or not a patient has an infection, using the representative
assays provided herein. According to one method, a biological
sample is obtained from a patient suspected of having or known to
have an influenza A infection. In preferred embodiments, the
biological sample includes cells from the patient. The sample is
contacted with a human monoclonal anti-M2e or anti-HA antibody,
e.g., for a time and under conditions sufficient to allow the human
monoclonal anti-M2e or anti-HA antibody to bind to infected cells
present in the sample. For instance, the sample is contacted with a
human monoclonal anti-M2e or anti-HA antibody for 10 seconds, 30
seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6
hours, 12 hours, 24 hours, 3 days or any point in between. The
amount of bound human monoclonal anti-M2e or anti-HA antibody is
determined and compared to a control value, which may be, e.g., a
pre-determined value or a value determined from normal tissue
sample. An increased amount of antibody bound to the patient sample
as compared to the control sample is indicative of the presence of
infected cells in the patient sample.
[1367] In a related method, a biological sample obtained from a
patient is contacted with a human monoclonal anti-M2e or anti-HA
antibody for a time and under conditions sufficient to allow the
antibody to bind to infected cells. Bound antibody is then
detected, and the presence of bound antibody indicates that the
sample contains infected cells. This embodiment is particularly
useful when the human monoclonal anti-M2e or anti-HA antibody does
not bind normal cells at a detectable level.
[1368] Different human monoclonal anti-M2e or anti-HA antibodies
possess different binding and specificity characteristics.
Depending upon these characteristics, particular human monoclonal
anti-M2e or anti-HA antibodies are used to detect the presence of
one or more strains of Influenza A. For example, certain antibodies
bind specifically to only one or several strains of Influenza
virus, whereas others bind to all or a majority of different
strains of Influenza virus. Antibodies specific for only one strain
of Influenza A are used to identify the strain of an infection.
[1369] In certain embodiments, antibodies that bind to an infected
cell preferably generate a signal indicating the presence of an
infection in at least about 20% of patients with the infection
being detected, more preferably at least about 30% of patients.
Alternatively, or in addition, the antibody generates a negative
signal indicating the absence of the infection in at least about
90% of individuals without the infection being detected. Each
antibody satisfies the above criteria; however, antibodies of the
present invention are used in combination to improve
sensitivity.
[1370] The present invention also includes kits useful in
performing diagnostic and prognostic assays using the antibodies of
the present invention. Kits of the invention include a suitable
container comprising a human monoclonal anti-M2e or anti-HA
antibody of the invention in either labeled or unlabeled form. In
addition, when the antibody is supplied in a labeled form suitable
for an indirect binding assay, the kit further includes reagents
for performing the appropriate indirect assay. For example, the kit
includes one or more suitable containers including enzyme
substrates or derivatizing agents, depending on the nature of the
label. Control samples and/or instructions are also included.
Therapeutic/Prophylactic Uses
[1371] Passive immunization has proven to be an effective and safe
strategy for the prevention and treatment of viral diseases. (See
Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall,
Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10
(1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of
which are incorporated herein by reference)). Passive immunization
using human monoclonal antibodies provide an immediate treatment
strategy for emergency prophylaxis and treatment of influenza
[1372] Human monoclonal anti-M2e or anti-HA antibodies and
fragments thereof, and therapeutic compositions, of the invention
specifically bind or preferentially bind to infected cells, as
compared to normal control uninfected cells and tissue. Thus, these
human monoclonal anti-M2e or anti-HA antibodies are used to
selectively target infected cells or tissues in a patient,
biological sample, or cell population. In light of the
infection-specific binding properties of these antibodies, the
present invention provides methods of regulating (e.g., inhibiting)
the growth of infected cells, methods of killing infected cells,
and methods of inducing apoptosis of infected cells. These methods
include contacting an infected cell with a human monoclonal
anti-M2e or anti-HA antibody of the invention. These methods are
practiced in vitro, ex vivo, and in vivo.
[1373] In various embodiments, antibodies of the invention are
intrinsically therapeutically active. Alternatively, or in
addition, antibodies of the invention are conjugated to a cytotoxic
agent or growth inhibitory agent, e.g., a radioisotope or toxin,
which is used in treating infected cells bound or contacted by the
antibody.
[1374] In one embodiment, the invention provides methods of
treating or preventing infection in a patient, including the steps
of providing a human monoclonal anti-M2e or anti-HA antibody of the
invention to a patient diagnosed with, at risk of developing, or
suspected of having an Influenza A infection. The methods of the
invention are used in the first-line treatment of the infection,
follow-on treatment, or in the treatment of a relapsed or
refractory infection. Treatment with an antibody of the invention
is a stand alone treatment. Alternatively, treatment with an
antibody of the invention is one component or phase of a
combination therapy regime, in which one or more additional
therapeutic agents are also used to treat the patient.
[1375] Subjects at risk for an influenza virus-related diseases or
disorders include patients who have come into contact with an
infected person or who have been exposed to the influenza virus in
some other way. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the
influenza virus-related disease or disorder, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression.
[1376] In various aspects, the human monoclonal anti-M2e or anti-HA
is administered substantially contemporaneously with or following
infection of the subject, i.e., therapeutic treatment. In another
aspect, the antibody provides a therapeutic benefit. In various
aspects, a therapeutic benefit includes reducing or decreasing
progression, severity, frequency, duration or probability of one or
more symptoms or complications of influenza infection, virus titer,
virus replication or an amount of a viral protein of one or more
influenza strains. still another aspect, a therapeutic benefit
includes hastening or accelerating a subject's recovery from
influenza infection.
[1377] Methods for preventing an increase in influenza virus titer,
virus replication, virus proliferation or an amount of an influenza
viral protein in a subject are further provided. In one embodiment,
a method includes administering to the subject an amount of a human
monoclonal anti-M2e or anti-HA antibody effective to prevent an
increase in influenza virus titer, virus replication or an amount
of an influenza viral protein of one or more influenza strains or
isolates in the subject.
[1378] Methods for protecting a subject from infection or
decreasing susceptibility of a subject to infection by one or more
influenza strains/isolates or subtypes, i.e., prophylactic methods,
are additionally provided. In one embodiment, a method includes
administering to the subject an amount of human monoclonal anti-M2e
or anti-HA antibody that specifically binds influenza M2 or HA,
respectively, effective to protect the subject from infection, or
effective to decrease susceptibility of the subject to infection,
by one or more influenza strains/isolates or subtypes.
[1379] Optionally, the subject is further administered with a
second agent such as, but not limited to, an influenza virus
antibody, an anti-viral drug such as a neuraminidase inhibitor, a
HA inhibitor, a sialic acid inhibitor or an M2 ion channel
inhibitor, a viral entry inhibitor or a viral attachment inhibitor.
The M2 ion channel inhibitor is for example amantadine or
rimantadine. The neuraminidase inhibitor for example zanamivir, or
oseltamivir phosphate.
[1380] Symptoms or complications of influenza infection that can be
reduced or decreased include, for example, chills, fever, cough,
sore throat, nasal congestion, sinus congestion, nasal infection,
sinus infection, body ache, head ache, fatigue, pneumonia,
bronchitis, ear infection, ear ache or death.
[1381] For in vivo treatment of human and non-human patients, the
patient is usually administered or provided a pharmaceutical
formulation including a human monoclonal anti-M2e or anti-HA
antibody of the invention. When used for in vivo therapy, the
antibodies of the invention are administered to the patient in
therapeutically effective amounts (i.e., amounts that eliminate or
reduce the patient's viral burden). The antibodies are administered
to a human patient, in accord with known methods, such as
intravenous administration, e.g., as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. The antibodies
may be administered parenterally, when possible, at the target cell
site, or intravenously. Intravenous or subcutaneous administration
of the antibody is preferred in certain embodiments. Therapeutic
compositions of the invention are administered to a patient or
subject systemically, parenterally, or locally.
[1382] For parenteral administration, the antibodies are formulated
in a unit dosage injectable form (solution, suspension, emulsion)
in association with a pharmaceutically acceptable, parenteral
vehicle. Examples of such vehicles are water, saline, Ringer's
solution, dextrose solution, and 5% human serum albumin. Nonaqueous
vehicles such as fixed oils and ethyl oleate are also used.
Liposomes are used as carriers. The vehicle contains minor amounts
of additives such as substances that enhance isotonicity and
chemical stability, e.g., buffers and preservatives. The antibodies
are typically formulated in such vehicles at concentrations of
about 1 mg/ml to 10 mg/ml.
[1383] The dose and dosage regimen depends upon a variety of
factors readily determined by a physician, such as the nature of
the infection and the characteristics of the particular cytotoxic
agent or growth inhibitory agent conjugated to the antibody (when
used), e.g., its therapeutic index, the patient, and the patient's
history. Generally, a therapeutically effective amount of an
antibody is administered to a patient. In particular embodiments,
the amount of antibody administered is in the range of about 0.1
mg/kg to about 50 mg/kg of patient body weight. Depending on the
type and severity of the infection, about 0.1 mg/kg to about 50
mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. The progress of this therapy is readily
monitored by conventional methods and assays and based on criteria
known to the physician or other persons of skill in the art.
[1384] In one particular embodiment, an immunoconjugate including
the antibody conjugated with a cytotoxic agent is administered to
the patient. Preferably, the immunoconjugate is internalized by the
cell, resulting in increased therapeutic efficacy of the
immunoconjugate in killing the cell to which it binds. In one
embodiment, the cytotoxic agent targets or interferes with the
nucleic acid in the infected cell. Examples of such cytotoxic
agents are described above and include, but are not limited to,
maytansinoids, calicheamicins, ribonucleases and DNA
endonucleases.
[1385] Other therapeutic regimens are combined with the
administration of the HuM2e antibody of the present invention. The
combined administration includes co-administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Preferably such
combined therapy results in a synergistic therapeutic effect.
[1386] In certain embodiments, it is desirable to combine
administration of an antibody of the invention with another
antibody directed against another antigen associated with the
infectious agent.
[1387] Aside from administration of the antibody protein to the
patient, the invention provides methods of administration of the
antibody by gene therapy. Such administration of nucleic acid
encoding the antibody is encompassed by the expression
"administering a therapeutically effective amount of an antibody".
See, for example, PCT Patent Application Publication WO96/07321
concerning the use of gene therapy to generate intracellular
antibodies.
[1388] In another embodiment, human monoclonal anti-M2e or anti-HA
antibodies of the invention are used to determine the structure of
bound antigen, e.g., conformational epitopes, the structure of
which is then used to develop a vaccine having or mimicking this
structure, e.g., through chemical modeling and SAR methods. Such a
vaccine could then be used to prevent Influenza A infection.
[1389] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet are
incorporated herein by reference, in their entirety.
EXAMPLES
Example 1
Screening and Characterization of M2e-Specific Antibodies Present
in Human Plasma Using Cells Expressing Recombinant M2e Protein
[1390] Fully human monoclonal antibodies specific for M2 and
capable of binding to influenza A infected cells and the influenza
virus itself were identified in patient serum, as described
below.
Expression of M2 in Cell Lines
[1391] An expression construct containing the M2 full length cDNA,
corresponding to the derived M2 sequence found in Influenza subtype
H3N2, was transfected into 293 cells.
[1392] The M2 cDNA is encoded by the following polynucleotide
sequence and SEQ ID NO: 53:
TABLE-US-00769 ATGAGTCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGGTG
CAGATGCAACGATTCAAGTGATCCTCTTGTTGTTGCCGCAAGTATCATTG
GGATCCTGCACTTGATATTGTGGATTCTTGATCGTCTTTTTTTCAAATGC
ATTTATCGTCTCTTTAAACACGGTCTGAAAAGAGGGCCTTCTACGGAAGG
AGTACCAGAGTCTATGAGGGAAGAATATCGAAAGGAACAGCAGAGTGCTG
TGGATGCTGACGATAGTCATTTTGTCAACATAGAGCTGGAG
[1393] The cell surface expression of M2 was confirmed using the
anti-M2e peptide specific MAb 14C2. Two other variants of M2, from
A/Hong Kong/483/1997 (HK483) and A/Vietnam/1203/2004 (VN1203), were
used for subsequent analyses, and their expression was determined
using M2e-specific monoclonal antibodies of the present invention,
since 14C2 binding may be abrogated by the various amino acid
substitutions in M2e.
Screening of Antibodies in Peripheral Blood
[1394] ver 120 individual plasma samples were tested for antibodies
that bound M2. None of them exhibited specific binding to the M2e
peptide. However, 10% of the plasma samples contained antibodies
that bound specifically to the 293-M2 H3N2 cell line. This
indicates that the antibodies could be categorized as binding to
conformational determinants of an M2 homotetramer, and binding to
conformational determinants of multiple variants of the M2
homotetramer; they could not be specific for the linear M2e
peptide.
Characterization of Anti-M2 MAbs
[1395] The human MAbs identified through this process proved to
bind to conformational epitopes on the M2 homotetramer. They bound
to the original 293-M2 transfectant, as well as to the two other
cell-expressed M2 variants. The 14C2 MAb, in addition to binding
the M2e peptide, proved to be more sensitive to the M2 variant
sequences. Moreover, 14C2 does not readily bind influenza virions,
while the conformation specific anti-M2 MAbs did.
[1396] These results demonstrate that the methods of the invention
provide for the identification of M2 MAbs from normal human immune
responses to influenza without a need for specific immunization of
M2. If used for immunotherapy, these fully human MAbs have the
potential to be better tolerated by patients that humanized mouse
antibodies. Additionally, and in contrast to 14C2 and the Gemini
Biosciences MAbs, which bind to linear M2e peptide, the MAbs of the
invention bind to conformational epitopes of M2, and are specific
not only for cells infected with A strain influenza, but also for
the virus itself. Another advantage of the MAbs of the invention is
that they each bind all of the M2 variants yet tested, indicating
that they are not restricted to a specific linear amino acid
sequence.
Example 2
Identification of M2-Specific Antibodies
[1397] Mononuclear or B cells expressing three of the MAbs
identified in human serum as described in Example 1 were diluted
into clonal populations and induced to produce antibodies. Antibody
containing supernatants were screened for binding to 293 FT cells
stably transfected with the full length M2E protein from influenza
strain Influenza subtype H3N2. Supernatants which showed positive
staining/binding were re-screened again on 293 FT cells stably
transfected with the full length M2E protein from influenza strain
Influenza subtype H3N2 and on vector alone transfected cells as a
control.
[1398] The variable regions of the antibodies were then rescue
cloned from the B cell wells whose supernatants showed positive
binding. Transient transfections were performed in 293 FT cells to
reconstitute and produce these antibodies. Reconstituted antibody
supernatants were screened for binding to 293 FT cells stably
transfected with the full length M2E protein as detailed above to
identify the rescued anti-M2E antibodies. Three different
antibodies were identified: 8i10, 21B15 and 23K12. A fourth
additional antibody clone was isolated by the rescue screens, 4C2.
However, it was not unique and had the exact same sequence as clone
8i10 even though it came from a different donor than clone
8i10.
[1399] The sequences of the kappa and gamma variable regions of
these antibodies are provided below.
Clone 8i10 (Corresponds to TCN-032):
[1400] The Kappa LC variable region of the anti M2 clone 8i10 was
cloned as Hind III to BsiW1 fragment (see below), and is encoded by
the following polynucleotide sequences, and SEQ ID NO: 54 (top) and
SEQ ID NO: 55 (bottom):
TABLE-US-00770 HindIII
AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTG
TTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCAC
CCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCA
GGTCTACACTGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGT
TCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCC
AGTGAACGGCCCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGGG
CTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGAT
GATTCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGTCACCTA
CTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC
GACCCTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAATGATGACAGTTG
BsiWI AGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAACGTACG
TCTCAATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTCTAGTTTGCATGC
[1401] The translation of the 8i10 Kappa LC variable region is as
follows, polynucleotide sequence (above, SEQ ID NO: 54, top) and
amino acid sequence (below, corresponding to residues 1-131 of SEQ
ID NO: 56):
TABLE-US-00771 HindIII
AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTG
M D M R V L A Q L L G L L L L W L R G
CCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCA
A R C D I Q M T Q S P S S L S A S V G D R V T
TCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCC
I T C R A S Q N I Y K Y L N W Y Q Q R P G K A
CTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGAT
P K G L I S A A S G L Q S G V P S R F S G S G
CTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC
S G T D F T L T I T S L Q P E D F A T Y Y C Q BsiWI
AGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAACGTACG Q S Y S
P P L T F G G G T R V E I K R T
[1402] The amino acid sequence of the 8i10 Kappa LC variable region
is as follows, with specific domains identified below (CDR
sequences defined according to Kabat methods):
TABLE-US-00772 M D M R V L A Q L L G L L L L W L R G A R C VK
leader (SEQ ID NO: 57) D I Q M T Q S P S S L S A S V G D R V T I T
C FR1 (SEQ ID NO: 58) R A S Q N I Y K Y L N CDR1(SEQ ID NO: 59) W Y
Q Q R P G K A P K G L I S FR2 (SEQ ID NO: 60) A A S G L Q S CDR2
(SEQ ID NO: 61) G V P S R F S G S G S G T D F T L T I T S L Q P E D
F A T Y Y C FR3 (SEQ ID NO: 62) Q Q S Y S P P L T CDR3 (SEQ ID NO:
63) F G G G T R V E I K FR4 (SEQ ID NO: 64) R T Start of Kappa
constant region
[1403] The following is an example of the Kappa LC variable region
of 8i10 cloned into the expression vector pcDNA3.1 which already
contained the Kappa LC constant region (upper polynucleotide
sequence corresponds to SEQ ID NO: 65, lower polynucleotide
sequence corresponds to SEQ ID NO: 66, amino acid sequence
corresponds to SEQ ID NO: 56). Bases in black represents pcDNA3.1
vector sequences, underlined bases represent the cloned antibody
sequences. The antibodies described herein have also been cloned
into the expression vector pCEP4.
TABLE-US-00773 NheI(894) PmeI (909) HindIII(910)
TCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTCCACCATG
AGCTTTAATTATGCTGAGTGATATCCCTCTGGGTTCGACCGATCGCAAATTTGAATTCGAAGGTGGTAC
M GACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGTG
CTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCACGGTCTACAC D
M R V L A Q L L G L L L L W L R G A R C
ACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAC
TGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGTAGTG D
I Q M T Q S P S S L S A S V G D R V T I T
TTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCC
AACGGCCCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGG C
R A S Q N I Y K Y L N W Y Q Q R P G K A
CCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCA
GGATTCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGT P
K G L I S A A S G L Q S G V P S R F S G
GTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTA
CACCTAGACCCTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAAT S
G S G T D F T L T I T S L Q P E D F A T Y
CTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAA
GATGACAGTTGTCTCAATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTCTAGTTT Y
C Q Q S Y S P P L T F G G G T R V E I K BsiWI
CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCT
GCATGCCACCGACGTGGTAGACAGAAGTAGAAGGGCGGTAGACTACTCGTCAACTTTAGACCTTGACGGA
R T V A A P S V F I F P P S D E Q L K S G T A
CTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
GACAACACACGGACGACTTATTGAAGATAGGGTCTCTCCGGTTTCATGTCACCTTCCACCTATTGCGGGA
S V V C L L N N F Y P R E A K V Q W K V D N A L hu Kappa constant
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC
GGTTAGCCCATTGAGGGTCCTCTCACAGTGTCTCGTCCTGTCGTTCCTGTCGTGGATGTCGGAGTCGTCG
Q S G N S Q E S V T E Q D S K D S T Y S L S S
ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC
TGGGACTGCGACTCGTTTCGTCTGATGCTCTTTGTGTTTCAGATGCGGACGCTTCAGTGGGTAGTCCCGG
T L T L S K A D Y E K H K V Y A C E V T H Q G DraI (1641) kbaI
(1636) ApaI (1642)
TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAGGGTCTAGAGGGCCCGTTTAAA
ACTCGAGCGGGCAGTGTTTCTCGAAGTTGTCCCCTCTCACAATCTCCCAGATCTCCCGGGCAAATTT
L S S P V T K S F N R G E C
[1404] The 8i10 Gamma HC variable region was cloned as a Hind III
to Xho 1 fragment, and is encoded the following polynucleotide
sequences, and SEQ ID NO: 67 (top) and SEQ ID NO: 68 (bottom).
TABLE-US-00774 HindIII
AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGT
TTCGAAGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCA
CCTGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTG
GGACAGGGTCCACGTTAACGTCCTCAGCCCGGGTCCTGACCACTTCGGAAGCCTCTGGGAC
TCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGC
AGGGAGTGGACGTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCG
AGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTA
TCAGGGGTCCCTTCCCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCAT
CAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCC
GTTAGGGAGGGAGTTCTCGGCGCAGTGGTATAGTGTTCTGTGAAGGTTCTCAGTCCAGAGG
CTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTT
GACTGCTACTCGAGACACTGGCGACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAA XhoI
GTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGA
CATCACCACCAATGACATAGGAACTGATGACCCCGGTCCCTTGGGACCAGTGGCAGAGCTC
[1405] The translation of the 8i10 Gamma HC is as follows,
polynucleotide sequence (above, SEQ ID NO: 67, top) and amino acid
sequence (below, corresponding to residues 1-138 of SEQ ID NO:
69):
TABLE-US-00775 HindIII
AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTC M K
H L W F F L L L V A A P S W V
CTGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTG L S Q
V Q L Q E S G P G L V K P S E T L
TCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGG S L T
C T V S G S S I S N Y Y W S W I R
CAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAG Q S P
G K G L E W I G F I Y Y G G N T K
TACAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTC Y N P
S L K S R V T I S Q D T S K S Q V
TCCCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCG S L T
M S S V T A A E S A V Y F C A R A XhoI
TCTTGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC S C S
G G Y C I L D Y W G Q G T L V T V TCGAG s
[1406] The amino acid sequence of the 8i10 Gamma HC is as follows
with specific domains identified below (CDR sequences defined
according to Kabat methods):
TABLE-US-00776 M K H L W F F L L L V A A P S W V L S VH leader (SEQ
ID NO: 70) Q V Q L Q E S G P G L V K P S E T L S L T C T V S G S S
I S FR1 (SEQ ID NO: 71) N Y Y W S CDR1(SEQ ID NO: 72) W I R Q S P G
K G L E W I G FR2 (SEQ ID NO: 73) F I Y Y G G N T K Y N P S L K S
CDR2 (SEQ ID NO: 74) R V T I S Q D T S K S Q V S L T M S S V T A A
E S A V Y F C A R FR3 (SEQ ID NO: 75) A S C S G G Y C I L D CDR3
(SEQ ID NO: 76) Y W G Q G T L V T V S FR4 (SEQ ID NO: 77)
YWGQGTLVTVSS Long FR4 (SEQ ID NO: 266)
[1407] The following is an example of the Gamma HC variable region
of 8i10 cloned into the expression vector pcDNA3.1 which already
contained the Gamma HC constant region (upper polynucleotide
sequence corresponds to SEQ ID NO: 78, lower polynucleotide
sequence corresponds to SEQ ID NO: 79, amino acid sequence
corresponds to SEQ ID NO: 69). Bases in black represents pcDNA3.1
vector sequences, underlined bases represent the cloned antibody
sequences.
TABLE-US-00777 PmeI (900) NheI (894) HindIII (910)
TGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCT
ACCGAATAGCTTTAATTATGCTGAGTGATATCCCTCTGGGTTCGACCGATCGCAAATTTGAATTCGA
TCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTCCTGTCCC
AGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCAGGACAGGG M
K H L W F F L L L V A A P S W V L S
AGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTG
TCCACGTTAACGTCCTCAGCCCGGGTCCTGACCACTTCGGAAGCCTCTGGGACAGGGAGTGGAC Q
V Q L Q E S G P G L V K P S E T L S L T C
CACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAG
GTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCGTCAGGGGTCCCTTC T
V S G S S I S N Y Y W S W I R Q S P G K
GGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGA
CCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCATGTTAGGGAGGGAGTTCT G
L E W I G F I Y Y G G N T K Y N P S L K
GCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTGAC
CGGCGCAGTGGTATAGTGTTCTGTGAAGGTTCTCAGTCCAGAGGGACTGCTACTCGAGACACTG S
R V T I S Q D T S K S Q V S L T M S S V T
CGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTTACTGTATCCTT
GCGACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAACATCACCACCAATGACATAGGAA A
A E S A V Y F C A R A S C S G G Y C I L XhoI (1331)
GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGAGCCTCCACCAAGGGCCCATCGGTCTTC
CTGATGACCCCGGTCCCTTGGGACCAGTGGCAGAGCTCTCGGAGGTGGTTCCCGGGTAGCCAGAAG
D Y W G Q G T L V T V S R A S T K G P S V F
CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT
GGGGACCGTGGGAGGAGGTTCTCGTGGAGACCCCCGTGTCGCCGGGACCCGACGGACCAGTTCCTGA
P L A P S S K S T S G G T A A L G C L V K D
ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
TGAAGGGGCTTGGCCACTGCCACAGCACCTTGAGTCCGCGGGACTGGTCGCCGCACGTGTGGAAGGG
Y F P E P V T V S W N S G A L T S G V H T F P
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG
CCGACAGGATGTCAGGAGTCCTGAGATGAGGGAGTCGTCGCACCACTGGCACGGGAGGTCGTCGAAC
A V L Q S S G L Y S L S S V V T V P S S S L
GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTG
CCGTGGGTCTGGATGTAGACGTTGCACTTAGTGTTCGGGTCGTTGTGGTTCCACCTGTTCTCTCAAC
G T Q T Y I C N V N H K P S N T K V D K R V
AGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC
TCGGGTTTAGAACACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGTGGACTTGAGGACCCCCCTGG
E P K S C D K T H T C P P C P A P E L L G G P
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA
CAGTCAGAAGGAGAAGGGGGGTTTTGGGTTCCTGTGGGAGTACTAGAGGGCCTGGGGACTCCAGTGT
S V F L F P P K P K D T L M I S R T P E V T
TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG
ACGCACCACCACCTGCACTCGGTGCTTCTGGGACTCCAGTTCAAGTTGACCATGCACCTGCCGCACC
C V V V D V S H E D P E V K F N W Y V D G V
AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT
TCCACGTATTACGGTTCTGTTTCGGCGCCCTCCTCGTCATGTTGTCGTGCATGGCACACCAGTCGCA
E V H N A K T K P R E E Q Y N S T Y R V V S V
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC
GGAGTGGCAGGACGTGGTCCTGACCGACTTACCGTTCCTCATGTTCACGTTCCAGAGGTTGTTTCGG
L T V L H Q D W L N G K E Y K C K V S N K A
CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA
GAGGGTCGGGGGTAGCTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTGGTGTCCACATGT
L P A P I E K T I S K A K G Q P R E P Q V Y
CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
GGGACGGGGGTAGGGCCCTCCTCTACTGGTTCTTGGTCCAGTCGGACTGGACGGACCAGTTTCCGAA
T L P P S R E E M T K N Q V S L T C L V K G F
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
GATAGGGTCGCTGTAGCGGCACCTCACCCTCTCGTTACCCGTCGGCCTCTTGTTGATGTTCTGGTGC
Y P S D I A V E W E S N G Q P E N N Y K T T
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGT
GGAGGGCACGACCTGAGGCTGCCGAGGAAGAAGGAGATATCGTTCGAGTGGCACCTGTTCTCGTCCA
P P V L D S D G S F F L Y S K L T V D K S R
GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA
CCGTCGTCCCCTTGCAGAAGAGTACGAGGCACTACGTACTCCGAGACGTGTTGGTGATGTGCGTCTT
W Q Q G N V F S C S V M H E A L H N H Y T Q K ApaI (2339) DraII
(2338) XhoI (2333) PmeI (2345)
GAGCCTCTCCCTGTCTCCGGGTAAATGAGTTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGA
CTCGGAGAGGGACAGAGGCCCATTTACTCAAGATCTCCCGGGCAAATTTGGGCGACTAGTCGGAGCT
S L S L S P G K CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGC
GACACGGAAGATCAACGGTCGGTAGACAACAAACG
[1408] The framework 4 (FR4) region of the Gamma HC normally ends
with two serines (SS), so that the full framework 4 region should
be WGQGTLVTVSS (SEQ ID NO: 80). The accepting Xho 1 site and one
additional base downstream of the Xho 1 site in the vector, in
which the Gamma HC constant region that the Gamma HC variable
regions are cloned, supplies the last bases, which encode this
final amino acid of framework 4. However, the original vector did
not adjust for the silent mutation made when the Xho1 site (CTCGAG,
SEQ ID NO: 81) was created and contained an "A" nucleotide
downstream of the Xho 11 site, which caused an amino acid change at
the end of framework 4: a serine to arginine (S to R) substitution
present in all the working Gamma HC clones. Thus, the full
framework 4 region reads WGQGTLVTVSR (SEQ ID NO: 82). Future
constructs are being created wherein the base downstream of the Xho
1 site is a "C" nucleotide. Thus, the creation of the Xho 1 site
used for cloning of the Gamma HC variable region sequences in
alternative embodiments is a silent mutation and restores the
framework 4 amino acid sequence to its proper WGQGTLV TVSS (SEQ ID
NO: 80). This is true for all M2 Gamma HC clones described
herein.
Clone 21B15:
[1409] The Kappa LC variable region of the anti M2 clone 21B15 was
cloned as Hind III to BsiW1 fragment, and is encoded by the
following polynucleotide sequences and SEQ ID NO: 83 and SEQ ID NO:
84:
TABLE-US-00778 HindIII
AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGC
TTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCACG
CAGATGTGACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC
GTCTACACTGTAGGTCCACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGTAG
ACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCCTA
TGAACGGCGCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGGGGAT
AGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGG
TCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGTCACCTAGACC
GACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGT
CTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAATGATGACAGTTGTCTCA
BsiWI TACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGATATCAAACGTACG
ATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTATAGTTTGCATGC
[1410] The translation of the 21B15 Kappa LC variable region is as
follows, polynucleotide sequence (above, SEQ ID NO: 83, top) and
amino acid sequence (below, corresponding to SEQ ID NO: 298):
TABLE-US-00779 HindIII
AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGT
M D M R V L A Q L L G L L L L W L R G
GCCAGATGTGACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC
A R C D I Q V T Q S P S S L S A S V G D R V T
ATCACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCC
I T C R A S Q N I Y K Y L N W Y Q Q R P G K A
CCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGA
P K G L I S A A S G L Q S G V P S R F S G S G
TCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAA
S G T D F T L T I T S L Q P E D F A T Y Y C Q BsiWI
CAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGATATCAAACGTACG Q S Y
S P P L T F G G G T R V D I K R T
[1411] The amino acid sequence of the 21B15 Kappa LC variable
region is as follows, with specific domains identified below (CDR
sequences defined according to Kabat methods):
TABLE-US-00780 (SEQ ID NO: 57) M D M R V L A Q L L G L L L L W L R
G A R C VK leader (SEQ ID NO: 58) D I Q V T Q S P S S L S A S V G D
R V T I T C FR1 (SEQ ID NO: 59) R A S Q N I Y K Y L N CDR1 (SEQ ID
NO: 60) W Y Q Q R P G K A P K G L I S FR2 (SEQ ID NO: 61) A A S G L
Q S CDR2 (SEQ ID NO: 62) G V P S R F S G S G S G T D F T L T I T S
L Q P E D F A T Y Y C FR3 (SEQ ID NO: 63) Q Q S Y S P P L T CDR3
(SEQ ID NO: 64) F G G G T R V D I K FR4 R T Start of Kappa constant
region
[1412] The primer used to clone the Kappa LC variable region
extended across a region of diversity and had wobble base position
in its design. Thus, in the framework 4 region a D or E amino acid
could occur. In some cases, the amino acid in this position in the
rescued antibody may not be the original parental amino acid that
was produced in the B cell. In most kappa LCs the position is an E.
Looking at the clone above (21B15) a D in framework 4 (DIKRT) (SEQ
ID NO: 544) was observed. However, looking at the surrounding amino
acids, this may have occurred as the result of the primer and may
be an artifact. The native antibody from the B cell may have had an
E in this position.
[1413] The 21B15 Gamma HC variable region was cloned as a Hind III
to Xho 1 fragment, and is encoded by the following polynucleotide
sequences and SEQ ID NO: 85 (top), and SEQ ID NO: 86 (bottom):
TABLE-US-00781 HindIII
AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTCC
TTCGAAGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCAGG
TGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCC
ACAGGGTCCACGTTAACGTCCTCAGCCCGGGTCCTGACCACTTCGGAAGCCTCTGGGACAGGG
TCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCC
AGTGGACGTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCGTCAGGG
CAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCT
GTCCCTTCCCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCATGTTAGGGA
CCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGA
GGGAGTTCTCGGCGCAGTGGTATAGTGTTCTGTGAAGGTTCTCAGTCCAGAGGGACTGCTACT
GCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTT
CGAGACACTGGCGACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAACATCACCACCAA
XhoI ACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAG
TGACATAGGAACTGATGACCCCGGTCCCTTGGGACCAGTGGCAGAGCTC
[1414] The translation of the 21B15 Gamma HC is as follows,
polynucleotide sequence (above, SEQ ID NO: 87, top) and amino acid
sequence (below, corresponding to residues 1-138 of SEQ ID NO:
69):
TABLE-US-00782 HindIII
AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTC M K
H L W F F L L L V A A P S W V
CTGTCCCAGGTGCAATTGCAGGAGTOGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCC L S
Q V Q L Q E S G P G L V K P S E T L S
CTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCC L T
C T V S G S S I S N Y Y W S W I R Q S
CCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCC P G
K G L E W I G F I Y Y G G N T K Y N P
TCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATG S L
K S R V T I S Q D T S K S Q V S L T M
AGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGT S S
V T A A E S A V Y F C A R A S C S G G XhoI
TACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAG Y C I L D Y W G
Q G T L V T V S
[1415] The amino acid sequence of the 21B15 Gamma HC is as follows,
with specific domains identified below (CDR sequences defined
according to Kabat methods):
TABLE-US-00783 (SEQ ID NO: 70) M K H L W F F L L L V A A P S W V L
S VH leader (SEQ ID NO: 71) Q V Q L Q E S G P G L V K P S E T L S L
T C T V S G S S I S FR1 (SEQ ID NO: 72) N Y Y W S CDR1 (SEQ ID NO:
73) W I R Q S P G K G L E W I G FR2 (SEQ ID NO: 74) F I Y Y G G N T
K Y N P S L K S CDR2 (SEQ ID NO: 75) R V T I S Q D T S K S Q V S L
T M S S V T A A E S A V Y F C A R FR3 (SEQ ID NO: 76) A S C S G G Y
C I L D CDR3 (SEQ ID NO: 77) Y W G Q G T L V T V S FR4 (SEQ ID NO:
266) YWGQGTLVTVSS Long FR4
Clone 23K12 (Corresponds to TCN-031):
[1416] The Kappa LC variable region of the anti M2 clone 23K12 was
cloned as Hind III to BsiW1 fragment (see below), and is encoded by
the following polynucleotide sequences SEQ ID NO: 88 (top) and SEQ
ID NO: 89 (below).
TABLE-US-00784 HindIII
AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGG
TTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCC
TGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTC
ACGGTCTACACTGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAG
ACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGA
TGGTAGTGAACGGCCTGTTCAGTCTCGTAATCGTCGATAAATTTAACCATAGTCGTCTTTGGTCCCT
AAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGG
TTCGGGGATTTGAGGACTAGATACGACGTAGGTCAAACGTTTCACCCCAGGGTAGTTCCAAGTCACC
CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACCTAC
GTCACCTAGACCCTGTCTAAAGTGAGAGTGGTAGTCGCCAGACGTTGGACTTCTAAAACGTTGGATG
BsiWI
TACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACG
ATGACAGTTGTCTCAATGTCATACGGACGGAAACCGGTCCCCTGGTTCGACCTCTAGTTTGCATGC
[1417] The translation of the 23K12 Kappa LC variable region is as
follows, polynucleotide sequence (above, SEQ ID NO: 90, top) and
amino acid sequence (below, corresponding to SEQ ID NO: 91).
TABLE-US-00785 HindIII
AAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGG
M D M R V L A Q L L G L L L L W L R G
TGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTC
A R C D I Q M T Q S P S S L S A S V G D R V
ACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGA
T I T C R T S Q S I S S Y L N W Y Q Q K P G
AAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGG
K A P K L L I Y A A S S L Q S G V P S R F S G
CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACCTAC
S G S G T D F T L T I S G L Q P E D F A T Y BsiWI
TACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACG
Y C Q Q S Y S M P A F G Q G T K L E I K R T
[1418] The amino acid sequence of the 23K12 Kappa LC variable
region is as follows, with specific domains identified below (CDR
sequences defined according to Kabat methods):
TABLE-US-00786 (SEQ ID NO: 57) M D M R V L A Q L L G L L L L W L R
G A R C VK leader (SEQ ID NO: 58) D I Q M T Q S P S S L S A S V G D
R V T I T C FR1 (SEQ ID NO: 92) R T S Q S I S S Y L N CDR1 (SEQ ID
NO: 93) W Y Q Q K P G K A P K L L I Y FR2 (SEQ ID NO: 94) A A S S L
Q S G V P S R F CDR2 (SEQ ID NO: 95) S G S G S G T D F T L T I S G
L Q P E D F A T Y Y C FR3 (SEQ ID NO: 96) Q Q S Y S M P A CDR3 (SEQ
ID NO: 114) F G Q G T K L E I K FR4 R T Start of Kappa LC constant
region
[1419] The 23K12 Gamma HC variable region was cloned as a Hind III
to Xho 1 fragment, and is encoded by the following polynucleotide
sequences and SEQ ID NO: 97 (top) and SEQ ID NO: 98 (bottom).
TABLE-US-00787 HindIII
AAGCTTCCACCATGGAGTTGGGGCTGTGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGT
TTCGAAGGTGGTACCTCAACCCCGACACGACCCAAAAGGAACAACGATAAAATTTTCCACAGGTCA
GTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTCCT
CACTCCACGTCGACCACCTCAGACCCCCTCCGAACCAGGTCGGACCCCCCAGGGACTCTTAGAGGA
GTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGG
CACGTCGGAGACCTAAGTGGCAGTCATCGTTGATGTACTCAACCCAGGCGGTCCGAGGTCCCTTCC
GGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCA
CCGACCTCACCCAGAGTCAATAAATATCACCACCATCGTGTATGATGCGTCTGAGGCACTTCCCGT
GATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAGAGCCG
CTAAGAGGAAGAGGTCTCTGTTGAGGTTCTTGTGTCACAAAGAAGTTTACTTGTCGGACTCTCGGC
AGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGGGTTACGGTTTAGACGTCT
TCCTGTGCCGACACATAATGACACGCTCTACAGACTCGTCCTACGCCCCAATGCCAAATCTGCAGA
XhoI GGGGCCAAGGGACCACGGTCACCGTCTCGAG
CCCCGGTTCCCTGGTGCCAGTGGCAGAGCTC
[1420] The translation of the 23K12 Gamma HC variable region is as
follows, polynucleotide sequence (above, SEQ ID NO: 99, top), and
amino acid sequence (below, corresponding to SEQ ID NO: 100):
TABLE-US-00788 HindIII
AAGCTTCCACCATGGAGTTGGGGCTGTGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAG M
E L G L C W V F L V A I L K G V Q
TGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTCC
C E V Q L V E S G G G L V Q P G G S L R I S
TGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAG
C A A S G F T V S S N Y M S W V R Q A P G K
GGGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC
G L E W V S V I Y S G G S T Y Y A D S V K G
AGATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAGAGCC
R F S F S R D N S K N T V F L Q M N S L R A
GAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGGGTTACGGTTTAGACGTC
E D T A V Y Y C A R C L S R M R G Y G L D V XhoI
TGGGGCCAAGGGACCACGGTCACCGTCTCGAG W G Q G T T V T V S
[1421] The amino acid sequence of the 23K12 Gamma HC variable
region is as follows, with specific domains identified below (CDR
sequences defined according to Kabat methods):
TABLE-US-00789 (SEQ ID NO: 101) M E L G L C W V F L V A I L K G V Q
C VH leader (SEQ ID NO: 102) E V Q L V E S G G G L V Q P G G S L R
I S C A A S G F T V S FR1 (SEQ ID NO: 103) S N Y M S CDR1 (SEQ ID
NO: 104) W V R Q A P G K G L E W V S FR2 (SEQ ID NO: 105) V I Y S G
G S T Y Y A D S V K CDR2 (SEQ ID NO: 106) G R F S F S R D N S K N T
V F L Q M N S L R A E D T A V Y Y C A R FR3 (SEQ ID NO: 107) C L S
R M R G Y G L D V CDR3 (SEQ ID NO: 108) W G Q G T T V T V S FR4
(SEQ ID NO: 111) WGQGTTVTVSS Long FR4
Example 3
Identification of Conserved Antibody Variable Regions
[1422] The amino acid sequences of the three antibody Kappa LC and
Gamma HC variable regions were aligned to identify conserved
regions and residues, as shown below.
[1423] Amino acid sequence alignment of the Kappa LC variable
regions of the three clones (SEQ ID NOs 673-675, respectively, in
order of appearance):
TABLE-US-00790 10 20 Translation of mp 73 21B15 A S T M D M R V L A
Q L L G L L L L W L R G A Translation of mp 147 8I10 A S T M D M R
V L A Q L L G L L L L W L R G A Translation of mp 137 23K12 A S T M
D M R V L A Q L L G L L L L W L R G A 30 40 Translation of mp 73
21B15 R C D I Q V T Q S P S S L S A S V G D R V T I Translation of
mp 147 8I10 R C D I Q M T Q S P S S L S A S V G D R V T I
Translation of mp 137 23K12 R C D I Q M T Q S P S S L S A S V G D R
V T I 50 60 Translation of mp 73 21B15 T C R A S Q N I Y K Y L N W
Y Q Q R P G K A P Translation of mp 47 8I10 T C R A S Q N I Y K Y L
N W Y Q Q R P G K A P Translation of mp 137 23K12 T C R T S Q S I S
S Y L N W Y Q Q R P G K A P 70 80 90 Translation of mp 73 21B15 K G
L I S A A S G L Q S G V P S R F S G S G S Translation of mp 147
8I10 K G L I S A A S G L Q S G V P S R F S G S G S Translation of
mp 137 23K12 K L L I Y A A S S L Q S G V P S R F S G S G S 100 110
Translation of mp 73 21B15 G T D F T L T I T S L Q P E D F A T Y Y
C Q Q Translation of mp 147 8I10 G T D F T L T I T S L Q P E D F A
T Y Y C Q Q Translation of mp 137 23K12 G T D F T L T I S G L Q P E
D F A T Y Y C Q Q 120 130 Translation of mp 73 21B15 S Y S P P L T
F G G G T R V D I K R T Translation of mp 147 8I10 S Y S P P L T F
G G G T R V E I K R T Translation of mp 137 23K12 S Y S M P -- A F
G Q G T K L E I K R T
[1424] Amino acid sequence alignment of the Gamma HC variable
regions of the three clones (SEQ ID Nos 676-678, respectively, in
order of appearance):
TABLE-US-00791 10 20 Translation of mp 81 21B15 A S T M K H L W F F
L L L V A A P S W V L S Translation of mp 145 23K12 A S T M E L G L
C W V F L V A I L K G V Q C Translation of mp 153 8I10 A S T M K H
L W F F L L L V A A P S W V L S 30 40 Translation of mp 81 21B15 Q
V Q L Q E S G P G L V K P S E T L S L T C Translation of mp 145
23K12 E V Q L V E S G G G L V Q P G G S L R I S C Translation of mp
153 8I10 Q V Q L Q E S G P G L V K P S E T L S L T C 50 60
Translation of mp 81 21B15 T V S G S S I S N Y Y W S W I R Q S P G
K G Translation of mp 145 23K12 A A S G F T V S S N Y M S W V R Q A
P G K G Translation of mp 153 8I10 T V S G S S I S N Y Y W S W I R
Q S P G K G 70 80 Translation of mp 81 21B15 L E W I G F I Y Y G G
N T K Y N P S L K S R Translation of mp 145 23K12 L E W V S V I Y S
G G S T Y Y A D S V K G R Translation of mp 153 8I10 L E W I G F I
Y Y G G N T K Y N P S L K S R 90 100 110 Translation of mp 81 21B15
V T I S Q D T S K S Q V S L T M S S V T A A Translation of mp 145
23K12 F S F S R D N S K N T V F L Q M N S L R A E Translation of mp
153 8I10 V T I S Q D T S K S Q V S L T M S S V T A A 120 130
Translation of mp 81 21B15 E S A V Y F C A R A S C S G G Y C I L D
Y W Translation of mp 145 23K12 D T A V Y Y C A R C L S R M R G Y G
L D V W Translation of mp 153 8I10 E S A V Y F C A R A S C S G G Y
C I L D Y W 140 Translation of mp 81 21B15 G Q T L V T V S
Translation of mp 145 23K12 G Q T T V T V S Translation of mp 153
8I10 G Q T L V T V S
[1425] Clones 8I10 and 21B15 came from two different donors, yet
they have the same exact Gamma HC and differ in the Kappa LC by
only one amino acid at position 4 in the framework 1 region (amino
acids M versus V, see above), (excluding the D versus E wobble
position in framework 4 of the Kappa LC).
[1426] Sequence comparisons of the variable regions of the
antibodies revealed that the heavy chain of clone 8i10 was derived
from germline sequence IgHV4 and that the light chain was derived
from the germline sequence IgKV1.
[1427] Sequence comparisons of the variable regions of the
antibodies revealed that the heavy chain of clone 21B15 was derived
from germline sequence IgHV4 and that the light chain was derived
from the germline sequence IgKV1.
[1428] Sequence comparisons of the variable regions of the
antibodies revealed that the heavy chain of clone 23K12 was derived
from germline sequence IgHV3 and that the light chain was derived
from the germline sequence IgKV1.
Example 4
Production and Characterization of M2 Antibodies
[1429] The antibodies described above were produced in milligram
quantities by larger scale transient transfections in 293 PEAK
cells. Crude un-purified antibody supernatants were used to examine
antibody binding to influenza A/Puerto Rico/8/1932 (PR8) virus on
ELISA plates, and were compared to the binding of the control
antibody 14C2, which was also produced by larger scale transient
transfection. The anti-M2 recombinant human monoclonal antibodies
bound to influenza while the control antibody did not (FIG. 9).
[1430] Binding was also tested on MDCK cells infected with the PR8
virus (FIG. 10). The control antibody 14C2 and the three anti M2E
clones: 8I10, 21B15 and 23K12, all showed specific binding to the
M2 protein expressed on the surface of PR8-infected cells. No
binding was observed on uninfected cells.
[1431] The antibodies were purified over protein A columns from the
supernatants. FACs analysis was performed using purified antibodies
at a concentration of 1 ug per ml to examine the binding of the
antibodies to transiently transfected 293 PEAK cells expressing the
M2 proteins on the cell surface. Binding was measured testing
binding to mock transfected cells and cells transiently transfected
with the Influenza subtype H3N2, A/Vietnam/1203/2004 (VN1203), or
A/Hong Kong/483/1997 HK483 M2 proteins. As a positive control the
antibody 14C2 was used. Unstained and secondary antibody alone
controls helped determined background. Specific staining for cells
transfected with the M2 protein was observed for all three clones.
Furthermore, all three clones bound to the high path strains
A/Vietnam/1203/2004 and A/Hong Kong/483/1997 M2 proteins very well,
whereas the positive control 14C2 which bound well to H3N2 M2
protein, bound much weaker to the A/Vietnam/1203/2004 M2 protein
and did not bind the A/Hong Kong/483/1997 M2 protein. See FIG.
11.
[1432] Antibodies 21B15, 23K12, and 8I10 bound to the surface of
293-HEK cells stably expressing the M2 protein, but not to vector
transfected cells (see FIG. 1). In addition, binding of these
antibodies was not competed by the presence of 5 mg/ml 24-mer M2
peptide, whereas the binding of the control chimeric mouse
V-region/human IgG1 kappa 14C2 antibody (hu14C2) generated against
the linear M2 peptide was completely inhibited by the M2 peptide
(see FIG. 1). These data confirm that these antibodies bind to
conformational epitopes present in M2e expressed on the cell or
virus surface, as opposed to the linear M2e peptide.
Example 5
Viral Binding of Human Anti-Influenza Monoclonal Antibodies
[1433] UV-inactivated influenza A virus (A/PR/8/34) (Applied
Biotechnologies) was plated in 384-well MaxiSorp plates (Nunc) at
1.2 .mu.g/ml in PBS, with 25 .mu.l/well, and was incubated at
4.degree. C. overnight. The plates were then washed three times
with PBS, and blocked with 1% Nonfat dry milk in PBS, 50
.mu.l/well, and then were incubated at room temp for 1 hr. After a
second wash with PBS, MAbs were added at the indicated
concentrations in triplicate, and the plates were incubated at room
temp for 1 hour. After another wash with PBS, to each well was
added 25 .mu.l of a 1/5000 dilution of horseradish peroxidase (HRP)
conjugated goat anti-human IgG Fc (Pierce) in PBS/1% Milk, and the
plates were left at room temp for 1 hr. After the final PBS wash,
the HRP substrate 1-Step.TM. Ultra-TMB-ELISA (Pierce) was added at
25 .mu.l/well, and the reaction proceeded in the dark at room temp.
The assay was stopped with 25 .mu.l/well 1N H.sub.2SO.sub.4, and
light absorbance at 450 nm (A450) was read on a SpectroMax Plus
plate reader. Data are normalized to the absorbance of MAb 8I10
binding at 10 .mu.g/ml. Results are shown in FIGS. 2A and 2B.
Example 6
Binding of Human Anti-Influenza Monoclonal Antibodies to
Full-Length M2 Variants
[1434] M2 variants (including those with a high pathology phenotype
in vivo) were selected for analysis. See FIG. 3A for sequences.
[1435] M2 cDNA constructs were transiently transfected in HEK293
cells and analyzed as follows: To analyze the transient
transfectants by FACS, cells on 10 cm tissue culture plates were
treated with 0.5 ml Cell Dissociation Buffer (Invitrogen), and
harvested. Cells were washed in PBS containing 1% FBS, 0.2%
NaN.sub.3 (FACS buffer), and resuspended in 0.6 ml FACS buffer
supplemented with 100 .mu.g/ml rabbit IgG. Each transfectant was
mixed with the indicated MAbs at 1 .mu.g/ml in 0.2 ml FACS buffer,
with 5.times.10.sup.5 to 10.sup.6 cells per sample. Cells were
washed three times with FACS buffer, and each sample was
resuspended in 0.1 ml containing 1 .mu.g/ml alexafluor (AF)
647-anti human IgG H&L (Invitrogen). Cells were again washed
and flow cytometry was performed on a FACSCanto device
(Becton-Dickenson). The data is expressed as a percentage of the
mean fluorescence of the M2-D20 transient transfectant. Data for
variant binding are representative of 2 experiments. Data for
alanine mutants are average readouts from 3 separate experiments
with standard error. Results are shown in FIGS. 3B and 3C.
Example 7
Alanine Scanning Mutagenesis to Evaluate M2 Binding
[1436] To evaluate the antibody binding sites, alanine was
substituted at individual amino acid positions as indicated by
site-directed mutagenesis.
[1437] M2 cDNA constructs were transiently transfected in HEK293
cells and analyzed as described above in Example 6. Results are
shown in FIGS. 4A and 4B. FIG. 8 shows that the epitope is in a
highly conserved region of the amino terminus of the M2
polypeptide. As shown in FIGS. 4A, 4B and FIG. 8, the epitope
includes the serine at position 2, the threonine at position 5 and
the glutamic acid at position 6 of the M2 polypeptide.
Example 8
Epitope Blocking
[1438] To determine whether the MAbs 8I10 and 23K12 bind to the
same site, M2 protein representing influenza strain A/HK/483/1997
sequence was stably expressed in the CHO (Chinese Hamster Ovary)
cell line DG44. Cells were treated with Cell Dissociation Buffer
(Invitrogen), and harvested. Cells were washed in PBS containing 1%
FBS, 0.2% NaN.sub.3 (FACS buffer), and resuspended at 10.sup.7
cells/ml in FACS buffer supplemented with 100 .mu.g/ml rabbit IgG.
The cells were pre-bound by either MAb (or the 2N9 control) at 10
.mu.g/ml for 1 hr at 4.degree. C., and were then washed with FACS
buffer. Directly conjugated AF647-8I10 or -23K12 (labeled with the
AlexaFluor.RTM. 647 Protein Labeling kit (Invitrogen) was then used
to stain the three pre-blocked cell samples at 1 .mu.g/ml for
10.sup.6 cells per sample. Flow cytometric analyses proceeded as
before with the FACSCanto. Data are average readouts from 3
separate experiments with standard error. Results are shown in FIG.
5.
Example 9
Binding of Human Anti-Influenza Monoclonal Antibodies to M2
Variants and Truncated M2 Peptides
[1439] The cross reactivity of mAbs 8i10 and 23K12 to other M2
peptide variants was assessed by ELISA. Peptide sequences are shown
in FIGS. 6A and 6B. Additionally, a similar ELISA assay was used to
determine binding activity to M2 truncated peptides.
[1440] In brief, each peptide was coated at 2 .mu.g/mL to a flat
bottom 384 well plate (Nunc) in 25 .mu.L/well of PBS buffer
overnight at 4.degree. C. Plates were washed three times and
blocked with 1% Milk/PBS for one hour at room temperature. After
washing three times, MAb titers were added and incubated for one
hour at room temperature. Diluted HRP conjugated goat anti-human
immunoglobulin FC specific (Pierce) was added to each well after
washing three times. Plates were incubated for one hour at room
temperature and washed three times. 1-Step.TM. Ultra-TMB-ELISA
(Pierce) was added at 25 .mu.l/well, and the reaction proceeded in
the dark at room temp. The assay was stopped with 25 .mu.l/well 1N
H.sub.2SO.sub.4, and light absorbance at 450 nm (A450) was read on
a SpectroMax Plus plate reader. Results are shown in FIGS. 6A and
6B.
Example 10
In Vivo Evaluation of the Ability of Human Anti-Influenza
Monoclonal Antibodies to Protect from Lethal Viral Challenge
[1441] The ability of antibodies, 23K12 and 8I10, to protect mice
from lethal viral challenge with a high path avian influenza strain
was tested.
[1442] Female BALB/c mice were randomized into 5 groups of 10. One
day prior (Day -1 (minus one)) and two days post infection (Day +2
(plus two), 200 ug of antibody was given via 200 ul
intra-peritoneal injection. On Day 0 (zero), an approximate LD90
(lethal dose 90) of A/Vietnam/1203/04 influenza virus, in a volume
of 30 .mu.l was given intra-nasally. Survival rate was observed
from Day 1 through Day 28 post-infection. Results are shown in FIG.
7.
Example 11
Characterization of M2 Antibodies TCN-032 (8I10), 21B15, TCN-031
(23K12), 3241_G23, 3244_I10, 3243_J07, 3259_J21, 3245_O19,
3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420_I23, 3139_P23,
3248_P18, 3253_P10, 3260_D19, 3362_B11, and 3242_P05
FACS
[1443] Full length M2 cDNA (A/Hong Kong/483/97) were synthesized
(Blue Heron Technology) and cloned into the plasmid vector pcDNA3.1
which was then transfected into CHO cells with Lipofectamine
(Invitrogen) to create a stable pool of CHO-HK M2-expressing cells.
For the panel of anti-M2 Mabs, 20 .mu.l samples of supernatant from
transient transfections from each of the IgG heavy and light chain
combinations was used to stain the CHO-HK M2 stable cell line.
Bound anti-M2 mabs were visualized on viable cells with Alexafluor
647-conjugated goat anti-Human IgG H&L antibody (Invitrogen).
Flow cytometry was performed with a FACSCanto, and analysis on the
accompanying FACSDiva software (Becton Dickenson).
ELISA
[1444] Purified Influenza A (A/Puerto Rico/8/34) inactivated by
.beta.-propiolactone (Advanced Biotechnologies, Inc.) was
biotinylated (EZ-Link Sulfo-NHS-LC-Biotin, Pierce) and adsorbed for
16 hours at 4.degree. C. to 384-well plates in 25 .mu.l PBS that
were pre-coated with neutravidin (Pierce). Plates were blocked with
BSA in PBS, samples of supernatant from transient transfections
from each of the IgG heavy and light chain combinations were added
at a final dilution of 1:5, followed by HRP-conjugated goat
anti-human Fc antibody (Pierce), and developed with TMB substrate
(ThermoFisher).
[1445] The results of this analysis are shown below in Table 2.
TABLE-US-00792 TABLE 2 FACS Virus Sequence ID M2-HK ELISA
Transfection no. BCC well ID Gamma Light MFI OD A450 322 3241_G23
G4_005 K1_004 1697 3.02 352 3244_I10 G4_007 K2_006 434 3.01 339
3243_J07 G4_007 K1_007 131 2.94 336 3259_J21 G4_005 K2_005 1673
2.40 348 3245_O19 G3_004 K1_001 919 3.51 345 3244_H04 G3_003 K1_006
963 3.31 346 Pos Cont (HC) Pos Cont (LC) 754 2.69 347 Neg Cont (HC)
Neg Cont (LC) 11 0.15 374 3136_G05 G4_007 K1_007 109 ND 386
3252_C13 G4_013 K1_002 449 ND 390 3255_J06 G4_013 K2_007 442 ND 400
3420_I23 G4_004 K1_003 112 ND 432 3139_P23 G4_016 K1_007a 110 1.02
412 3248_P18 G4_009 K1_006 967 0.56 413 3253_P10 G4_007 K1_004 43
0.50 434 3260_D19 G3_004a K2_001 846 2.46 439 3362_B11 G4_010a
K1_007 218 1.83 408 3242_P05 G3_005 K2_004 596 0.50 451 Pos Cont
(HC) Pos Cont (LC) 1083 1.87 452 Neg Cont (HC) Neg Cont (LC) 6 0.05
Positive control: supernatant from tranisent transfection with the
IgG heavy and light chain combination of mAb 8I10 Negative control:
supernatant from tranisent transfection with the IgG heavy and
light chain combination of mAb 2N9 MFI = mean fluorescence
intensity
Example 12
Human Antibodies Reveal a Protective Epitope that is Highly
Conserved Among Human and Non-Human Influenza A Viruses
[1446] Influenza remains a serious public health threat throughout
the world. Vaccines and antivirals are available that can provide
protection from infection. However, new viral strains emerge
continuously because of the plasticity of the influenza genome
which necessitates annual reformulation of vaccine antigens, and
resistance to antivirals can appear rapidly and become entrenched
in circulating virus populations. In addition, the spread of new
pandemic strains is difficult to contain due to the time required
to engineer and manufacture effective vaccines. Monoclonal
antibodies that target highly conserved viral epitopes might offer
an alternative protection paradigm. Herein we describe the
isolation of a panel of monoclonal antibodies derived from the
IgG.sup.+ memory B cells of healthy, human subjects that recognize
a previously unknown conformational epitope within the ectodomain
of the influenza matrix 2 protein, M2e. This antibody binding
region is highly conserved in influenza A viruses, being present in
nearly all strains detected to date including highly pathogenic
viruses that infect primarily birds and swine, and the current 2009
swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human
anti-M2e monoclonal antibodies protect mice from lethal challenges
with either H5N1 or H1N1 influenza viruses. These results suggest
that viral M2e can elicit broadly cross-reactive and protective
antibodies in humans. Accordingly, recombinant forms of these human
antibodies may provide useful therapeutic agents to protect against
infection from a broad spectrum of influenza A strains.
Introduction
[1447] Seasonal influenza epidemics hospitalize more than 200,000
people each year in the US and kill an estimated 500,000 worldwide
(Thompson, W. W. et al. (2004) JAMA 292:1333-1340). The immune
system affords only partial protection from seasonal strains in
most individuals because of constantly arising point mutations in
the viral genome which lead to structural variability known as
antigenic drift. Pandemic strains encounter even less immune
resistance due to genomic reassortment events among different
viruses which result in more radical shifts in viral antigenic
determinants. Consequently, pandemic influenza has the potential to
cause widespread illness, death, and economic disruption. Vaccines
and antiviral agents are available to counter the threat of
influenza epidemics and pandemics. However, the strain composition
of influenza vaccines must be determined prior to the influenza
season on an annual basis, and predicting in advance which strains
will become dominant is challenging. Moreover, the emergence of
strains that evade vaccine-induced, protective immune responses is
relatively rapid which often results in inadequate protection
(Carrat F, Flahault A. (2007) Influenza vaccine: the challenge of
antigenic drift. Vaccine 25:6852-6862). Antiviral drugs include
oseltamivir and zanamivir which inhibit the function of the viral
protein neuraminidase (NA), and adamantanes which inhibit the ion
channel function of the viral M2 protein (Gubareva L V, et al.
(2000) Lancet 355:827-835; Wang C, et al. (1993) J Virol
67:5585-5594). Antiviral agents are effective for sensitive virus
strains but viral resistance can develop quickly and has the
potential to render these drugs ineffective. In the 2008-2009 US
influenza season nearly 100% of seasonal H1N1 or H3N2 influenza
isolates tested were resistant to oseltamivir or adamantane
antivirals, respectively (CDC Influenza Survey: weekly archives
2008-2009, weekly 23).
[1448] Passive immunotherapy using anti-influenza antibodies
represents an alternative paradigm for preventing or treating viral
infection. Evidence for the utility of this approach dates back
nearly 100 years when passive serum transfer was used during the
1918 influenza pandemic with some success (Luke T C, et al. (2006)
Ann Intern Med 145:599-609). While protection provided by
anti-influenza monoclonal antibodies (mAbs) is typically narrow in
breadth because of the antigenic heterogeneity of influenza
viruses, several groups have recently reported protective mAbs that
bind to conserved epitopes within the stem region of viral
hemagglutinin (HA) (Okuno Y, et al. (1993) J Virol 67:2552-2558;
Throsby M, et al. (2008) PLoS One. 3: e3942; Sui J, et al. (2009)
Nat Struct Mol Biol 16:265-273; Corti D, et al. (2010) J Clin
Invest doi: 10.1172/JCI41902). These epitopes appear to be
restricted to a subset of influenza viruses; these anti-HA mAbs
would not be expected to provide protection against viruses of the
H3 and H7 subtypes. Of these, the former comprises an important
component of circulating human strains (Russell C A, et al. (2008)
Science 320:340-346) while the latter includes highly pathogenic
avian strains which have caused mortality in humans (Fouchier R A,
et al. (2004) Proc Natl Acad Sci USA 101:1356-1361; Belser J A, et
al. (2009) Emerg Infect Dis 15:859-865).
[1449] Of the three antibody targets present on the surface of the
influenza virus, the ectodomain of the viral M2 protein (M2e) is
much more highly conserved than either HA or NA, which makes it an
attractive target for broadly protective mAbs. Monoclonal
antibodies to M2e have been shown to be protective in vivo (Wang R,
et al. (2008) Antiviral Res 80:168-177; Liu W, et al. (2004)
Immunol Lett 93:131-6; Fu T M, et al. (2008) Virology 385:218-226;
Treanor J J, et al. (1990) J Virol 64:1375-1357; Beerli R, et al.
(2009) Virology J 6:224-234), and several groups have demonstrated
protection against infection with vaccine strategies based on M2e
(Fu T M, et al. (2009) Vaccine 27:1440-1447; Fan J, et al. (2004)
Vaccine 22:2993-3003; Slepushkin V A, et al. (1995) Vaccine
13:1399-1402; Neirynck S, et al. (1999) Nat Med 5:1157-1163;
Tompkins S M, et al. (2007) Emerg Infect Dis 13:426-435;
Mozdzanowska K, et al. (2003) Vaccine 21:2616-2626). In these
cases, purified M2 protein or peptides derived from M2e sequence
have been used as immunogens to generate anti-M2e antibodies in
animals or as vaccine candidates. In the present study, mAbs were
isolated directly from human B cells that bind to the M2 protein
displayed on virus particles and on virus-infected cells. Further,
we demonstrate that these antibodies protect mice from a lethal
influenza A virus challenge and that they can recognize M2 variants
derived from a wide range of human and animal influenza A virus
isolates. This combination of properties may enhance the utility of
these antibodies to prevent and treat influenza A virus
infections.
Results and Discussion
[1450] Isolation of a Family of Anti-M2e mAbs from Human B Cells.
To explore the humoral immune response to natural influenza
infection in humans, we have isolated antibodies from IgG.sup.+
memory B cells of M2e-seropositive subjects. Serum samples from 140
healthy adult, United States-sourced donors were tested for
reactivity with M2e expressed on the surface of HEK293 cells that
were transfected with a viral M2 gene (derived from A/Fort Worth/50
H1N1). IgG.sup.+ memory B cells from 5 of the 23 M2e-seropositive
subjects were cultured under conditions where they proliferated and
differentiated into IgG-secreting plasma cells. B cell culture
wells were screened for IgG reactivity to cell-surface M2e and
immunoglobulin heavy and light chain variable region (VH and VL)
genes were rescued by RT-PCR from 17 positive wells and
incorporated into a human IgG1 constant region background for
recombinant expression and purification. VH and VL sequences of 15
of the 17 anti-M2e mAbs cluster into two related groups (Table 3)
(IMGT.RTM., the International ImMunoGeneTics Information
System.RTM.). In group A, assignment of the germline VH gene
segment is IGHV4-59*01 while in the group B, the germline gene
segment is IGHV3-66*01. The two more distantly related mAbs 62B11
and 41G23 (group C) utilize the germline V gene segment IGHV4-31*03
which has only 5 amino acid residue differences from the germline V
gene segment IGHV4-59*01 of group A. All of these mAbs utilize the
same light chain V gene, IGKV1-39*01 or its allele IGKVID-39*01 and
show evidence of somatic hypermutation from the germline heavy or
kappa chain sequence (FIG. 12). Competitive binding experiments
showed that all of these human mAbs appear to bind similar sites on
native M2e expressed on the surface of Chinese hamster ovary (CHO)
cells (FIG. 13). We selected for further characterization one mAb
from each of groups A and B, designated TCN-031 and TCN-032,
respectively.
TABLE-US-00793 TABLE 3 Immunoglobulin gene segment usage of human
anti-M2e antibodies. Heavy chain germline gene segments Light chain
germline gene segments mAb Variable Diversity Joining Variable
Joining Group A TCN-032 IGHV4-59*01 IGHD2-15*01 IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01 IGKJ4*01 43I7 IGHV4-59*07 IGHD1-26*01
IGHJ4*02 IGKV1-39*01, or IGKV1D-39*01 IGKJ2*01 53P10 IGHV4-59*07
IGHD1-26*01 IGHJ4*02 IGKV1-39*01, or IGKV1D-39*01 IGKJ2*01 44I10
IGHV4-59*07 IGHD1-26*01 IGHJ4*02 IGKV1-39*01, or IGKV1D-39*01
IGKJ2*01 55J6 IGHV4-59*01 IGHD5-18*01 IGHJ4*02 IGKV1-39*01, or
IGKV1D-39*01 IGKJ5*01 52C13 IGHV4-59*01 IGHD5-18*01 IGHJ4*02
IGKV1-39*01, or IGKV1D-39*01 IGKJ5*01 39P23 IGHV4-59*01 IGHD4-23*01
IGHJ4*01 IGKV1-39*01, or IGKV1D-39*01 IGKJ1*01 36G5 IGHV4-59*01
IGHD2-8*01 IGHJ6*04 IGKV1-39*01, or IGKV1D-39*01 IGKJ3*01 48P18
IGHV4-59*01 IGHD2-15*01 IGHJ6*02 IGKV1-39*01, or IGKV1D-39*01
IGKJ4*01 59J21 IGHV4-59*01 IGHD2-15*01 IGHJ6*02 IGKV1-39*01, or
IGKV1D-39*01 IGKJ4*01 20I23 IGHV4-59*01 IGHD6-6*01 IGHJ6*02
IGKV1-39*01, or IGKV1D-39*01 IGKJ5*01 Group C 62B11 IGHV4-31*03
IGHD4-23*01 IGHJ6-02(a) IGKV1-39*01, or IGKV1D-39*01 IGKJ5*01 41G23
IGHV4-31*03 IGHD3-16*01 IGHJ6*02 IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01 Group B TCN-031 IGHV3-66*01 IGHD3-10*01 IGHJ3*01
IGKV1-39*01, or IGKV1D-39*01 IGKJ2*01 44H4 IGHV3-66*01 Cannot
assign IGHJ6*02 IGKV1-39*01, or IGKV1D-39*01 IGKJ5*01 45O19
IGHV3-66*01 Cannot assign IGHJ6*02 IGKV1-39*01, or IGKV1D-39*01
IGKJ5*01 60D19 IGHV3-66*01 Cannot assign IGHJ6*02 IGKV1-39*01, or
IGKV1D-39*01 IGKJ2*01 Reference sequences for each mAb heavy and
light chain were analysed using IMGT/V-QUEST to determine gene
usage.
[1451] High Affinity Binding to the Surface of Influenza Virus.
[1452] Both TCN-031 and TCN-032 bound directly to an H1N1 virus
(A/Puerto Rico/8/34) with high avidity, with half-maximal binding
at about 100 ng/mL (FIG. 14a). Fab fragments prepared from TCN-031
and TCN-032 bound virus with affinities (KD) of 14 and 3 nM,
respectively, as determined by surface plasmon resonance (Table 4).
The human mAbs did not bind appreciably to a 23 amino acid
synthetic peptide corresponding to the M2e domain of an H1N1 virus
(A/Fort Worth/1/50) (FIG. 14b). A chimeric derivative of the murine
anti-M2e mAb 14C2 (ch14C2), which was originally generated by
immunization with purified M2 (Zebedee S L and Lamb R A. (1988) J
Virol 62:2762-2772), exhibited the opposite behavior to that
observed with the human mAbs, with little binding to virus but
robust binding to the isolated 23mer M2e peptide with half-maximal
binding to peptide at 10 ng/mL (FIGS. 14a and 14b). Interestingly,
both the human mAbs and ch14C2 bound to the surface of Madin-Darby
canine kidney (MDCK) cells infected with H1N1 virus (A/Puerto
Rico/8/34) with similar avidities (FIG. 14c). It thus appears that
viral epitopes recognized by the human anti-M2e mAbs are present
and accessible on the surface of both virus and infected cells,
while the epitope bound by ch14C2 is accessible only on the surface
of infected cells. Our observation that the human anti-M2e mAbs do
not bind appreciably to immobilized synthetic peptides derived from
M2e, and further that such peptides do not compete for binding of
these antibodies to M2e expressed on the surface of mammalian cells
(FIG. 14d), supports the idea that secondary structure within the
M2e epitope is important for binding by the human antibodies. That
ch14C2 binds peptide immobilized on plastic suggests a lesser
importance of higher order structure for binding of this mAb.
TABLE-US-00794 TABLE 4 Affinity of anti-M2e Fab fragments for
influenza virus. Fab ka (M.sup.-1 * s.sup.-1) kd (s.sup.-1) KD
TCN-031 1.0e6 1.4e-2 14 nM TCN-032 7.4e5 2.3e-3 3.2 nM cH14C2 5.0e2
1.8e-3 4.0 .mu.M
[1453] Protection from Lethal Challenges with H5N1 and H1N1
Viruses.
[1454] We next examined the protective efficacy of the human
anti-M2e mAbs TCN-031 and TCN-032 in a lethal challenge model of
influenza infection in mice. Animals were challenged intranasally
with 5.times.LD50 units of a high-pathogenicity H5N1 virus
(A/Vietnam/1203/04) and both human mAbs were protective when
treatment was initiated one day after viral challenge. In contrast,
mice that were subjected to similar treatment regimens with a
subclass-matched, irrelevant control mAb 2N9, which targets the AD2
epitope of the gp116 portion of the human cytomegalovirus gB, or
with a vehicle control were protected to a lesser extent, or not at
all, resulting in 70-80% survival for mice treated with human mAbs
versus 20% survival for control mAb and 0% survival for vehicle
(FIG. 15a). The anti-M2e mAb ch14C2 did not confer substantial
protection in this model (20% survival; FIG. 15a), though this mAb
has been shown to reduce the titer of virus in the lungs of mice
infected with other strains of influenza virus (Treanor J J, et al.
(1990) J Virol 64:1375-1357). All of the animals, including those
in the TCN-031 and TCN-032 treatment groups, exhibited weight loss
from days 4 to 8 post infection followed by a gradual increase in
weight in the surviving animals through the end of the study on day
14 (FIG. 15b), indicating that the human anti-M2e mAbs afforded
protection by reducing the severity or extent of infection rather
than by completely preventing infection. Indeed, results of
immunohistological and viral load analyses of lung, brain and liver
tissue from additional animals in each treatment cohort are
consistent with a reduction in the spread of virus beyond the lung
to the brain and also possibly liver in animals that were treated
with the human anti-M2e mAbs, but not with ch14C2 or the
subclass-matched control mAb 2N9. The effect of the human anti-M2e
mAbs on viral load in the lung versus the control mAbs was,
however, more moderate (Table 5 and FIG. 16, respectively).
[1455] To test whether protection conferred by the human anti-M2e
mAbs mirrors their broad binding behavior, we performed a similar
in vivo challenge study with a mouse-adapted isolate of the
relatively divergent H1N1 virus A/Puerto Rico/8/34. One hundred
percent of PBS-treated or subclass-matched, control
antibody-treated mice were killed by this virus, while a majority
of the animals treated with the human anti-M2e mAbs TCN-031 and
TCN-032 survived (60%; FIG. 15c). With this virus mice treated with
ch14C2 provided a similar survival benefit to that of the human
anti-M2e mAbs (FIG. 15c). Weight changes in each treatment group
throughout the course of infection and its subsequent resolution
followed a pattern that was similar to that of mice infected with
the H5N1 virus (FIG. 15d).
[1456] The human anti-M2e mAbs and ch14C2 bound to cell
surface-expressed M2e from A/Vietnam/1203/04 and A/Puerto Rico/8/34
viruses (FIG. 19b, Table 6) and cells infected with A/Puerto
Rico/8/34 (FIG. 14c). Mechanisms for antibody-mediated protection
could include killing of infected host cells by antibody-dependent
cell-mediated cytotoxicity or complement-dependent cytotoxicity
(Wang R, et al. (2008) Antiviral Res 80:168-177; Jegerlehner A, et
al. (2004) J Immunol 172:5598-5605). We found in vitro evidence for
both of these mechanisms with the human anti-M2e mAbs and ch14C2
(FIG. 17 and Table 6). An explanation for the enhanced in vivo
protection observed with the human anti-M2e mAbs as compared to
ch14C2 following challenge by the high-pathogenicity avian virus
A/Vietnam/1203/04 as compared with A/Puerto Rico/8/34 could be due
to the unique capability of the human mAbs to bind virus directly
whereas ch14C2 does not appear to bind influenza virions (FIG.
14a). Protective properties of antibodies that bind to virus might
be expected to include mechanisms such as antibody-dependent
virolysis (Nakamura M, et al. (2000) Hybridoma 19:427-434) and
clearance via opsonophagocytosis by host cells (Huber V C, et al.
(2001) J Immunol 166:7381-7388). Some of these mechanisms require
efficient interaction between antibodies and host Fc receptors. In
our mouse challenge experiments all of the mAbs tested had human
constant regions; however other studies have shown that human
antibodies can interact productively with murine Fc receptors
(Clynes R A, et al. (2000) Nat Med 6:443-446).
TABLE-US-00795 TABLE 5 Pathological assessment of lung, liver, and
brain of mice treated with anti- M2e mAbs TCN-031 and TCN-032 after
challenge with H5N1 A/Vietnam/1203/04. Organs Mouse TCN-031 TCN-032
2N9 CH14C2 PBS UT/C Lung 1 ++/++ ++/++ ++/++ ++/++ ++/++ ++/+++ 2
++/++ ++/++ ++/+++ ++/++ ++/++ ++/++ 3 ++/++ ++/++ ++/++ ++/++
++/+++ ++/++ Brain 1 -/- -/- +/+ -/- .sup. +/+++ ++/+++ 2 -/-
.+-./+ .sup. +/+++ +/+ -/- +/+ 3 -/- -/- +/+ +/++ .sup. +/+++
++/+++ Liver 1 -/- -/- +/+ +/- +/+ +/+ 2 -/- -/- +/+ +/- +/- +/- 3
-/- +/- +/+ +/+ +/+ +/+ Pathological changes/viral antigens: +++
severe/many, ++ moderate/moderate, + mild/few, .+-. scant/rare, -
not observed/negative.
Pathological changes and viral antigens were detected in the lungs
of all virus-challenged mice. The mice had similar lung lesions
across all groups, although mice in the TCN-031 and TCN-032 groups
had a tendency toward less viral antigen expression in the lung. In
the brain and liver, lesions were not detected in mice in the
TCN-31 group and only one of three mice in the TCN-032 group showed
some evidence of viral antigens in the brain.
TABLE-US-00796 TABLE 6 Amino acids 1-23 of the M2 extracellular
domain 1 A/Brevig Mission/1/1918 S L L T E V E T P T R N E W G C R
C N D S S D H1N1 2 A/Fort Monmouth/1/1947 H1N1 K E 3
A/Singapore/02/2005 H3N2 I E 4 A/Wisconsin/10/1998 H1N1 I G E 5
A/Wisconsin/301/1976.H1N1 I S 6 A/Panama/1/1966 H2N2 F P I 7 A/New
York/321/1999 H3N2 I N 8 A/Caracas/1/1971 H3N2 I K 9
A/Taiwan/3/1971 H3N2 F I S 10 A/Wuhan/359/1995 H3N2 P I S 11 A/Hong
Kong/1144/1999 H3N2 P I 12 A/Hong Kong/1180/1999 H3N2 P I G 13
A/Hong Kong/1774/199 H3N2 G E S G 14 A/New York/217/2002 H1N2 I E Y
15 A/New York/300/2003 H1N2 I E Y S 16 A/swine/Spain/54008/2004 G E
Y S H3N2 17 A/Guangzhou/333/99 H9N2 F L G E S 18 A/Hong
Kong/1073/1999 H9N2 L G E K R 19 A/Hong Kong/1/1968 H3N2 I 20
A/swine/Hong Kong/126/1982 I S G H3N2 21 A/New York/703/1995 H3N2 I
E G 22 A/swine/Quebec/192/1981 P I H1N1 23 A/Puerto Rico/9/1934
H1N1 I G 24 A/Hong Kong/485/1997 H5N1 D L G S 25 A/Hong
Kong/542/1997 H5N1 L K G S 26 A/silky chicken/Shantou/ G E K S
1826/2004 H9N2 27 A/chicken/Taiwan/0305/2004 H G E K S H6N1 28
A/Quail/Arkansas/16309-7/ K G E K S 1994 H7N3 29 A/Hong
Kong/486/1997 H5N1 L G S 30 A/chicken/Pennsylvania/ D G E K S
13552-1/1998 H7N2 31 A/chicken/Heilongjiang/48/ G S 2001 H9N2 32
A/swine/Korea/S5/2005 H1N2 G E K 33 A/Hong Kong/1073/1999 H9N2 L G
E K S 34 A/Wisconsin/3523/1988 H1N1 I K 35 A/X-31 Vaccine strain
H3N2 F I G 36 A/Chicken/Rostock/8/1934 G E H7N1 37
A/environment/New York/ I K G E N S 16326-1/2005 H7N2 38
A/chicken/Hong Kong/SF1/ G H G K S 2003 H9N2 39 A/chicken/Hong
Kong/YU427/ P H G S 2003 H9N2 40 A/Indonesia/560H/2006 H5N1 E HK
A/Hong Kong/483/1997 H5N1 L G S VN A/Vietnam/1203/2004 H5N1 E S D20
A/A/FW/1/1950 H1N1 I
The M2e sequence at the top is from A/Brevig Mission/1/18 (H1N1)
and is used as the reference sequence for alignment of the M2
ectodomain amino acids 1-23 of 43 wild-type variants. Grey boxes
denote amino acid identity with the reference sequence and white
boxes are amino acid replacement mutations. This list of
non-identical sequences, except for HK, VN, and D20, was derived
from M2 sequences used in references 11 and 27. Sequence data are
from The Influenza Virus Resource at the National Center for
Biotechnology Information
[1457] Binding to the Highly Conserved N-Terminal Segment of
M2e.
[1458] To better understand the unique viral binding property of
the human anti-M2e mAbs we mapped their binding sites within the
M2e domain. The lack of appreciable binding of the human mAbs to
M2e-derived linear peptides precluded a synthetic peptide approach
to fine structure mapping of their epitopes. Instead, binding of
the mAbs to M2e alanine substitution mutants and naturally
occurring M2 variants that were expressed on the surface of
cDNA-transfected mammalian cells was quantified by flow cytometry.
Binding experiments with a panel of M2 mutant proteins where each
position in the 23 amino acid M2 ectodomain was substituted with
alanine revealed that the first (S), fourth (T), and fifth (E)
positions of the mature (methionine-clipped) M2 polypeptide were
critical for binding of both TCN-031 and TCN-032 (FIG. 19a). In
contrast, the binding of ch14C2 was selectively diminished when
alanine was substituted at position 14 of mature M2 (FIG. 19a).
These observations were confirmed in studies with a panel of
divergent, naturally occurring M2 variants; substitution with
proline at position 4 (Table 6: A/Panama/1/1966 H2N2, A/Hong
Kong/1144/1999 H3N2, A/Hong Kong/1180/1999 H3N2, and A/chicken/Hong
Kong/YU427/2003 H9N2) and glycine at position 5 (Table 6:
A/chicken/Hong Kong/SF1/2003 H9N2) correlated with diminished
binding of the human anti-M2e mAbs but not ch14C2 (FIG. 19b, Table
6). These results suggest that both TCN-031 and TCN-032 recognize a
core sequence of SLLTE at positions 1-5 of the N-terminus of mature
M2e. This is supported by data which show that these mAbs compete
effectively with each other for binding to M2e expressed on the
surface of CHO cells (FIG. 20). In contrast, our results indicate
that ch14C2 binds to a site that is spatially distinct and
downstream of the SLLTE core that is recognized by the human
anti-M2e mAbs. Indeed, previous studies have shown that 14C2 binds
a relatively broad, linear epitope with the sequence EVERTPIRNEW at
positions 5-14 of processed M2e (Wang R, et al. (2008) Antiviral
Res 80:168-177).
[1459] While the epitopes recognized by TCN-031 and TCN-032 are
likely very similar, there were some differences between these
human mAbs in their binding to several of the M2e mutants. For
instance, TCN-031 appears to have a greater dependence than TCN-032
on residues 2 (L) and 3 (L) of the mature M2e sequence (FIG. 19a).
The VH regions of these two human mAbs utilize different variable,
diversity, and joining gene segments which may explain the minor
differences in binding observed between these mAbs. Interestingly,
despite the differences in their VH make-up these human mAbs
utilize the same germline kappa chain V gene segments, albeit with
distinct kappa chain joining segments.
[1460] Localization of the binding region of the human anti-M2e
mAbs at the N-terminal region of M2e is especially significant in
light of the remarkably high sequence conservation in this part of
the polypeptide among influenza A viruses. The viral M gene segment
that encodes M2 also encodes the internal viral protein M1 via
differential splicing. However, the splice site is located
downstream of the shared N-terminus of M2 and M1 resulting in two
distinct mature polypeptides with an identical 8 amino acid
N-terminal sequence (Lamb R A and Choppin P W (1981) Virology
112:729-737). Options for viral escape from host anti-M2e
antibodies that bind this region might be limited as escape
mutations in the N-terminal region would result in changes to not
just M2 but also the M1 protein. Indeed, this N-terminal 8 amino
acid segment of M2e shows nearly complete identity in the 1364
unique full-length M2 variants catalogued in the NCBI Influenza
Database while much lower levels of conservation are seen in M2e
sequences downstream of this region (FIG. 19c). In fact, the core
human anti-M2e antibody epitope SLLTE is present in .about.98% of
the 1364 unique full-length M2e sequences catalogued in the NCBI
Influenza Database, including 97%, 98% and 98% of the human, swine
and avian viruses, respectively. This contrasts to the much lower
conservation within the linear binding sites of anti-M2e mAbs
elicited by immunization with M2e peptides or proteins. For
instance, 14C2 and Z3G1 (Wang R, et al. (2008) Antiviral Res
80:168-177) bind sequences that are conserved in less than 40% of
influenza A viruses, and conservation within this region is even
lower in avian and swine viruses (Table 7).
[1461] The linear M2e epitopes recognized by peptide-elicited
antibodies may be more sensitive to escape mutations and natural
substitutions that are present in some viral isolates. For example,
P10L and P10H escape mutations to mAb 14C2 have been mapped to the
central portion of M2e (Zharikova D, et al. (2005). J Virol
79:6644-6654) and those same substitutions also occur in M2e
variants from some highly pathogenic H5N1 strains. We have found
that the human mAbs TCN-031 and TCN-032 but not ch14C2 bind to the
M2 variant from the H5N1 virus A/Hong Kong/483/97 (HK) which
contains the P10L substitution (FIG. 19b, Table 6). Thus,
monoclonal antibodies with specificities similar to that of 14C2
are likely to have limited utility as broad spectrum therapeutic
agents.
[1462] In the examination of 5 human subjects we found 17 unique
anti-M2e antibodies that bind the conserved N-terminal region of
M2e, but did not observe IgG-reactivity with M2e-derived peptides
that contain the linear epitopes recognized by 14C2 and other
peptide-elicited antibodies. In contrast to the apparently uniform
antibody response to M2e in naturally infected or vaccinated
humans, mice immunized with M2e-derived peptides produced
antibodies with a range of specificities within M2e, including the
conserved N-terminus and also downstream regions (Fu T M, et al.
(2008) Virology 385:218-226). It is tempting to speculate that the
human immune system has evolved a humoral response that exclusively
targets the highly conserved N-terminal segment of M2e rather than
the more divergent, and thus less sustainably protective,
downstream sites. Despite the lack of evidence for human antibodies
that recognize this internal region of M2e, analysis of the
evolution of the M gene suggests that this region of M2e is under
strong positive selection in human influenza viruses (Furuse Y, et
al. (2009) J Virol 29:67). One explanation for this finding is that
selective pressure is being directed at this internal region by
immune mechanisms other than antibodies. For instance, human T cell
epitopes have been mapped to these internal M2e sites (Jameson J,
et al. (1998) J Virol 72:8682-8689).
TABLE-US-00797 TABLE 7 Conservation of the viral binding site for
human anti-M2e mAbs compared with those for mAbs derived from
immunized mice, in influenza A. Human Swine Avian All mAb (n = 506)
(n = 193) (n = 665) (n = 1364) TCN-031, TCN-032 97 98 98 98
[1-SLLTE-5] Z3G1 79 39 7 38 [2-LLTEVETPIR-11] (Ref. 11) 14C2 75 19
2 31 [5-EVETPIRNEW-14] (Ref. 11)
[1463] Recognition of 2009 H1N1 S-OIV.
[1464] Broadly protective anti-influenza mAbs could be used in
passive immunotherapy to protect or treat humans in the event of
outbreaks from highly pathogenic, pandemic viral strains. A
critical test of the potential for such mAbs as immunotherapeutic
agents is whether they are capable of recognizing virus strains
that may evolve from future viral reassortment events. As a case in
point, the human anti-M2e mAbs TCN-031 and TCN-032 were tested for
their ability to recognize the current H1N1 swine-origin pandemic
strain (S-OIV). These mAbs were derived from human blood samples
taken in 2007 or earlier, prior to the time that this strain is
thought to have emerged in humans (Neumann G, et al. (2009) Nature
459:931-939). Both human mAbs bound to MDCK cells infected with
A/California/4/2009 (S-OIV H1N1, pandemic) and A/Memphis/14/1996
(H1N1, seasonal) whereas ch14C2 bound only to cells infected with
the seasonal virus (FIG. 21). If this broad binding behavior proves
to correlate with protection, as was the case with
A/Vietnam/1203/2004 and A/Puerto Rico/8/34, then these human mAbs
might be useful to prevent or treat the S-OIV pandemic strain or
possibly other pandemic strains that might emerge in the
future.
[1465] While it is remarkable that humans have the capability to
make antibodies that may confer nearly universal protection against
influenza infection, the discovery of this heretofore un-described
class of antibodies raises the question of why this virus is able
to mount a productive infection in immunocompetent individuals at
all. This apparent paradox may be explained by the nature of the
protective M2e epitope and its relative immunogenicity. It has been
noted by others that M2e appears to exhibit low immunogenicity in
humans (Feng J, et al. (2006) Virol J 3:102; Liu W, et al. (2003)
FEMS Immunol Med Microbio 35:141-146), especially when compared to
the immunodominant virus glycoproteins HA and NA. Therefore,
protective anti-M2e antibodies may exist in many individuals but at
suboptimal titers. In support of this notion is our observation
that most individuals did not display a detectable humoral response
to M2e. We observed that fewer than 20% (23/140) of the individuals
that we sampled in our cohort of healthy subjects had detectable
serum levels of anti-M2e antibodies. The reasons for this
phenomenon are not clear but a similar situation exists in HCMV
where only a minority of HCMV seropositive subjects has measurable
antibodies to the broadly conserved, neutralizing AD2 epitope
within the gB complex of HCMV (Meyer H, et al. (1992) J Gen Virol
73:2375-2383; Ayata M, et al. (1994) J Med Virol 43:386-392;
Navarro D, et al. (1997) J Med Virol 52:451-459).
[1466] An important requirement for an immunotherapeutic solution
to the influenza threat will be the identification of protective
epitopes that are conserved in pre-existing and emerging viruses.
Using large-scale sampling of the human immune response to native
influenza M2 we have identified a naturally immunogenic and
protective epitope within the highly conserved N-terminal region of
M2e. Human antibodies directed to this epitope, including those
described in the present study, may be useful for the prevention
and treatment of pandemic and seasonal influenza.
Methods
[1467] Memory B Cell Culture.
[1468] Whole blood samples were collected from normal donors under
IRB approved informed consent and peripheral blood mononuclear
cells (PBMC) were purified by standard techniques. B cell cultures
were set up using PBMC, B cells enriched by selection with
M2-expressing cells, or IgG.sup.+ memory B cells enriched from PBMC
via negative depletion of nonIgG.sup.+ cells with antibodies to
CD3, CD14, CD16, IgM, IgA, and IgD on magnetic beads (Miltenyi,
Auburn, Calif.) as previously described (Walker L, et al. (2009)
Science 326:289-293). Briefly, to promote B cell activation,
proliferation, terminal differentiation and antibody secretion,
cells were seeded in 384-well microtiter plates in the presence of
feeder cells and conditioned media generated from
mitogen-stimulated human T cells from healthy donors. The culture
supernatants were collected 8 days later and screened in a high
throughput format for binding reactivity to M2 protein expressed on
HEK 293 cells stably transfected with influenza virus M2 (A/Fort
Worth/50 H1N1) using fluorescent imaging (FMAT system, Applied
Biosystems).
[1469] Reconstitution of Recombinant mAbs from B Cell Cultures.
[1470] mRNA was isolated from lysed B-cell cultures using magnetic
beads (Ambion). After reverse transcription (RT) with gene-specific
primers, variable domain genes were PCR amplified using VH, V, and
V.lamda. family-specific primers with flanking restriction sites
(Walker L, et al. (2009) Science 326:289-293). PCR reactions
producing an amplicon of the expected size were identified using
96-well E-gels (Invitrogen) and the variable domain amplicons were
cloned into the pTT5 expression vector (National Research of
Canada, Ottawa, Canada) containing human IgG1, Ig.kappa., or
Ig.lamda. constant regions. Each VH pool was combined with the
corresponding V.kappa., or V.lamda. pools from individual BCC wells
and was transiently transfected in 293-6E cells to generate
recombinant antibody. Conditioned media was harvested 3-5 days
after transfection and assayed for antibody binding to M2 protein
expressed on HEK 293 cells. Individual clones were isolated from
positive pools and unique VH and VL genes were identified by
sequencing. From these, monoclonal antibodies were subsequently
expressed and re-assayed for binding activity.
[1471] ELISA.
[1472] To detect viral antigen, either 10.2 .mu.g/mL UV-inactivated
H1N1 A/Puerto Rico/8/34 (PR8) virus (Advanced Biotechnologies,
Inc.) was passively adsorbed to 384-well plates in 25 .mu.L
PBS/well for 16 hr at 4.degree. C., or PR8 inactivated by
.beta.-propiolactone (Advanced Biotechnologies, Inc.) was
biotinylated (EZ-Link Sulfo-NHS-LC-Biotin, Pierce) and likewise
adsorbed to plates coated with neutravidin (Pierce). Virus-coated
and biotinylated virus-coated plates were blocked with PBS
containing 1% milk or BSA, respectively. Binding of mAbs at the
indicated concentrations was detected with HRP-conjugated goat
anti-human Fc antibody (Pierce) and visualized with TMB substrate
(ThermoFisher). The M2e peptide, SLLTEVETPIRNEWGCRCNDSSD (SEQ ID
NO: 680) (Genscript) was passively adsorbed at 1 .mu.g/mL and
antibody binding to the peptide was detected by the same
method.
[1473] FACS Analysis of Virally Infected Cells.
[1474] To detect M2e following in vitro infection, MDCK cells were
treated with PR8 at multiplicity of infection (MOI) of 60:1 for 1
hr at 37.degree. C. after which the culture media was replaced. The
infected MDCK cells were further cultured for 16 hr before
harvesting for cell staining with the indicated mAbs. Bound anti-M2
mAbs were visualized on viable cells with Alexafluor 647-conjugated
goat anti-Human IgG H&L antibody (Invitrogen). Flow cytometry
was performed on FACSCanto equipped with the FACSDiva software
(Becton Dickenson). For the panel of anti-M2 mAbs, 20 .mu.L samples
of supernatant from transient transfections from each of the IgG
heavy and light chain combinations was used to stain the 293 stable
cell line expressing M2 of A/Hong Kong/483/97 FACS analysis was
performed as above.
[1475] M2 Variant Analyses.
[1476] Individual full length M2 cDNA mutants were synthesized with
single ala mutations at each position of the ectodomain
representing A/Fort Worth/1/1950 (D20), as well as were the
forty-three naturally occurring variants of M2 (Blue Heron
Technology). They were cloned into the plasmid vector pcDNA3.1.
After transient transfection with Lipofectamine (Invitrogen),
HEK293 cells were treated with 1 .mu.g/mL of the indicated mAbs in
PBS supplemented with 1% fetal bovine serum and 0.2% NaN3 (FACS
buffer). Bound anti-M2 mAbs were visualized on viable cells with
Alexafluor 647-conjugated goat anti-Human IgG H&L antibody
(Invitrogen). Flow cytometry was performed with FACSCanto equipped
with the FACSDiva software (Becton Dickenson). The relative binding
to the naturally occurring variants was expressed as the percentage
of the respective mAb staining of the D20 transiently transfected
cells, using the formula of Normalized MFI (%)
100.times.(MFIexperimental-MFImock transfected)/(MFID20-MFImock
transfected).
[1477] Therapeutic Efficacy Studies in Mice.
[1478] Animal studies were conducted under Institutional Animal
Care and Use Committee protocols. We inoculated 6 groups of 10 mice
(female 6-8 week old BALB/C) intranasally with 5.times..sub.LD50 of
A/Vietnam/1203/04 (FIG. 15a and b) or 6 groups of 5 mice
intranasally with 5.times..sub.LD50 A/Puerto Rico/8/34 (FIG. 15c
and d). At 24, 72, and 120 hours post-infection the mice received
intraperitoneal injections of 400 .mu.g/200 .mu.L dose of the
anti-M2e mAbs TCN-031 TCN-032, control human mAb 2N9, control
chimeric mAb ch14C2, PBS, or were left untreated. Mice were weighed
daily for 2 weeks and were euthanized when weight loss exceeded 20%
(H5N1 study shown in FIG. 15a and 15b and H1N1 study shown in FIG.
15c and 15d) of the pre-infection body weight.
[1479] Antibody Reactivity to a/California/4/2009 Infected
Cells.
[1480] MDCK cells were infected with media alone or media
containing A/California/4/2009 (H1N1) or A/Memphis/14/1996 (H1N1)
at an MOI of approximately 1 and were cultured for 24 hours at
37.degree. C. The cells were detached from the tissue culture
plates with trypsin, washed extensively, and then fixed in 2%
paraformaldehyde for 15 minutes. The cells were incubated with 1
.mu.g/ml of the indicated antibodies and the primary antibody
binding was detected with Alexafluor 647-conjugated goat anti-Human
IgG H&L antibody (Invitrogen). The cells were analyzed with a
Becton Dickinson FACSCalibur and data were processed using FlowJo
software.
[1481] Competition Analysis of Antibody Binding.
[1482] Transient transfection supernatant containing antibody was
screened for binding to 293 cells stably transfected with M2 from
H1N1 (A/Fort Worth/50 H1N1), or mock transfected cells, in the
presence or absence of the M2e peptide SLLTEVETPIRNEWGCRCNDSSD
(Genscript) at 5 .mu.g/mL. Bound anti-M2 mAbs were detected with
anti-huIgG Fc FMAT Blue at 700 ng/ml in DMEM with 10% FCS and
visualized by fluorescent imaging (FMAT system, Applied
Biosystems).
Example 13
Screening and Characterization of HA-Specific Antibodies Present in
Human Plasma that Bind Purified Whole Inactivated Influenza A
Virions, Bind Recombinant Homotrimeric HA Proteins, and Neutralize
Infectious Influenza A
[1483] Fully human monoclonal antibodies specific for HA and
capable of binding purified whole inactivated Influenza A Virions,
binding recombinant homotrimeric HA proteins, and neutralizing live
influenza A were identified in patient plasma, as described
below.
Expression of Recombinant Soluble HA
[1484] An expression construct was generated containing a cDNA
encoding an HA precursor (HA0) polypeptide corresponding to the
derived HA sequence found in the Influenza subtypes, for example,
as listed in Table 9. Recombinant HA0 precursor polypeptides of the
invention lack an integral membrane or transmembrane domain, and
contain additional amino acids at the C-terminus of the HA0
ectodomain, for instance, corresponding to the sequence:
TABLE-US-00798 (SEQ ID NO: 726) SGR PGSGYIPEAPRDGQAYVRKDGEWVLLSTFL
HHHHHH,
wherein the thrombin cleavage site is bolded and italicized, the
bacteriophage T4 fibritin "foldon" or trimerization domain is
underlined, the last amino acid of the trimerization domain, "G",
is the start of the boxed "V5" epitope tag, which is followed by
the hexa histadine (HIS) epitope tag in bold. The hexa-HIS tag
within the preceding C-terminal region is used for purification of
recombinant HA proteins of the invention. Thus, recombinant HA0
precursor proteins that contain a trimerization domain are
considered recombinant HA homotrimeric proteins of the
invention.
[1485] Recombinant HA homotrimeric proteins of the invention retain
the native signal sequence to allow efficient secretion from
art-recognized cell lines maintained in vitro, e.g. 293 HEK cells
as done by Immune Technology Corp. Moreover, within these
recombinant HA homotrimeric proteins, or the HA0 precursors
thereof, the native HA1/HA2 viral protease cleavage site was
maintained, for instance, in all of the sequences provided in Table
9, except SEQ ID NO: 737, in which the native cleavage site
positioned at amino acids 337-347 and consisting of the sequence
"PQREGGRRRKR" (SEQ ID NO: 1250) was substituted with the sequence
"PQTETR" (SEQ ID NO: 1251).
[1486] Furthermore, exemplary receptor binding domains of
recombinant HA homotrimeric proteins, or the HA0 precursors
thereof, include the following structural elements: a 190
.alpha.-helix, a 130-loop, and a 220-loop (see, sequence of
Influenza A strain A/Vietnam/1203/2004) (or equivalent HA
structures in other Influenza A strains that the ordinarily skilled
artisan could readily obtain by accessing public databases,
including GenBank, and The Influenza Sequence Database, and
downloading sequences) (Stevens et al. 2006. Science 312: 404-410).
In other embodiments of the invention, in which the recombinant HA
homotrimeric protein, or HA0 precursor thereof, encoded by this
expression construct is partially or entirely expressed and
administered to a subject, these receptor binding domains may be
modified. The term "modified" is meant to describe the removal of
one or more structural elements. Alternatively, or in addition,
"modified" is meant to describe the addition, deletion,
substitution, inversion, or translocation of one or more amino
acids within a structural element of a receptor-binding domain of
HA. For instance, a linear or discontinuous epitope to which a
HuMHA antibody of the invention binds is administered to a subject
at risk of contracting an influenza infection to prevent the
infection. Alternatively or in addition, a linear or discontinuous
epitope to which a HuMHA antibody of the invention binds is
administered to a subject prior to exposure to an influenza virus
to prevent influenza infection. In other embodiments a structural
mimic of the conformational or discontinuous epitope is
administered to a subject. When the above proteins are used for
prophylactic purposes, for instance, as a vaccine, it may be
advantageous to modify one or more receptor binding domains to
control the resultant immune response in the subject. Exemplary
structural elements of HA that are optionally modified include, but
are not limited to, the 190 .alpha.-helix, the 130-loop, and the
220-loop of HA.
[1487] Recombinant homotrimeric HA proteins of the invention are
encoded by the following amino acid sequences, wherein the native
sequence is bolded and the sequence of SEQ ID NO: 726 is normal
(see also, Table 9):
[1488] A/California/4/09 (SEQ ID NO: 727)
TABLE-US-00799 DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPL
HLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEE
LREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIW
LVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVF
VGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVV
PRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITI
GKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYG
YHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHL
EKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVR
SQLKNNAKEIGNGCFEFYHKCDNTCMESVICNGTYDYPKYSEEAKLNREE
IDGVKLESTRIYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLST
FLGKPIPNPLLGLDSTGHHHHHH
[1489] A/Solomon Islands/3/06--H1N1 (SEQ ID NO: 728)
TABLE-US-00800 DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCLLKGIAPL
QLGNCSVAGWILGNPECELLISRESWSYIVEKPNPENGTCYPGHFADYEE
LREQLSSVSSFERFEIFPKESSWPNHTTTGVSASCSHNGESSFYKNLLWL
TGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQRALYHKENAYVSV
VSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTHFEANGNLIAPR
YAFALSRGFGSGIINSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGE
CPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYH
HQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLER
RMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQ
LKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDG
VKLESMGVYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG
KPIPNPLLGLDSTGHHHHHH
[1490] A/South Carolina/1/18--(SEQ ID NO: 729)
TABLE-US-00801 MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL
EDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETS
NSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVT
AACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGVHHPP
TGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTL
LEPGDTITFEATGNLIAPWYAFALNRGSGSGHTSDAPVHDCNTKCQTPHG
AINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIA
GFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVIE
KMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERT
LDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGTY
DYPKYSEESKLNREEIDGVKLESMGVYQSGRLVPRGSPGSGYIPEAPRDG
QAYVRKDGEWVLLSTFLGKPIPNPLLGLDSTGHHHHHH
[1491] A/Japan/305/57--H2N2 (SEQ ID NO: 730),
TABLE-US-00802 DQICIGYHANNSTEKVDTNLERNVTVTHAKDILEKTHNGKLCKLNGIPPL
ELGDCSIAGWLLGNPECDRLLSVPEWSYIMEKENPRDGLCYPGSFNDYEE
LKHLLSSVKHFEKVKILPKDRWTQHTTTGGSRACAVSGNPSFFRNMVWLT
KEGSDYPVAKGSYNNTSGEQMLIIWGVHHPIDETEQRTLYQNVGTYVSVG
TSTLNKRSTPEIATRPKVNGQGGRMEFSWTLLDMWDTINFESTGNLIAPE
YGFKISKRGSSGIMKTEGTLENCETKCQTPLGAINTTLPFHNVHPLTIGE
CPKYVKSEKLVLATGLRNVPQIESRGLFGAIAGFIEGGWQGMVDGWYGYH
HSNDQGSGYAADKESTQKAFDGITNKVNSVIEKMNTQFEAVGKEFGNLER
RLENLNKRMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRMQ
LRDNVKELGNGCFEFYHKCDDECMNSVKNGTYDYPKYEEESKLNRNEIKG
VKLSSMGVYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG
KPIPNPLLGLDSTGHHHHHH
[1492] A/Wisconsin/67/05--H3N2 (SEQ ID NO: 731)
TABLE-US-00803 QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGG
ICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPY
DVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSSCKRRSNNSFFSR
LNWLTQLKFKYPALNVTMPNNEKFDKLYIWGVHHPVTDNDQIFLYAQASG
RITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGN
LIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRI
TYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGW
YGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFS
EVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFER
TKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRF
QIKGVELKSGSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG
KPIPNPLLGLDSTGHHHHHH
[1493] A/Swine/Ontario/01911-2/99--H4N6 (SEQ ID NO: 732)
TABLE-US-00804 QNYTGNPVICLGHHAVSNGTMVKTLTDDQIEVVTAQELVESQHLPELCPS
PLRLVDGQTCDIVNGALGSPGCDHLNGAEWDVFIERPTAVDTCYPFDVPD
YQSLRSILANNGKFEFIAEEFQWNTVKQNGKSGACKRANVNDFFNRLNWL
TKSDGNAYPLQNLTKVNNGDYARLYIWGVHHPSTDTEQTNLYKNNPGRVT
VSTQTSQTSVVPNIGSRPWVRGLSSRISFYWTIVEPGDLIVFNTIGNLIA
PRGHYKLNSQKKSTILNTAVPIGSCVSKCHTDKGSISTTKPFQNISRISI
GDCPKYVKQGSLKLATGMRSILEKATRGLFGAIAGFIENGWQGLIDGWYG
FRHQNAEGTGTAADLKSTQAAIDQINGKLNRLIGKPNEKYHQIEKEFEQV
EGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERVR
HQLRENAEDKGNGCFEIFHQCDNSCIESIRNGTYDHDIYRDEAINNRFQI
QGVKLIQGYKDSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFL
GKPIPNPLLGLDSTGHHHHHH
[1494] A/Hong Kong/156/97--H5N1 (SEQ ID NO: 733)
TABLE-US-00805 MERTVLLLATVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILE
RTHNGKLCDLNGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAS
PANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSNHDASSGVSSA
CPYLGRSSFFRNVVWLIKKNSAYPTIKRSYNNTNQEDLLVLWGVHHPNDA
AEQTKLYQNPTTYISVGTSTLNQRLVPEIATRPKVNGQSGRMEFFWTILK
PNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGA
INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNTPQRERRRKKRGLFG
AIAGFIEGGWQGMVDGWYGYHHSNEQGSCYSADKESTQKAIDGVTNKVNS
IINKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN
ERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKN
GTYDYPQYSEEARLNREEISGVKLESMGTYQSGRLVPRGSPGSGYIPEAP
RDGQAYVRKDGEWVLLSTFLGKPIPNPLLGLDSTGHHHHHH
[1495] A/Vietnam/1203/04--H5N1 (SEQ ID NO: 734)
TABLE-US-00806 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPL
ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEE
LKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWL
IKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISV
GTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAP
EYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIG
ECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDG
WYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREF
NNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYD
KVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKR
EEISGVKLESIGIYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLL
STFLGKPIPNPLLGLDSTGHHHHHH
[1496] A/Indonesia/5/05--H5N1 (SEQ ID NO: 735)
TABLE-US-00807 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL
ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEE
LKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWL
IKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISI
GTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAP
EYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIG
ECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDG
WYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREF
NNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYD
KVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKR
EEISGVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLL
STFLGKPIPNPLLGLDSTGHHHHHH
[1497] A/Egypt/3300-NAMRU3/08--H5N1 (SEQ ID NO: 736)
TABLE-US-00808 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL
ILRDCSVAGWLLGNPMCDEFLNVSEWSVIVEKINPANDLCYPGNFNNYEE
LKHLLSRINRFEKIQIIPKSSWPDHEASLGVSSACPYQGGPSFYRNVVWL
IKKNNTYPTIKKSYHNTNQEDLLVLWGIHHPNDEAEQTRIYKNPTTYISV
GTSTLNQRLVPKIATRSKVNGQSGRVEFFWTILKSNDTINFESNGNFIAP
ENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIG
ECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDG
WYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREF
NNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYD
KVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKR
EEISGVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLL
STFLGKPIPNPLLGLDSTGHHHHHH
[1498] A/Common Magpie/Hong Kong/5052/07--H5N1 (SEQ ID NO: 737)
TABLE-US-00809 DHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPL
ILKDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEE
LKHLLSRINHFEKIQIIPKDSWSDHEASLGVSSACPYQGNSSFFRNVVWL
IKKGNAYPTIKKSYNNTNQEDLLVLWGIHHPNDEAEQTRLYQNPTTYISI
GTSTLNQRLVPKIATRSKVNGQSGRIDFFWTILKPNDAINFESNGNFIAP
EYAYKIVKKGDSTIMKSEVEYGNCNTRCQTPMGAINSSMPFHNIHPLTIG
ECPKYVKSNKLVLATGLRNSPQRERRRKRGLFGAIAGFIEGGWQGMVDGW
YGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFN
NLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDK
VRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKRE
EISGVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLS
TFLGKPIPNPLLGLDSTGHHHHHH
[1499] A/Anhui/1/05--H5N1 (SEQ ID NO: 738)
TABLE-US-00810 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL
ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEE
LKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWL
IKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPTTYISV
GTSTLNQRLVPKIATRSKVNGQSGRMDFFWTILKPNDAINFESNGNFIAP
EYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIG
ECPKYVKSNKLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGW
YGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFN
NLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDK
VRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKRE
EISGVKLESIGTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLS
TFLGKPIPNPLLGLDSTGHHHHHH
[1500] A/chicken/Vietnam/NCVD-016/08--H5N1 (SEQ ID NO: 739)
TABLE-US-00811 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPL
ILKDCSVAGWLLGNPMCDEFLNVSEWSYIVEKASPANGLCYPGDFNDYEE
LKHLLSRINHLKKIKIIPKSYWSNHEASSGVSAACSYLGEPSFFRNVVWL
IKKNNTYPPIKVNYTNTNQEDLLVLWGIHHPNDEKEQIRIYQNPNTSISV
GTSTLNQRLVPKIATRPKVNGQSGRMEFFWTILKPNDSINFDSNGNFIAP
EYAYKIAKKGDSVIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIG
ECPKYVKSNRLVLATGLRNAPQTETRGLFGAIAGFIEGGWQGMVDGWYGY
HHSNEQGSGYAADKESTQKAIDGITNKVNSIIDKMNTQFEIVGREFNNLE
RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYEKVRL
QLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLSREEIS
GVKMESMVTYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFL
GKPIPNPLLGLDSTGHHHHHH
[1501] A/Northern Shoveler/California/HKWF115/2007--H6N1 (SEQ ID
NO: 740)
TABLE-US-00812 DKICIGYHANNSTTQVDTILEKNVTVTHSVELLENQKEERFCKILNKAPL
DLRGCTIEGWILGNPQCDLLLGDQSWSYIVERPTAQNGICYPGALNEVEE
LKALIGSGERVERFEMFPKSTWTGVDTSSGVTKACPYNSGSSFYRNLLWI
IKTKSAAYPVIKGTYNNTGSQPILYFWGVHHPPDTNEQNTLYGSGDRYVR
MGTESMNFAKSPEIAARPAVNGQRGRIDYYWSVLKPGETLNVESNGNLIA
PWYAYKFVSTNNKGAIFKSNLPIENCDATCQTIAGVLRTNKTFQNVSPLW
IGECPKYVKSESLRLATGLRNVPQIETRGLFGAIAGFIEGGWTGMIDGWY
GYHHENSQGSGYAADKESTQKAIDGITNKVNSIIDKMNTQFEAVDHEFSN
LERRIDNLNKRMEDGFLDVWTYNAELLVLLENERTLDLHDANVKNLYEKV
KSQLRDNANDLGNGCFEFWHKCDNECIESVKNGTYDYPKYQDESKLNRQE
IESVKLDNLGVYQSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLST
FLGKPIPNPLLGLDSTGHHHHHH
[1502] A/Netherlands/219/03--H7N7 (SEQ ID NO: 741)
TABLE-US-00813 DKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNVPRICSKGKRTVD
LGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQ
ILRESGGIDKETMGFTYSGIRTNGTTSACRRSGSSFYAEMKWLLSNTDNA
AFPQMTKSYKNTRKDPALIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNY
QQSFVPSPGARPQVNGQSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASF
LRGKSMGIQSEVQVDANCEGDCYHSGGTIISNLPFQNINSRAVGKCPRYV
KQESLLLATGMKNVPEIPKRRRRGLFGAIAGFIENGWEGLIDGWYGFRHQ
NAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFTEVERQI
GNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVKRQLR
ENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAIQNRIQIDPVK
LSSGYKDSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPI
PNPLLGLDSTGHHHHHH
[1503] H8N4--A/Duck/Yangzhou/02/2005 (SEQ ID NO: 742)
TABLE-US-00814 DRICIGYQSNNSTDTVNTLIEQKVPVTQTMELVETEKHPAYCNTDLGAPL
ELRDCKIEAVIYGNPKCDIHLKDQGWSYIVERPSAPEGMCYPGSVENLEE
LRFVFSSAASYKRIRLFDYSRWNVTSSGTSKACNASTGGQSFYRSINWLT
KKKPDTYDFNEGTYVNNEDGDIIFLWGIHHPPDTKEQTTLYKNANTLSSV
TTNTINRSFQPNIGPRPLVRGQQGRMDYYWGILKRGETLKIRTNGNLIAP
EFGYLLKGESHGRTIQNEDIPIGNCYTKCQTYAGAINSSKPFQNASRHYM
GECPKYVKKASLRLAVGLRNTPSVEPRGLFGAIAGFIEGGWSGMIDGWYG
FHHSNSEGTGMAADQKSTQEAIDKITNKVNNIVDKMNREFEVVNHEFSEV
EKRINMINDKIDDQIEDLWAYNAELLVLLENQKTLDEHDSNVKNLFDEVK
RRLSANAIDAGNGCFDILHKCDNECMETIKNGTYDHKEYEEEAKLERSKI
NGVKLEENTTYKSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTF
LGKPIPNPLLGLDSTGHHHHHH
[1504] A/Hong Kong/2108/03--H9N2 (SEQ ID NO: 743)
TABLE-US-00815 DKICIGYQSTNSTETVDTLTKTNVPVTQAKELLHTEHNGMLCATNLGHPL
ILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGMCYPGNVENLEE
LRLLFSSASSYQRVQIFPDTIWNVTYSGTSSACSNSFYRSMRWLTQKDNT
YPVQDAQYTNNRGKSILFMWGINHPPTDTVQTNLYTRTDTTTSVTTEDIN
RAFKPVIGPRPLVNGLQGRIDYYWSVLKPGQTLRVRSNGNLIAPWYGHIL
SGESHGRILKSDLNSGNCVVQCQTERGGLNTTLPFHNVSKYAFGNCPKYV
GVKSLKLAVGMRNVPARSSRGLFGAIAGFIEGGWPGLVAGWYGFQHSNDQ
GVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEIETRLNMI
NNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNA
MEDGKGCFELYHKCDDRCMETIRNGTYNRGKYKEESRLERQKIEGVKLES
EGTYKSGRLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKPIPN
PLLGLDSTGHHHHHH
[1505] A/Hong Kong/1073/99--H9N2 (SEQ ID NO: 744)
TABLE-US-00816 METISLITILLVVTASNADKICIGHQSTNSTETVDTLTETNVPVTHAKEL
LHTEHNGMLCATSLGHPLILDTCTIEGLVYGNPSCDLLLGGREWSYIVER
SSAVNGTCYPGNVENLEELRTLFSSASSYQRIQIFPDTTWNVTYTGTSRA
CSGSFYRSMRWLTQKSGFYPVQDAQYTNNRGKSILFVWGIHHPPTYTEQT
NLYIRNDTTTSVTTEDLNRTFKPVIGPRPLVNGLQGRIDYYWSVLKPGQT
LRVRSNGNLIAPWYGHVLSGGSHGRILKTDLKGGNCVVQCQTEKGGLNST
LPFHNISKYAFGTCPKYVRVNSLKLAVGLRNVPARSSRGLFGAIAGFIEG
GWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQ
YEIIDHEFSEVETRLNMINNKIDDQIQDVWAYNAELLVLLENQKTLDEHD
ANVNNLYNKVKRALGSNAMEDGKGCFELYHKCDDQCMETIRNGTYNRRKY
REESRLERQKIEGVKLESEGTYKSGRLVPRGSPGSGYIPEAPRDGQAYVR
KDGEWVLLSTFLGKPIPNPLLGLDSTGHHHHHH
[1506] The recombinant and soluble HA expression constructs of SEQ
ID NO: 727-744 were transfected into 293 HEK cells. Recombinant HA0
protein or HA cleaved into its respective subunits HA1 and HA2 and
disulphide linked was purified from culture supernatant by standard
procedures using the hexa-HIS tag at the C-terminal. The purified
protein was analyzed by size exclusion chromatography and/or
denaturing coomasie gel to confirm a recombinant protein of the
expected size was present.
Example 14
Screening of Antibodies in Peripheral Blood
[1507] One hundred and twenty-six individual serum or plasma
samples were screened for the presence of IgG antibody that bound
to recombinant soluble homotrimeric HA proteins (Table 9) using a
micro-array scanning system, bind to whole inactivated Influenza A
virions (Table 10) using standard ELISA techniques, and inhibits or
neutralizes virus infection of MDCK cells with Influenza A H1N1
A/Solomon Islands/3/06 or H3N2 A/Wisconsin/67/05. A portion of the
plasma samples contained IgG antibodies that bound specifically to
the recombinant soluble HA homotrimeric protein, bound to
inactivated virions, and neutralized virus infectivity. This
indicates that the antibodies could be binding linear or
discontinuous epitopes in the HA homotrimer, as well as binding to
conformational determinants of multiple variants of the HA
homotrimer. Soluble targets include, but are not limited to,
exemplary recombinant HA proteins derived from the influenza virus
strains listed in Table 2 and the inactivated virus strains listed
in Table 11.
TABLE-US-00817 TABLE 11 Inactivated whole virions used in ELISA
binding assays. Influenza A Subtype Strain designation H1N1
A/Solomon Islands/3/06 H2N2 A/Japan/305/57 H3N2
A/Wisconsin/67/05
Example 15
Identification and Rescue of HA-Specific Antibodies
[1508] IgG+ memory B cells purified from a human blood sample were
cultured for 9 days in order to activate, proliferate, and
differentiate these memory B cells into IgG secreting plasma cells.
The B cell culture media containing the IgG was screened for the
presence of IgG antibody that bound to recombinant soluble
homotrimeric HA proteins (Table 9) using a micro-array scanning
system, bind to whole Influenza A virus (Table 10) using standard
ELISA techniques, and inhibits or neutralizes virus infection of
MDCK cells with Influenza A H1N1 A/Solomon Islands/3/06 or H3N2
A/Wisconsin/67/05. As shown in Tables 12, 13, and 14, thirty-nine
BCC wells were identified that contained an IgG antibody with
several virus, HA, or neutralizing profiles and the variable
regions of the antibody were then cloned from the corresponding B
cell cultures.
[1509] Transient transfections with monoclonal heavy and light
chain pairs from each BCC well were performed in 293 6E cells to
reconstitute and produce the antibody. Reconstituted antibody
supernatants were then screened for IgG that binds to recombinant
soluble homotrimeric HA proteins (Table 9) using a micro-array
scanning system, bind to whole Influenza A virus (Table 11) using
standard ELISA techniques, and inhibits or neutralizes virus
infection of MDCK cells with Influenza A H1N1 A/Solomon
Islands/3/06, and/or H1N1 A/California/04/09, and/or H3N2
A/Wisconsin/67/05 to identify the rescued anti-HA antibody. Binding
and neutralization of the human IgG antibodies to the preceding
targets is compared to the binding of the proprietary positive
control antibody TCN-032 (specific for the N-terminal of the matrix
2 protein of influenza A, U.S. patent application Ser. No.
12/795,618) or to the binding of the broadly HA specific,
non-neutralizing mAb TCN-504 (also known as 3251K17, and described
herein), and the proprietary negative control antibody TCN-202
(specific for the AD2 site I epitope on human cytomegalovirus gB)
to these same targets.
[1510] The sequences of the rescued antibodies are determined. In
addition, the sequence of each of the polynucleotides encoding the
antibody sequences is determined.
Example 16
Binding Profiles of IgG in B Cell Culture Supernatant or Monoclonal
Transfection Supernatant Using Inactivated Whole Influenza a
Virions
[1511] To determine whether the human mAbs in BCC SN or monoclonal
transfection supernatant bind to purified virus, Enzyme-Linked
ImmunoSorbent Assays (ELISAs) were performed according to the
following methods. Briefly, various purified Influenza A virus
subtype strains were coated directly onto an ELISA plate. A single
dilution of BCC supernatant of the mAbs shown in Table 12 or of the
monoclonal transfection supernatant shown in Table 15 and various
dilutions of a positive control antibody (TCN-032) were then added
to the virus coated wells. Unbound antibody was washed away and the
bound antibody was detected with an anti-human antibody conjugated
to HRP. The presence of anti-influenza antibodies was detected when
the chromogen (TMB) is oxidized by the HRP conjugate, resulting in
a bluecolor. The reaction is stopped by the addition of HCl,
turning antibody positive wells yellow. The yellow color has a
maximal absorbance at 450 nm.
[1512] Methods: [1513] Coat Microlon.TM. plates with 25 ul/well of
3 ug/ml inactivated influenza A virions [1514] Incubate plates
overnight at 4.degree. C. [1515] Remove plates from 4.degree. C.
and wash four times with phosphate buffered saline with Calicum and
Magnesium (PBS w/Ca.sup.2+, Mg.sup.2+), using EL405x (Wash program:
ELISA.sub.--4x_wash). [1516] Add 20 ul/well of 1% milk/PBS to
plates. [1517] Prepare control mAb curves by 1:3 dilutions of 6
ug/ml [1518] 5 ul of each BCC SN or monoclonal transient
transfection supernatant and control mAb curves are stamped onto
the plate according to the plate map. [1519] Incubate 2 hour (hr)
at room temperature (RT). [1520] Wash plates four times with PBS
w/Ca.sup.2+, Mg.sup.2+, using EL405x (Wash program:
ELISA.sub.--4x_wash) [1521] Add 25 ul/well of polyclonal antibody
(pAb) goat anti-human (aHuman) IgG Fc horseradish peroxidase (HRP)
at a dilution of 1:5000. [1522] Incubate 1 hr at RT. [1523] Wash
plates four times with PBS w/Ca.sup.2+, Mg.sup.2+, using EL405x
(Wash program: ELISA.sub.--4x_wash) [1524] Add 25 ul/well of
Ultra-TMB (3,3',5,5'-tetramethylbenzidine) at Neat. [1525] Develop
30 minutes (min) at RT. [1526] Stop by adding 25 ul/well of
hydrochloric acid (HCl) at a concentration of 0.3M. [1527] Read the
plate at 450 nm with the Spectromax.
[1528] One or more of the following control antibodies were used in
these experiments: TCN-504 (also known as 3251_K17, and described
herein) TCN-032 (also known as 8I10, specific for the influenza A
M2 protein), and TCN-202 (also known as 2N9, specific for the AD2
site I epitope on human cytomegalovirus gB) protein.
[1529] The following purified viruses were used in these
experiments: A/Solomon Islands/3/2006 (H1N1) (SEQ ID NO: 728),
A/Japan/305/1957 (H2N2) (SEQ ID NO: 729), A/Wisconsin/67/2005
(H3N2) (SEQ ID NO: 730). As shown in Table 15 the human monoclonal
antibodies in the transient transfection supernatant bind strongly
to one or more of the H1N1, H2N2, and/or H3N2 viruses reproducing
the virus binding profile of the IgG antibody in the original BCC
SN (Table 12).
Example 17
Binding Profiles of IgG in B Cell Culture Supernatant or Monoclonal
Transfection Supernatant Using Trimeric HA
[1530] To determine whether the human mAbs in BCC SN or monoclonal
transfection supernatant bind to one or more of the recombinant
homotrimeric HA proteins, a micro-array scanning system was used
according to the following methods. Twenty Nexterion P (Schott)
glass slides are incubated overnight in a humid chamber with 3
mg/ml goat anti-human, Fc--specific antibody. Nexterion P slides
contain a hydrogel that terminates in an NHS ester reactive group
that binds the capture antibody. The reaction is then quenched
using 50 mM ethanolamine in 50 mM NaBorate (pH 8.0) buffer for 1
hour with agitation followed by 3 washes with ultrapure water.
Transfection supernatants are transferred to 384-well array source
plates. Control array source plates are made in 8-point, 3-fold
serial dilutions of antibody starting at 3 ug/ml and ending 0 ug/ml
in BCC or mock transfection media. Both BCC or transfection
supernatant array source plates and control plates are loaded onto
an Aushon 2470 microarray printer along with the 20 prepared
slides.
[1531] Microarrays are printed in 48 subarray blocks with variable
number of features and replicates depending on number of
transfection supernatants are being printed. Printed microarrays
are allowed to sit for at least 1 hour after printing (to
overnight) in the printer's humidity controlled chamber (80%). They
are then quickly removed from the printer, washed, and spun dry to
prepare the slides for sample incubations. A LifterSlip is placed
on each slide and 90 uls of sample is added to each. Each slide
receives either one of 18 V5-tagged HA trimerics at predetermined
concentrations, a horseradish peroxidase goat anti-human H+L
specific antibody at a 1 ug/ml, or a blank for a total of 20
slides. The slides are then incubated overnight in a humid chamber
at room temperature. After washing, spin drying, and new LifterSlip
application, 19 slides, excluding slide incubated with anti-human
IgG, are incubated with anti-V5 conjugated to biotin at a 1:1000
dilution for 1 hour. Subsequently the slides are again washed,
dried and prepared for an 1-hour incubation with a 1:300 dilution
of NeutrAvidin-HRP. After further washing, drying and preparation
all 20 slides are incubated for 1 hour with a Tyramide-AlexaFluor
reagent according to kit instructions. After final washing and
drying the slides are scanned on an Axon Genepix 4300A at an
excitation wavelength of 594 nm and with an emission band ranging
from 619 nm to 641 nm. Data is recovered using the Axon Genepix
software and is then analyzed for binding profiles.
[1532] Methods:
1. Dilute goat anti-human, FC-specific antibody (JacksonImmuno, 10
mg/ml) to 3 mg/ml in PBS 0.05&Tween and apply to 20 Nexterion P
Slides (Schott) using 90 uls under lifterslips (Thermo). 2.
Incubate in a humid chamber overnight at room temperature 3.
Removing the lifterslip quench all slides in 50 mM ethanolamine
(Fisher) in 50 mM Sodium tetraborate decahydrate (Fisher, S248-500)
at a pH of 8.0 4. Quench for 1 hour with incubation at room
temperature 5. Wash all slides (3.times.2 min MilliQ water washes
with agitation). 6. Load all slides onto Aushon 2470 MicroArray
printer. 7. Prepare control plates using by spiking control
antibodies into transfection media to form an 8 point 3-fold serial
dilution starting at 3 ug/ml and ending in 0 ug/ml. Transfer
control dilutions to 4 array source plates (Thermo). 8. Load all
array source plates, control and sample, onto Aushon 2470
microarray printer and begin deposition after checking all required
fluid levels. Number of replicates is based on number of
transfection supernatants being printed. Typically 1 to 10
replicates. 9. Allow slides to sit for at least an hour, to
overnight, after deposition. 10. Immediately wash (PBS with 2%
tween 20, 5 min; MilliQ water, 2 min, 3.times.) and spin dry (2000
RPM for 1 minute) 11. Using lifterslips and apply 90 uls of HA at
the following concentration:
TABLE-US-00818 Homotrimeric HA - strain [ug/ml] A/California/4/09 -
H1N1 5 A/Solomon Islands/3/06 - H1N1 5 A/Japan/305/57 - H2N2 5
A/Wisconsin/67/05 - H3N2 20 A/swine/Ontario/01911-2/99 - H4N6 5
A/Vietnam/1203/04 - H5N1 20 A/Indonesia/5/05 - H5N1 0.5
A/Egypt/3300-NAMRU3/08 - H5N1 0.5 A/Common magpie/Hong Kong/5052/07
- H5N1 0.5 A/Anhui/1/05 - H5N1 0.5 A/chicken/Vietnam/NCVD-016/08 -
H5N1 0.5 A/northern shoveler/California/HKWF115/2007 - H6N1 0.5
A/Netherlands/219/03 - H7N7 0.5 A/duck/Yangzhou/02/2005 - H8N4 5
A/Hong Kong/2108/03 - H9N2 5 A/South Carolina/1/18 - H1N1 20 A/Hong
Kong/1073/99 20 A/Hong Kong/156/97 - H5N1 5
[1533] Use 1.times.PBS, 0.05% Tween 20, 10% Blocker Casein (Thermo,
#37528) to bring the homotrimeric HA to the desired
concentration.
12. Incubate overnight in a humid chamber at room temperature 13.
Immediately wash (PBS with 2% tween 20, 5 min; MilliQ water, 2 min,
3.times.) and spin dry (2000 RPM for 1 minute) 14. Using
lifterslips incubate all but slide previously incubated with
anti-human IgG(H&L)--HRP with 90 uls of anti-V5-biotin (AbD
Serotec, MCA1360B) at 1 ug/ml in 1.times.PBS 0.05% Tween 20 for 1
hour at room temperature in a humid chamber. 15. Immediately wash
(PBS with 2% tween 20, 5 min; MilliQ water, 2 min, 3.times.) and
spin dry (2000 RPM for 1 minute) 16. Using lifterslips incubate
with 90 uls of horseradish peroxidase conjugated NeutrAvidin
(Pierce #31030) for 1 hour at room temperature in a humid chamber.
17. Immediately wash (PBS with 2% tween 20, 5 min; MilliQ water, 2
min, 3.times.) and spin dry (2000 RPM for 1 minute) 18. Prepare
Tyramide Signal Amplification reagent according to kit instructions
(TSA Kit #25, Invitrogen, T20935). Briefly dilute 1 ul of hydrogen
peroxide solution into 200 uls of amplification buffer. Take 20 uls
of hydrogen peroxide/amplification buffer solution and add to 1940
uls of fresh amplification buffer. Then add 40 uls of
tyramide-Alexa Fluor resulting in a total of 2 mls of amplification
reagent 19. Incubate all 20 slides with amplification reagent for 1
hour at room temperature in a humid chamber. 20. Immediately wash
(PBS with 2% tween 20, 5 min; MilliQ water, 2 min, 3.times.) and
spin dry (2000 RPM for 1 minute) 21. Scan all slides on an Axon
Genepix 4300A at an excitation wavelength of 594 nm and with an
emission band ranging from 619 nm to 641 nm or optical scanner with
similar capabilities. 22. Lay templates on each slide using GenePix
Pro 7 or similar software to recover feature data. 23. Analyze
feature data for binding profiles to each HA trimeric.
[1534] As shown in Table 16, the human monoclonal antibodies in the
transient transfection supernatant bind strongly to one or more of
the recombinant homotrimeric HA proteins reproducing the virus
binding profile of the IgG antibody in the original BCC SN (Table
12).
Example 18
Neutralization Profiles of IgG in B Cell Culture Supernatant or
Monoclonal Transfection Supernatant
[1535] MDCK cells were plated at 3.times.10.sup.3 cells/well in a
384-well plate in complete DMEM media (supplemented with 10% FBS,
1.times. penicillin/streptomycin, 1.times. Glutamax.TM., and
1.times. Sodium Pyruvate) and incubated at 37.degree. C.
overnight.
[1536] Influenza A virus was preincubated with either BCC
supernatant or monoclonal transfection supernatant or a positive
control neutralizing monoclonal antibody (MAb), which was diluted
in pooled BCC supernatant or in mock transfection supernatant at
the desired concentrations and incubated overnight (.about.16
hours) at 37.degree. C. Void volumes and dilutions were made in PBS
with Mg.sup.2+, Ca.sup.2+, 200 mM Mannose, and 1% BSA at 37.degree.
C. with a total volume of 30 .mu.l/well.
[1537] Each Sample Well Contained:
[1538] (a) 20 .mu.l/well BCC supernatant; or
[1539] (b) 20 ul/well of monoclonal transfection
[1540] (c) 3000 IU/well A/Solomon Islands/03/2006 (H1N1) in 10
.mu.l/well; or
[1541] (e) 3000 IU/well A/California/04/2009 (H1N1) in 10 ul/well;
or
[1542] (f) 3000 IU/well A/Wisconsin/67/05 (H3N2) in 10 ul/well
[1543] Prior to infection, the MDCK cells were washed once with a
solution containing PBS, Mg.sup.2+, and Ca.sup.2+ at 60 .mu.l/well.
After the wash, 25 ul of the virus/mAb mixture was transferred and
the infection proceeded for 4 hours at 37.degree. C.
[1544] After 4 hours of infection, the MDCK cells are washed twice
with complete DMEM. After the last wash, 25 .mu.l/well of complete
DMEM remained, and the plates were incubated overnight at
37.degree. C.
[1545] After the overnight incubation the culture media was removed
and 20 .mu.l of BD Cytofix/Cytoperm.TM. (cat#51-2090KZ) was added
to each well and incubated at room temperature (RT) for 30 minutes.
Next, the wells were washed three times using the M384 Atlas plate
washer.
[1546] After the final wash, 15 .mu.l/well of 100 .mu.g/ml Rabbit
IgG (Sigma) in PBS with Mg.sup.2+, Ca.sup.2+, and 1% BSA, was added
and incubated for at least 5 minutes at RT. Twenty .mu.l/well of
anti-M2e mAb TCN-032 at 2 .mu.g/ml in PBS with Mg.sup.2+,
Ca.sup.2+, and 1% BSA, were added to each well and incubated for at
least 30 mins at room temperature. The wells are washed one time
using the Atlas plate washer with PBS with Mg.sup.2+, Ca.sup.2+
Twenty .mu.l/well of 2 .mu.g/ml Alexa Fluor.RTM. 647 anti-Human IgG
H+L (Invitrogen.TM.) and 20 .mu.g/ml Hoechst 33342 (Invitrogen.TM.)
was added and incubated in the dark for 45 mins at room
temperature. Wells were washed three times using the Atlas plate
washer with PBS with Mg.sup.2+, Ca.sup.2+. Twenty microliters of
PBS with Mg.sup.2+, Ca.sup.2+, and 1% BSA was added to each well,
and plates were sealed with black sealing tape. Plates were
analyzed by scanning using an IN Cell analyzer. Specifically, the
scan was performed using the IN Cell Developer Software, Protocol
"384 GG Hoechst AF647 4x." Analysis of the scan was performed using
the Developer Tool Box software, Protocol "Cellular Binding Nuclei
GG Density 4."
[1547] The assay was able to detect well supernatants that
individually neutralized the Influenza A infection. If an arbitrary
cutoff was established at .ltoreq.150 nucleoprotein (NP)+ cells and
the wells with a disrupted cell monolayer were subtracted, a total
of 122 wells scored as positive.
[1548] Exemplary influenza neutralizing antibodies that were
identified using this method are TCN-522 (3212_I12), TCN-521
(3280_D18), TCN-523 (5248_A17), TCN-563 (5237_B21), TCN-526
(5084_C17), TCN-527 (5086_C06), TCN-528 (5087_P17), TCN-529
(5297_H01), TCN-530 (5248_H10a), TCN-531 (5091_H13), TCN-532
(5262_H18), TCN-533 (5256_A17), TCN-534 (5249_B02), TCN-535
(5246_P19), TCN-536 (5095_N01), TCN-537 (3194_D21), TCN-538
(3206_O17), TCN-539 (5056_A08), TCN-540 (5060_F05), TCN-541
(5062_M11), TCN-542 (5079_A16), TCN-543 (5081_G23), TCN-544
(5082_A19), TCN-545 (5082_I15), TCN-546 (5089_L08), TCN-547
(5092_F11), TCN-548 (5092_P01), TCN-549 (5092_P04), TCN-550
(5096_F06), TCN-551 (5243_D01), TCN-552 (5249_I23), TCN-553
(5261_C18), TCN-554 (5277_M05), TCN-555 (5246_L16), TCN-556
(5089_K12), TCN-557 (5081_A04), TCN-558 (5248_H10b), TCN-559
(5097_G08), TCN-560 (5084_P10), and TCN-504 (3251_K17). The
individual neutralization activities of some of these antibodies
are provided in Table 17.
[1549] Several antibodies were identified that may be
non-neutralizing, including, TCN-504 (3251_K17), TCN-556
(5089_K12), TCN-557 (5081_A04), TCN-559 (5097_G08), and TCN-560
(5084_P10). These antibodies, similar to the neutralizing
antibodies of the invention, bind to a broad range of HA proteins,
including sequence and conformational variants. In certain
embodiments of the invention, non-neutralizing antibodies,
including TCN-504 (3251_K17), TCN-556 (5089_K12), TCN-557
(5081_A04), TCN-559 (5097_G08), and TCN-560 (5084_P10) may be used
as antibody-drug conjugates.
TABLE-US-00819 TABLE 12 Summary of BCC SN screening by ELISA for
virus binding. ELISA: Virus Binding (OD.sub.450) A/Solomon A/Japan/
A/Wisconsin/ Theraclone BCC well Islands/ 305/57 67/05 mAb ID ID
3/06 H1N1 H2N2 H3N2 TCN-521 3280_D18 3.70 3.10 1.10 TCN-522
3212_I12 2.12 ND 0.07 TCN-523 5248_A17 3.47 1.62 0.08 TCN-563
5237_B21 3.62 1.23 0.07 TCN-526 5084_C17 0.06 0.07 3.65 TCN-527
5086_C06 3.03 1.48 0.07 TCN-528 5087_P17 3.62 2.82 0.24 TCN-529
5297_H01 0.07 ND 3.62 TCN-530 5248_H10 3.52 1.73 0.06 TCN-531
5091_H13 3.23 0.67 3.45 TCN-532 5262_H18 0.06 0.07 3.67 TCN-533
5256_A17 3.54 1.10 0.10 TCN-534 5249_B02 3.55 2.56 0.07 TCN-535
5246_P19 3.43 1.46 0.08 TCN-536 5095_N01 3.63 0.08 3.66 TCN-537
3194_D21 3.24 ND 0.06 TCN-538 3206_O17 3.47 ND 0.07 TCN-539
5056_A08 0.06 0.06 2.85 TCN-540 5060_F05 0.07 3.62 3.65 TCN-541
5062_M11 3.44 0.06 0.25 TCN-542 5079_A16 3.66 0.08 3.13 TCN-543
5081_G23 3.63 3.62 0.07 TCN-544 5082_A19 0.32 0.07 2.71 TCN-545
5082_I15 3.32 0.06 0.47 TCN-546 5089_L08 1.95 0.06 3.69 TCN-547
5092_F11 0.06 0.07 3.68 TCN-548 5092_P01 0.09 0.09 3.62 TCN-549
5092_P04 0.09 0.08 3.58 TCN-550 5096_F06 0.06 0.06 3.65 TCN-551
5243_D01 3.35 0.19 0.07 TCN-552 5249_I23 3.57 0.71 0.06 TCN-553
5261_C18 3.60 2.54 0.07 TCN-554 5277_M05 0.06 ND 1.09 TCN-555
5246_L16 2.89 0.60 0.06 TCN-556 5089_K12 2.70 2.41 0.06 TCN-557
5081_A04 2.32 2.70 0.07 TCN-559 5097_G08 3.68 1.25 0.70 TCN-560
5084_P10 3.63 2.07 0.07 TCN-202 2N9 ND ND ND TCN-504 3251_K17 ND ND
ND TCN-032 8I10 3.61 3.61 3.62
TABLE-US-00820 TABLE 13 Summary of BCC SN screening for virus
binding to recombinant homotrimeric HA. Trimeric HA Binding: RLU
A/swine/ A/Egypt/ A/Cali- A/Solomon A/South A/Japan/ A/Wiscon-
Ontario/ A/Vietnam/ A/Indo- 3300- Theraclone BCC fornia/ Islands/
Carolina/ 305/57 sin/67/ 01911-2/ 1203/04 nesia/ NAMRU3/ mAb ID
well ID 4/09 H1N1 3/06 H1N1 1/18 H1N1 H2N2 05 H3N2 99 H4N6 H5N1
5/05 H5N1 08 H5N1 TCN-521 3280_D18 25726 39644 ND 6732 2298 210 196
31473 47871 TCN-522 3212_I12 5019 38914 ND 989 302 156 145 67 ND
TCN-523 5248_A17 34721 37916 ND 5111 22568 1288 3383 45815 36471
TCN-563 5237_B21 45157 30005 ND 6612 40848 4501 4836 39533 27514
TCN-526 5084_C17 203 341 ND 246 49312 253 372 46027 ND TCN-527
5086_C06 27991 16507 ND 19261 5715 7686 21264 23838 ND TCN-528
5087_P17 48845 44804 ND 46393 48795 47500 45577 42801 ND TCN-529
5297_H01 434 179 ND 435 51631 235 101 44085 53394 TCN-530 5248_H10
46600 32801 ND 47049 36846 2743 7152 39774 30430 TCN-531 5091_H13
22207 51663 ND 410 7094 408 357 37443 ND TCN-532 5262_H18 135 327
ND 176 18046 119 440 27992 24500 TCN-533 5256_A17 29280 39186 ND
10806 13823 1582 5677 44063 34299 TCN-534 5249_B02 30109 44185 ND
50626 7978 683 3435 46352 41381 TCN-535 5246_P19 48576 39442 ND
26068 34320 5950 4740 45592 39412 TCN-536 5095_N01 151 150 ND 121
34996 79 146 3969 ND TCN-537 3194_D21 21918 44264 ND 44685 549 19
80 14858 38168 TCN-538 3206_O17 13808 33228 ND 43002 6900 406 60
26421 23553 TCN-539 5056_A08 1803 239 ND 468 715 1579 545 49 11420
TCN-540 5060_F05 57 64 ND 2969 728 178 63 31 1968 TCN-541 5062_M11
34 51 ND 83 2923 102 43 30 1162 TCN-542 5079_A16 3108 132 ND 142
1940 350 306 ND ND TCN-543 5081_G23 13080 511 ND 197 1358 428 488
ND ND TCN-544 5082_A19 281 179 ND 187 2090 316 349 3052 ND TCN-545
5082_I15 365 266 ND 157 575 289 235 ND ND TCN-546 5089_L08 350 256
ND 520 30209 587 349 18432 ND TCN-547 5092_F11 170 155 ND 40 16932
252 308 6986 ND TCN-548 5092_P01 234 283 ND 416 48600 240 270 17626
ND TCN-549 5092_P04 338 284 ND 387 30912 421 312 11445 ND TCN-550
5096_F06 454 204 ND 201 26315 195 277 9100 ND TCN-551 5243_D01
53362 53821 ND 22633 6840 733 4152 53543 47183 TCN-552 5249_I23
35312 39314 ND 23832 14769 493 2728 43559 39971 TCN-553 5261_C18
20281 16271 ND 25509 20043 2583 6560 24406 18828 TCN-554 5277_M05
173 115 ND 328 46531 78 113 32348 36126 TCN-555 5246_L16 44903
26404 ND 2131 9800 1953 876 34539 42676 TCN-556 5089_K12 14640 2846
ND 5611 9323 5604 13823 8454 ND TCN-557 5081_A04 17603 40699 ND
43367 10218 26967 47282 45165 ND TCN-559 5097_G08 539 7376 ND 16332
2402 1658 21670 18295 ND TCN-560 5084_P10 49166 38758 ND 46720
49078 41599 43990 35864 ND TCN-202 2N9 39 46 ND 85 252 75 53 24
1275 TCN-504 3251_K17 3326 24140 ND 4091 40516 14669 196 43259
44352 TCN-032 8I10 ND ND ND ND ND ND ND ND ND Trimeric HA Binding:
RLU A/common A/chicken/ A/northern magpie/Hong A/Anhui/ Vietnam/
A/Hong shoveler/ A/Nether- A/duck/ A/Hong A/Hong Theraclone
Kong/5052/ 1/05 NCVD-016/ Kong/156/ California/ lands/219/
Yangzhou/ Kong/2108/ Kong/1073/ mAb ID 07 H5N1 H5N1 08 H5N1 97 H5N1
HKWF115/07 H6N4 03 H7N7 02/05 H8N4 03 H9N2 99 H9N2 TCN-521 13528
487 32390 ND 47991 2496 11659 93 ND TCN-522 ND ND ND ND ND ND 502
639 ND TCN-523 45837 5705 48880 ND 46908 2336 28949 189 ND TCN-563
27500 9581 40953 ND 27328 7515 46729 225 ND TCN-526 ND 20916 179 ND
2100 1338 596 36629 ND TCN-527 ND 21369 27047 ND 30323 8892 28803
15956 ND TCN-528 ND 49772 38191 ND 46781 49251 38362 41966 ND
TCN-529 302 39376 410 ND 533 1165 771 42271 ND TCN-530 42460 11874
48341 ND 45339 2896 41661 486 ND TCN-531 ND 3606 20840 ND 43874
1969 3117 745 ND TCN-532 134 25219 46 ND 511 327 785 29816 ND
TCN-533 44511 8065 47704 ND 41449 2473 24745 172 ND TCN-534 32514
6861 56642 ND 52429 4340 46537 165 ND TCN-535 45937 6875 49961 ND
48192 4757 36650 861 ND TCN-536 ND 543 287 ND 2210 976 436 1566 ND
TCN-537 18702 335 8617 ND 5206 1052 7826 147 ND TCN-538 18746 689
37312 ND 17448 6531 6123 109 ND TCN-539 3957 91 100 ND 814 1698
1488 292 ND TCN-540 113 25 70 ND 131 381 117 22 ND TCN-541 71 17 40
ND 106 381 131 29 ND TCN-542 ND ND 375 ND 900 909 332 1907 ND
TCN-543 ND 325 347 ND 2128 661 737 131 ND TCN-544 ND ND 272 ND 1690
1618 464 296 ND TCN-545 ND ND 392 ND 1167 1182 656 227 ND TCN-546
ND 2037 421 ND 1354 1451 487 12983 ND TCN-547 ND 1476 198 ND 1005
401 278 3986 ND TCN-548 ND 5381 254 ND 314 758 184 3424 ND TCN-549
ND 1793 498 ND 1103 1134 844 5037 ND TCN-550 ND 1206 361 ND 964 939
407 2279 ND TCN-551 45688 4187 42301 ND 52491 786 9817 224 ND
TCN-552 42201 4156 46249 ND 38413 1241 13115 171 ND TCN-553 25979
12610 24158 ND 21732 1838 27998 273 ND TCN-554 224 10851 250 ND 296
610 583 28594 ND TCN-555 16060 1497 22302 ND 49507 3399 18390 262
ND TCN-556 ND 3703 8800 ND 11890 10606 16910 7413 ND TCN-557 ND
46033 45545 ND 24375 6779 50106 35309 ND TCN-559 ND 11888 12935 ND
7233 1983 10385 2791 ND TCN-560 ND 49133 45969 ND 42303 48519 44696
49385 ND TCN-202 162 25 69 ND 105 348 196 50 ND TCN-504 17038 26175
38456 ND 7869 47190 14137 454 ND TCN-032 ND ND ND ND ND ND ND ND
ND
TABLE-US-00821 TABLE 14 Summary of BCC SN screening for virus
neutralization % Neutralization A/Solomon Theraclone BCC
Islands/3/06 A/Wisconsin/ mAb ID well ID H1N1 67/05 H3N2 TCN-522
3212_I12 33 0 TCN-521 3280_D18 99 ND TCN-523 5248_A17 69 21 TCN-563
5237_B21 78 1 TCN-526 5084_C17 0 100 TCN-527 5086_C06 12 0 TCN-528
5087_P17 0 15 TCN-529 5297_H01 0 99 TCN-530 5248_H10 64 4 TCN-531
5091_H13 65 80 TCN-532 5262_H18 11 99 TCN-533 5256_A17 56 0 TCN-534
5249_B02 86 0 TCN-535 5246_P19 82 1 TCN-536 5095_N01 75 100 TCN-537
3194_D21 89 5 TCN-538 3206_O17 35 0 TCN-539 5056_A08 97 62 TCN-540
5060_F05 100 75 TCN-541 5062_M11 89 99 TCN-542 5079_A16 98 51
TCN-543 5081_G23 100 55 TCN-544 5082_A19 95 62 TCN-545 5082_I15 98
72 TCN-546 5089_L08 0 100 TCN-547 5092_F11 0 100 TCN-548 5092_P01 0
97 TCN-549 5092_P04 0 99 TCN-550 5096_F06 0 100 TCN-551 5243_D01
100 19 TCN-552 5249_I23 57 0 TCN-553 5261_C18 87 0 TCN-554 5277_M05
42 100 TCN-555 5246_L16 48 ND TCN-556 5089_K12 0 4 TCN-557 5081_A04
0 2 TCN-559 5097_G08 82 ND TCN-560 5084_P10 94 ND TCN-202 2N9 ND ND
TCN-504 3251_K17 ND ND TCN-032 8I10 ND ND
TABLE-US-00822 TABLE 15 Summary of monoclonal antibody transfection
supernatant screening by ELISA for virus binding. ELISA: Virus
Binding (OD.sub.450) A/Solomon A/ Theraclone Islands/3/06
A/Japan/305/57 Wisconsin/67/05 mAb ID BCC well ID H1N1 H2N2 H3N2
Monoclonal TCN-523 5248_A17 3.59 1.69 0.09 transfection set 1
TCN-504 3251_K17 3.65 3.65 3.65 TCN-202 2N9 0.07 0.07 0.07
Monoclonal TCN-522 3212_I12 3.48 0.61 0.07 transfection set 2
TCN-526 5084_C17 0.08 0.07 0.31 TCN-527 5086_C06 3.69 3.63 0.14
TCN-528 5087_P17 3.66 3.64 0.23 TCN-563 5237_B21 3.60 0.84 0.09
TCN-504 3251_K17 3.65 3.62 3.65 TCN-523 5248_A17 3.67 1.39 0.09
TCN-202 2N9 0.08 0.07 0.07 Monoclonal TCN-531 5091_H13 0.10 0.09
3.59 transfection set 3 TCN-530 5248_H10 3.62 3.43 0.21 TCN-529
5297_H01 0.16 0.10 3.65 TCN-533 5256_A17 3.61 3.65 0.22 TCN-532
5262_H18 0.13 0.08 3.43 TCN-504 3251_K17 3.63 3.64 3.65 TCN-523
5248_A17 3.59 3.47 0.15 TCN-202 2N9 0.10 0.08 0.08 Monoclonal
TCN-535 5246_P19 3.52 2.45 0.10 transfection set 4 TCN-534 5249_B02
3.50 2.45 0.08 TCN-536 5095_N01 3.52 0.06 3.61 TCN-504 3251_K17
3.52 3.51 3.59 TCN-523 5248_A17 3.43 1.73 0.09 TCN-202 2N9 0.10
0.08 0.07 Monoclonal TCN-537 3194_D21 3.56 3.48 0.11 transfection
set 5 TCN-538 3206_O17 3.59 3.37 0.12 TCN-539 5056_A08 0.07 0.07
0.13 TCN-540 5060_F05 0.09 3.61 3.62 TCN-541 5062_M11 3.63 0.07
0.07 TCN-542 5079_A16 3.60 0.07 0.23 TCN-543 5081_G23 3.63 3.64
2.23 TCN-544 5082_A19 0.07 0.07 3.65 TCN-545 5082_I15 1.36 0.10
3.62 TCN-546 5089_L08 3.67 0.07 3.67 TCN-547 5092_F11 0.09 0.09
0.13 TCN-548 5092_P01 0.13 0.09 3.63 TCN-549 5092_P04 0.09 0.08
0.46 TCN-550 5096_F06 0.13 0.08 3.62 TCN-551 5243_D01 3.65 0.10
0.27 TCN-552 5249_I23 3.61 2.68 0.27 TCN-553 5261_C18 3.56 3.28
0.17 TCN-554 5277_M05 0.18 0.11 3.40 TCN-555 5246_L16 3.57 2.10
0.13 TCN-556 5089_K12 3.64 3.64 0.34 TCN-557 5081_A04 3.58 3.59
3.47 TCN-558 5248_H10 3.52 3.31 0.20 TCN-559 5097_G08 1.16 2.29
2.39 TCN-560 5084_P10 3.52 3.55 0.30 TCN-504 3251_K17 3.60 3.59
3.60 TCN-523 5248_A17 3.62 2.56 0.14 TCN-202 2N9 0.07 0.07 0.07
TABLE-US-00823 TABLE 16 Summary of monoclonal antibody transfection
supernatant screening for virus binding to recombinant homotrimeric
HA. Trimeric HA Binding: RLU A/Cali- A/Solomon A/South A/Wis-
A/swine/ A/Indo- A/Egypt/ fornia/ Islands/ Carolina/ A/Japan/
consin/ Ontario/ A/Vietnam/ nesia/ 3300- Theraclone BCC 4/09 3/06
1/18 305/57 67/05 01911-2/ 1203/04 5/05 NAMRU3/ mAb ID well ID H1N1
H1N1 H1N1 H2N2 H3N2 99 H4N6 H5N1 H5N1 08 H5N1 Monoclonal TCN-523
5248_A17 17703 6785 26364 191 1646 49 80 18508 25494 transfection
TCN-504 3251_K17 18286 29844 21541 15059 14311 26491 1300 16640
16113 set 1 TCN-202 2N9 20 67 32 27 27 29 37 57 76 Monoclonal
TCN-522 3212_I12 37463 17281 39613 11936 3346 1048 48169 50472
55413 transfection TCN-526 5084_C17 3598 1468 2312 3592 27776 3487
9181 37831 39894 set 2 TCN-527 5086_C06 21498 19685 48062 20758
35796 30085 20600 23433 23595 TCN-528 5087_P17 44586 26908 25226
23083 46012 49173 23336 39704 36834 TCN-563 5237_B21 21231 26475
17808 11198 23686 2248 31549 47277 23915 TCN-504 3251_K17 15621
31447 24695 22573 21615 32496 14953 19717 21365 TCN-523 5248_A17
18654 3270 25504 882 2062 82 26227 24527 30672 TCN-202 2N9 52 16
195 32 46 15 121 8 18 Monoclonal TCN-531 5091_H13 2830 194 1557 489
4159 296 2411 54 363 transfection TCN-530 5248_H10 21176 13820
17166 5174 3048 123 25172 25546 25523 set 3 TCN-529 5297_H01 735 43
158 45 2939 45 415 1353 1673 TCN-533 5256_A17 6877 15526 16417
18618 16132 180 11677 28339 18587 TCN-532 5262_H18 23601 59 311 441
9967 29 452 2786 2821 TCN-504 3251_K17 18614 32672 27617 35957
21437 34456 13284 18559 17969 TCN-523 5248_A17 19366 11014 23710
4554 8558 1640 22129 21973 29080 TCN-202 2N9 205 57 731 42 51 54
967 43 63 Monoclonal TCN-535 5246_P19 25157 836 22433 21491 4450
138 25244 18202 25318 transfection TCN-534 5249_B02 27035 60 11830
14745 897 264 23675 948 8314 set 4 TCN-536 5095_N01 49 30 39 260
20576 168 182 80 97 TCN-504 3251_K17 549 14625 21713 19576 20627
21792 25665 18154 24796 TCN-523 5248_A17 28847 371 22983 3845 9742
850 30898 24534 33351 TCN-202 2N9 94 34 27 334 125 157 67 19 40
Monoclonal TCN-537 3194_D21 33793 760 46788 16197 1935 3282 50280
2420 33495 transfection TCN-538 3206_O17 15956 1965 8434 6624 2070
3354 52375 13748 40969 set 5 TCN-539 5056_A08 1053 871 5330 2565
58831 6511 53254 8947 24338 TCN-540 5060_F05 984 1176 1356 47894
52986 5419 42500 2569 15589 TCN-541 5062_M11 1557 2277 2583 5382
1238 4724 45517 2959 23234 TCN-542 5079_A16 667 48021 1116 5184 711
14340 29492 1761 14763 TCN-543 5081_G23 2338 43523 28290 2775 37048
2866 33145 1826 17583 TCN-544 5082_A19 706 997 1331 1003 38514 2391
38620 4400 19414 TCN-545 5082_I15 818 951 900 1355 32037 2936 42906
5269 26538 TCN-546 5089_L08 1085 1140 955 1730 28453 4609 47108
40866 53039 TCN-547 5092_F11 1474 1082 2016 1354 6968 1602 27666
12041 16784 TCN-548 5092_P01 818 750 1154 1016 34194 1624 23678
29142 37002 TCN-549 5092_P04 622 640 2340 1075 51626 1878 32265
8645 18581 TCN-550 5096_F06 628 758 1014 731 37809 1704 41900 9001
26791 TCN-551 5243_D01 46133 43523 4344 25191 936 3120 41915 1289
17368 TCN-552 5249_I23 23459 48559 37374 51986 18451 9084 18494
43382 21246 TCN-553 5261_C18 23323 49581 41300 39848 25248 12230
19214 31183 19470 TCN-554 5277_M05 4034 992 1261 2621 25329 5402
36582 44935 42395 TCN-555 5246_L16 20223 44571 48009 16743 14660
4522 19300 33927 20001 TCN-556 5089_K12 20865 23409 32732 22805
44715 19346 17421 15229 16603 TCN-557 5081_A04 44419 26960 44865
27282 46082 41525 21320 22246 26351 TCN-558 5248_H10 20220 36107
40709 34434 26611 10003 23527 24632 21861 TCN-559 5097_C08 8079
41254 2233 46993 15279 35761 37607 51869 47369 TCN-560 5084_P10
31134 22427 30462 25855 55383 16751 19475 16971 18730 TCN-504
3251_K17 17599 30086 31975 23254 38088 54789 21485 18485 20625
TCN-523 5248_A17 34415 37587 52010 22019 13690 1699 27819 55048
35484 TCN-202 2N9 733 525 5331 1883 581 2692 30002 1973 13876
Trimeric HA Binding: RLU A/common A/chicken/ A/northern magpie/Hong
Vietnam/ A/Hong shoveler/ A/Nether- A/duck/ A/Hong A/Hong Kong/
NCVD- Kong/ California/ lands/ Yangzhou/ Kong/ Kong/ Theraclone
5052/07 A/Anhui/ 016/08 156/97 HKWF115/ 219/03 02/05 2108/03
1073/99 mAb ID H5N1 1/05 H5N1 H5N1 H5N1 07 H6N4 H7N7 H8N4 H9N2 H9N2
Monoclonal TCN-523 6235 60 10813 ND 9046 28 229 41 21719
transfection TCN-504 29696 29046 27765 ND 5791 23828 28934 78 25703
set 1 TCN-202 21 20 14 ND 13 55 26 24 21 Monoclonal TCN-522 21393
2031 49343 ND 54364 2170 1646 203 11800 transfection TCN-526 1758
43991 3294 ND 3789 5659 4305 23267 490 set 2 TCN-527 43794 15238
20112 ND 19272 49149 17921 25527 557 TCN-528 45257 21207 22573 ND
28370 47229 19792 33621 322 TCN-563 52783 1568 36304 ND 21901 5423
6275 364 49230 TCN-504 16850 30975 27266 ND 1277 19272 17751 23
25832 TCN-523 7214 139 17590 ND 21214 407 383 18 31283 TCN-202 11 9
22 ND 30 26 19 7 33 Monoclonal TCN-531 37 76 44 ND 96 384 3220 158
370 transfection TCN-530 5576 97 17979 ND 23513 118 227 44 10219
set 3 TCN-529 33 1258 33 ND 110 33 48 1197 46 TCN-533 25210 301
24551 ND 7806 209 711 33 23808 TCN-532 20 2402 29 ND 78 42 57 2767
48 TCN-504 17814 29988 10261 ND 8335 19600 19647 36 27781 TCN-523
640 546 18903 ND 29404 3144 2017 51 24193 TCN-202 16 33 14 ND 73
247 66 30 57 Monoclonal TCN-535 477 24 20929 ND 18057 159 428 18
21197 transfection TCN-534 127 20 11352 ND 21287 361 79 27 731 set
4 TCN-536 27 18 19 ND 95 246 48 24 115 TCN-504 8675 14053 19176 ND
2609 6707 19872 7 18770 TCN-523 2131 48 23995 ND 25087 443 463 38
24575 TCN-202 21 13 42 ND 117 173 77 22 81 Monoclonal TCN-537 2068
2580 4476 10681 2376 3654 25529 transfection TCN-538 3141 2745 2948
5167 8895 4374 4065 set 5 TCN-539 1436 3244 3496 1450 2862 15729
7281 TCN-540 1799 2192 1514 1551 2461 4386 6078 TCN-541 1936 2998
11039 1975 2684 13649 21203 TCN-542 1674 7407 5458 786 2369 8049
11598 TCN-543 1070 2626 5877 1433 2512 12873 13130 TCN-544 1116
3770 3069 1491 2915 4647 9444 TCN-545 1388 4876 3230 1560 2319 3125
13197 TCN-546 1916 33344 2940 1243 2429 3743 11391 TCN-547 856 1238
1315 1955 2618 2089 3335 TCN-548 813 15578 1059 1555 2104 2131 5735
TCN-549 1340 4113 1601 1848 996 8475 9452 TCN-550 993 7680 1802 955
2305 3248 4713 TCN-551 1123 2643 792 792 1764 4837 3097 TCN-552
34619 9010 34451 36317 43861 7416 15937 TCN-553 39224 8548 34718
36337 32461 7532 12545 TCN-554 1734 42805 2708 1441 3352 4522 5808
TCN-555 33642 12236 35215 36302 28717 12656 8544 TCN-556 20140
13795 22676 24620 34817 43640 22400 TCN-557 54874 19980 23323 27061
49611 53747 24015 TCN-558 49655 9783 33423 47113 29396 5841 19531
TCN-559 12900 41835 33850 28953 6632 4852 42614 TCN-560 30485 18628
18174 29287 35866 32120 27927 TCN-504 37328 37168 34166 26597 39179
30626 35074 TCN-523 33905 6079 51296 28380 46941 6750 25599 TCN-202
1366 10033 2570 945 3036 18026 13796
TABLE-US-00824 TABLE 17 Summary of monoclonal antibody transfection
supernatant screening for virus neutralization. % Neutralization
A/Solo- A/Cali- A/Wis- mon Is- fornia/ consin/ Theraclone BCC
lands/3/ 4/09 67/05 mAb ID well ID 06 H1N1 H1N1 H3N2 Monoclonal
TCN-523 5248_A17 82.71 ND ND transfec- TCN-504 3251_K17 0.00 ND
0.00 tion set 1 TCN-202 2N9 0.00 ND 0.00 Monoclonal TCN-522
3212_I12 39.46 ND 0.84 transfec- TCN-526 5084_C17 10.58 ND 94.26
tion set 2 TCN-527 5086_C06 17.55 ND 0.00 TCN-528 5087_P17 23.92 ND
0.00 TCN-563 5237_B21 88.85 ND 0.00 TCN-504 3251_K17 7.19 ND 0.00
TCN-523 5248_A17 80.68 ND 0.00 TCN-202 2N9 7.00 ND 0.00 Monoclonal
TCN-531 5091_H13 0.00 ND 97.30 transfec- TCN-530 5248_H10 96.95 ND
34.54 tion set 3 TCN-529 5297_H01 0.00 ND 99.59 TCN-533 5256_A17
96.12 ND 32.51 TCN-532 5262_H18 0.00 ND 99.73 TCN-504 3251_K17 0.00
ND 10.74 TCN-523 5248_A17 62.19 ND 34.00 TCN-202 2N9 0.00 ND 17.77
Monoclonal TCN-535 5246_P19 88.60 97.28 0.00 transfec- TCN-534
5249_B02 65.66 96.37 0.00 tion set 4 TCN-536 5095_N01 81.63 18.76
99.88 TCN-504 3251_K17 29.68 0.00 0.00 TCN-523 5248_A17 65.54 93.19
0.00 TCN-202 2N9 ND ND ND Monoclonal TCN-537 3194_D21 0 71 0
transfec- TCN-538 3206_O17 21 66 0 tion set 5 TCN-539 5056_A08 0 0
99 TCN-540 5060_F05 3 0 98 TCN-541 5062_M11 88 0 4 TCN-542 5079_A16
99 15 8 TCN-543 5081_G23 98 0 73 TCN-544 5082_A19 0 0 97 TCN-545
5082_I15 88 0 99 TCN-546 5089_L08 98 0 100 TCN-547 5092_F11 0 0 88
TCN-548 5092_P01 0 0 96 TCN-549 5092_P04 0 0 100 TCN-550 5096_F06 0
0 100 TCN-551 5243_D01 99 74 6 TCN-552 5249_I23 0 81 14 TCN-553
5261_C18 44 83 5 TCN-554 5277_M05 0 0 100 TCN-555 5246_L16 49 89 5
TCN-556 5089_K12 0 0 15 TCN-557 5081_A04 7 10 0 TCN-558 5248_H10 84
96 7 TCN-559 5097_G08 7 10 12 TCN-560 5084_P10 0 0 4 TCN-504
3251_K17 0 0 0 TCN-523 5248_A17 39 86 7 TCN-202 2N9 0 0 0
Other Embodiments
[1550] Although specific embodiments of the invention have been
described herein for purposes of illustration, various
modifications may be made without deviating from the spirit and
scope of the invention. Accordingly, the invention is not limited
except as by the appended claims.
[1551] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[1552] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. Genbank and NCBI
submissions indicated by accession number cited herein are hereby
incorporated by reference. All other published references,
documents, manuscripts and scientific literature cited herein are
hereby incorporated by reference.
[1553] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150104459A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150104459A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References