Patent application title: ANTI-CD47 ANTIBODIES
Inventors:
John Lippincott (San Diego, CA, US)
Dana Duey (San Diego, CA, US)
Larry Green (San Diego, CA, US)
IPC8 Class: AC07K1628FI
USPC Class:
Class name:
Publication date: 2022-08-25
Patent application number: 20220267440
Abstract:
The present invention provides anti-CD47 monoclonal antibodies and
related compositions, which may be used in any of a variety of
therapeutic and diagnostic methods for the treatment of cancer,
ischemic-reperfusion injury, and other diseases.Claims:
1-15. (canceled)
16. A method of treating or preventing a disease or disorder associated with CD47 and/or SIRP.alpha. or aberrant expression thereof in a human subject in need thereof, the method comprising administrating to said subject a therapeutically effective amount of an anti-CD47 antibody, or an antigen-binding fragment thereof, comprising a) a heavy chain variable region comprising a VHCDR1, a VHCDR2, and a VHCDR3 and b) a light chain variable region comprising a VLCDR1, a VLCDR2, and a VLCDR3, wherein: i) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 43, VHCDR3 comprises the amino acid sequence of SEQ ID NO: 64 the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 106 the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 127, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 148; ii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 23, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 44, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 65, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 107, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 128, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 149; iii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 24, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 45, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 66, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 108, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 129, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 150; iv) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 25, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 46, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 67, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 109, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 130, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 151; v) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 26, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 47, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 68, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 110, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 131, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 152; vi) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 27, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 48, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 69, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 111, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 132, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 153; vii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 28, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 49, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 70, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 112, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 133, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 154; viii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 50, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 71, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 113, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 134, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 155; ix) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 30, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 51, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 72, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 114, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 135, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 156; x) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 31, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 52, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 73, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 115, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 136, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 157; xi) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 32, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 53, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 74, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 116, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 137, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 158; xii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 33, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 54, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 75, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 117, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 138, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 159; xiii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 34, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 55, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 76, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 118, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 139, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 160; xiv) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 35, the VHCDR2 comprising the amino acid sequence of SEQ ID NO: 56, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 77, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 119, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 140, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 161; xv) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 36, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 57, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 78, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 120, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 141, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 162; xvi) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 37, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 58, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 79, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 121, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 142, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 163; xvii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 38, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 59, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 80, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 122, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 143, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 164; xviii) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 39, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 60, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 81, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 123, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 144, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 165; xix) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 40, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 61, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 82, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 124, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 145, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 166; xx) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 41, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 62, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 83, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 125, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 146, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 167; or xxi) the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 42, the VHCDR2 comprises the amino acid sequence of SEQ ID NO: 63, the VHCDR3 comprises the amino acid sequence of SEQ ID NO: 84, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 126, the VLCDR2 comprises the amino acid sequence of SEQ ID NO: 147, and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 168.
17. The method of claim 16, wherein the disease or disorder is a proliferative disease or disorder.
18. The method of claim 17, wherein the proliferative disease or disorder is a neoplasm, a tumor or a metastasis.
19. The method of claim 17, wherein the proliferative disease or disorder is cancer.
20. The method of claim 16, wherein the disease or disorder is ischemic-reperfusion injury.
21. The method of claim 20, wherein the ischemic-reperfusion injury occurs during a transplant.
22. The method of claim 16, wherein the disease or disorder is an immunological disease.
23. The method of claim 22, wherein the immunological disease is an inflammatory disease.
24. The method of claim 22, wherein the immunological disease is an autoimmune disease.
25. The method of claim 16, wherein the disease or disorder is associated with CD47 overexpression.
26. The method of claim 16, wherein the anti-CD47 antibody is administered alone or in combination with one or more agents.
27. The method of claim 26, wherein the agent is selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, a radioisotope, and combinations thereof.
28. The method of claim 18, wherein the anti-CD47 antibody induces phagocytosis of tumor cells by macrophages.
29. The method of claim 16, wherein the anti-CD47 antibody induces little to zero hemagglutination of human erythrocytes.
30. The method of claim 29, wherein the anti-CD47 antibody induces zero hemagglutination of human erythrocytes.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Application No. 62/536,267, filed Jul. 24, 2017 and U.S. Provisional Application No. 62/419,158, filed Nov. 8, 2016, each of which is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is ABLX_008_02WO_ST25.txt. The text file is 124 KB, was created on Nov. 8, 2017, and is being submitted electronically via EFS-Web.
BACKGROUND
Technical Field
[0003] The present invention relates generally to anti-CD47 antibodies, compositions and methods of using same. Such antibodies are useful, for example, for treating a variety of diseases, such as oncological and immunological diseases.
Description of the Related Art
[0004] CD47 is a five-pass integral membrane protein with a large extracellular domain of 123 amino acids. The biology of CD47 is reviewed in Murata et al., J. Biochem (2014) 155: 335-344. CD47 is a tumor antigen that prevents phagocytosis by SIRP .alpha. expressing macrophages. CD47/SIRP .alpha. blocking antibodies can induce phagocytosis of tumor cells by macrophages.
[0005] Anti-CD47 antibodies that induce phagocytosis may have utility in the treatment of cancer and inflammatory and autoimmune diseases. In particular, CD47 mAbs may be useful for treating cancer as a monotherapy or in combination therapy. Macrophages that have been induced to phagocytose cancer cells by anti-CD47 monoclonal antibodies (mAbs) further stimulate an anti-cancer response by displaying tumor antigens in the MHC to stimulate a secondary immune response against the cancer cells. The importance of macrophages in cancer therapy is reviewed in Weiskopf and Weissman, MAbs (2015) 7: 303-310. CD47 is expressed on cells in normal human tissue in humans. In particular, it is expressed on human erythrocytes (red blood cells, RBCs). Some anti-CD47 antibodies can induce hemagglutination. There is a need for therapeutic candidate CD47 mAbs that exhibit both induction of macrophage phagocytosis of cancer cells and have very low to no hemagglutination at high concentrations.
SUMMARY OF THE INVENTION
[0006] The present invention relates to anti-CD47 antibodies. More specifically, it relates to chimeric anti-CD47 antibodies generated from an AlivaMab Mouse, fully human anti-CD47 antibodies produced therefrom, and methods of use thereof.
[0007] One aspect of the invention provides an isolated anti-CD47 antibody, or an antigen-binding fragment thereof, comprising i) a heavy chain variable region comprising a VHCDR1 disclosed herein, a VHCDR2 disclosed herein, and a VHCDR3 disclosed herein and ii) a light chain variable region comprising a VLCDR1 disclosed herein, a VLCDR2 disclosed herein, and a VLCDR3 disclosed herein.
[0008] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:22, the VHCDR2 of SEQ ID NO:43, the VHCDR3 of SEQ ID NO:64, the VLCDR1 of SEQ ID NO:106, the VLCDR2 of SEQ ID NO:127, and the VLCDR3 of SEQ ID NO:148.
[0009] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:23, the VHCDR2 of SEQ ID NO:44, the VHCDR3 of SEQ ID NO:65, the VLCDR1 of SEQ ID NO:107, the VLCDR2 of SEQ ID NO:128, and the VLCDR3 of SEQ ID NO:149.
[0010] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:24, the VHCDR2 of SEQ ID NO:45, the VHCDR3 of SEQ ID NO:66, the VLCDR1 of SEQ ID NO:108, the VLCDR2 of SEQ ID NO:129, and the VLCDR3 of SEQ ID NO:150.
[0011] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:25, the VHCDR2 of SEQ ID NO:46, the VHCDR3 of SEQ ID NO:67, the VLCDR1 of SEQ ID NO:109, the VLCDR2 of SEQ ID NO:130, and the VLCDR3 of SEQ ID NO:151.
[0012] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:26, the VHCDR2 of SEQ ID NO:47, the VHCDR3 of SEQ ID NO:68, the VLCDR1 of SEQ ID NO:110, the VLCDR2 of SEQ ID NO:131, and the VLCDR3 of SEQ ID NO:152.
[0013] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:27, the VHCDR2 of SEQ ID NO:48, the VHCDR3 of SEQ ID NO:69, the VLCDR1 of SEQ ID NO:111, the VLCDR2 of SEQ ID NO:132, and the VLCDR3 of SEQ ID NO:153.
[0014] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:28, the VHCDR2 of SEQ ID NO:49, the VHCDR3 of SEQ ID NO:70, the VLCDR1 of SEQ ID NO:112, the VLCDR2 of SEQ ID NO:133, and the VLCDR3 of SEQ ID NO:154.
[0015] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:29, the VHCDR2 of SEQ ID NO:50, the VHCDR3 of SEQ ID NO:71, the VLCDR1 of SEQ ID NO:113, the VLCDR2 of SEQ ID NO:134, and the VLCDR3 of SEQ ID NO:155.
[0016] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:30, the VHCDR2 of SEQ ID NO:51, the VHCDR3 of SEQ ID NO:72, the VLCDR1 of SEQ ID NO:114, the VLCDR2 of SEQ ID NO:135, and the VLCDR3 of SEQ ID NO:156.
[0017] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:31, the VHCDR2 of SEQ ID NO:52, the VHCDR3 of SEQ ID NO:73, the VLCDR1 of SEQ ID NO:115, the VLCDR2 of SEQ ID NO:136, and the VLCDR3 of SEQ ID NO:157.
[0018] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:32, the VHCDR2 of SEQ ID NO:53, the VHCDR3 of SEQ ID NO:74, the VLCDR1 of SEQ ID NO:116, the VLCDR2 of SEQ ID NO:137, and the VLCDR3 of SEQ ID NO:158.
[0019] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:33, the VHCDR2 of SEQ ID NO:54, the VHCDR3 of SEQ ID NO:75, the VLCDR1 of SEQ ID NO:117, the VLCDR2 of SEQ ID NO:138, and the VLCDR3 of SEQ ID NO:159.
[0020] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:34, the VHCDR2 of SEQ ID NO:55, the VHCDR3 of SEQ ID NO:76, the VLCDR1 of SEQ ID NO:118, the VLCDR2 of SEQ ID NO:139, and the VLCDR3 of SEQ ID NO:160.
[0021] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:35, the VHCDR2 of SEQ ID NO:56, the VHCDR3 of SEQ ID NO:77, the VLCDR1 of SEQ ID NO:119, the VLCDR2 of SEQ ID NO:140, and the VLCDR3 of SEQ ID NO:161.
[0022] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:36, the VHCDR2 of SEQ ID NO:57, the VHCDR3 of SEQ ID NO:78, the VLCDR1 of SEQ ID NO:120, the VLCDR2 of SEQ ID NO:141, and the VLCDR3 of SEQ ID NO:162.
[0023] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:37, the VHCDR2 of SEQ ID NO:58, the VHCDR3 of SEQ ID NO:79, the VLCDR1 of SEQ ID NO:121, the VLCDR2 of SEQ ID NO:142, and the VLCDR3 of SEQ ID NO:163.
[0024] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:38, the VHCDR2 of SEQ ID NO:59, the VHCDR3 of SEQ ID NO:80, the VLCDR1 of SEQ ID NO:122, the VLCDR2 of SEQ ID NO:143, and the VLCDR3 of SEQ ID NO:164.
[0025] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:39, the VHCDR2 of SEQ ID NO:60, the VHCDR3 of SEQ ID NO:81, the VLCDR1 of SEQ ID NO:123, the VLCDR2 of SEQ ID NO:144, and the VLCDR3 of SEQ ID NO:165.
[0026] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:40, the VHCDR2 of SEQ ID NO:61, the VHCDR3 of SEQ ID NO:82, the VLCDR1 of SEQ ID NO:124, the VLCDR2 of SEQ ID NO:145, and the VLCDR3 of SEQ ID NO:166.
[0027] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:41, the VHCDR2 of SEQ ID NO:62, the VHCDR3 of SEQ ID NO:83, the VLCDR1 of SEQ ID NO:125, the VLCDR2 of SEQ ID NO:146, and the VLCDR3 of SEQ ID NO:167.
[0028] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, comprises the VHCDR1 of SEQ ID NO:42, the VHCDR2 of SEQ ID NO:63, the VHCDR3 of SEQ ID NO:84, the VLCDR1 of SEQ ID NO:126, the VLCDR2 of SEQ ID NO:147, and the VLCDR3 of SEQ ID NO:168.
[0029] In one embodiment, the VH comprises an amino acid sequence of one of SEQ ID NOs:1-21. In another embodiment, the VL comprises an amino acid sequence of one of SEQ ID NOs:85-105. In another embodiment, the VH comprises an amino acid sequence of one of SEQ ID NOs:1-21, and the VL comprises an amino acid sequence of one of SEQ ID NOs:85-105. In one embodiment, the VH and the VL are from the same anti-CD47 AlivaMab Antibody.
[0030] In one embodiment, the anti-CD47 antibody, or antigen-binding fragment thereof, is human. In one embodiment, the antibody is chimeric. In certain embodiments, the antibody is selected from a single-variable domain antibody, single chain antibody, a scFv, a bispecific antibody, a multi-specific antibody, a Fab, a F(ab')2, and a whole antibody.
[0031] One aspect of the invention provides a recombinant polynucleotide encoding the anti-CD47 antibody, or antigen-binding fragment thereof, described above. Another aspect of the invention provides an expression vector comprising the recombinant polynucleotide. In another aspect of the invention provides an isolated host cell that comprises the expression vector. One aspect of the invention provides a composition comprising an anti-CD47 antibody, or antigen-binding fragment thereof, described herein and a physiologically acceptable carrier.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 shows that 16 of the 61 antibodies tested substantially or completely blocked binding of human CD47 to SIRP .alpha..
[0033] FIG. 2 shows anti-CD47 antibody binding to native human CD47 expressed on the surface of CCRF-CEM human tumor cell line, a T lymphoblastoid cell line (ATCC CCL-119) as detected by flow cytometry.
[0034] FIG. 3 shows anti-CD47 mAbs potently induced phagocytosis of CCRF-CEM human tumor cells by macrophages.
[0035] FIG. 4 shows anti-CD47 mAbs with little to zero induced hemagglutination of human erythrocytes (RBCs).
[0036] FIG. 5 shows the epitope binning of anti-CD47 mAbs.
BRIEF DESCRIPTION OF THE SEQUENCES
TABLE-US-00001
[0037] TABLE 1 Anti-CD47 AlivaMab Antibody Amino Acid Sequences VH VL (V-D-J) VHCD R1 VHCD R2 VHCD R3 (V-J) VLCD R1 VLCD R2 VLCD R3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Clone NO NO NO NO NO NO NO NO 5 1 22 43 64 85 106 127 148 10 2 23 44 65 86 107 128 149 14 3 24 45 66 87 108 129 150 85 4 25 46 67 88 109 130 151 87 5 26 47 68 89 110 131 152 90 6 27 48 69 90 111 132 153 91 7 28 49 70 91 112 133 154 101 8 29 50 71 92 113 134 155 102 9 30 51 72 93 114 135 156 106 10 31 52 73 94 115 136 157 107 11 32 53 74 95 116 137 158 139 12 33 54 75 96 117 138 159 143 13 34 55 76 97 118 139 160 144 14 35 56 77 98 119 140 161 151 15 36 57 78 99 120 141 162 153 16 37 58 79 100 121 142 163 161 17 38 59 80 101 122 143 164 164 18 39 60 81 102 123 144 165 201 19 40 61 82 103 124 145 166 204 20 41 62 83 104 125 146 167 205 21 42 63 84 105 126 147 168
TABLE-US-00002 TABLE 2 Anti-CD47 AlivaMab Antibody Polynucleotide Sequences VH VHCD R1 VHCD R2 VHCD R3 VL VLCD R1 VLCD R2 VLCD R3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Clone NO NO NO NO NO NO NO NO 5 169 190 211 232 253 274 295 316 10 170 191 212 233 254 275 296 317 14 171 192 213 234 255 276 297 318 85 172 193 214 235 256 277 298 319 87 173 194 215 236 257 278 299 320 90 174 195 216 237 258 279 300 321 91 175 196 217 238 259 280 301 322 101 176 197 218 239 260 281 302 323 102 177 198 219 240 261 282 303 324 106 178 199 220 241 262 283 304 325 107 179 200 221 242 263 284 305 326 139 180 201 222 243 264 285 306 327 143 181 202 223 244 265 286 307 328 144 182 203 224 245 266 287 308 329 151 183 204 225 246 267 288 309 330 153 184 205 226 247 268 289 310 331 161 185 206 227 248 269 290 311 332 164 186 207 228 249 270 291 312 333 201 187 208 229 250 271 292 313 334 204 188 209 230 251 272 293 314 335 205 189 210 231 252 273 294 315 336
[0038] SEQ ID NO:337 is a 3' IgG-specific primer.
[0039] SEQ ID NO:338 is a 3' Ig.kappa.-specific primer.
DETAILED DESCRIPTION
[0040] The present disclosure relates to anti-CD47 antibodies. Ablexis has used its proprietary AlivaMab Mouse technology (See WO 2010/039900 and WO 2011/123708, incorporated herein in their entirety) to generate panels of monoclonal antibodies (mAbs) against human CD47. Antibodies that potently neutralize SIRP.alpha., binding to CD47 were identified within the panel of anti-CD47 AlivaMab antibodies. In one embodiment, anti-CD47 AlivaMab antibodies potently induce phagocytosis of tumor cells by macrophages. In one embodiment, anti-CD47 AlivaMab antibodies that do not induce hemagglutination of erythrocytes, even at high concentration, were identified.
[0041] Embodiments of the invention pertain to the use of anti-CD47 antibodies, or antigen-binding fragments thereof, for the diagnosis, assessment and treatment of diseases and disorders associated with CD47 or aberrant expression thereof. The subject antibodies are used in the treatment or prevention of neoplasms, among other diseases. The subject antibodies may also be used in the blocking of ischemia-reperfusion injury.
[0042] Portions of variable regions from the AlivaMab antibodies may include all or a combination of the complementarity determining regions (CDRs) of the VH and/or VL. The variable regions may be formatted with constant regions, either native or desirably modified for induction of either up-regulation or down-regulation of various effector functions, in a standard antibody structure (two heavy chains with two light chains). The variable regions may also be formatted as multi-specific antibodies, e.g., bispecific antibodies binding to two different epitopes on CD47 or to two different antigens, one of which is CD47. The variable regions may also be formatted as antibody fragments, e.g., single-domain antibodies comprising a single VH or VL, Fab, Fab'2, or chimeric antigen receptor (CAR). The antibodies may also be used as antibody-drug conjugates, or carry other additions such as small molecule toxins, biologic toxins, cytokines, oligopeptides, RNAs, or CAR-T cells to increase therapeutic modality and/or increase safety.
[0043] 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., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); 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) and other like references.
[0044] Before describing certain embodiments in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular illustrative embodiments only, and is not intended to be limiting. The terms used in this specification generally have their ordinary meaning in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them. The scope and meaning of any use of a term will be apparent from the specific context in which the term is used. As such, the definitions set forth herein are intended to provide illustrative guidance in ascertaining particular embodiments of the invention, without limitation to particular compositions or biological systems.
[0045] As used in the present disclosure and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.
[0046] Throughout the present disclosure and the appended claims, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or group of elements but not the exclusion of any other element or group of elements.
[0047] The terms "antibody" and "immunoglobulin" (Ig) are used interchangeably herein. An antibody may be either membrane bound or secreted. As used herein, the term encompasses not only intact, or "whole", polyclonal or monoclonal antibodies, but also fragments thereof (such as single-variable domain (VH, VL or combination thereof) antibodies, Fab, Fab', F(ab')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, chimeric antigen receptors (CARs), and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity.
[0048] Antibody, or Ig, molecules are typically comprised of two identical heavy chains and two identical light chains linked together through disulfide bonds. Both heavy chains (IgH) and light chains (IgL) contain a variable (V) region or domain and a constant (C) region or domain. The portion of the IgH locus encoding the V region comprises multiple copies of variable (V), diversity (D), and joining (J) gene segments. The portion of the IgL loci encoding the V region comprises multiple copies of V and J gene segments. The V region encoding portion of the IgH and IgL loci undergo gene segment rearrangement, e.g., different combinations of a V, (D) and J gene segments arrange to form the IgH and IgL variable regions, to develop diverse antigen specificity in antibodies. Each variable region comprises three complementarity-determining regions (CDRs) interspersed between the less variable framework regions (FRs). The heavy chain comprises VHCDR1, VHCDR2, and VHCDR3. The light chain comprises VLCDR1, VLCDR2, and VLCDR3. The secreted form of the IgH C region is made up of three C domains, CH1, CH2, CH3, optionally CH4 (C.mu.), and a hinge region except for C.mu., which lacks a hinge region. The membrane-bound form of the IgH C region also has membrane and intra-cellular domains. The IgH constant region determines the isotype of the antibody, e.g. IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA and IgE. It will be appreciated that non-human mammals, such as an AlivaMab Mouse, encoding multiple Ig isotypes will be able to undergo isotype class switching. There are two types of human IgL, Ig.kappa. and Ig.lamda..
[0049] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chain that binds to CD47. In this regard, an antigen-binding fragment of the antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence set forth herein from anti-CD47 antibodies described herein. An antigen-binding fragment of the CD47-specific antibodies described herein is capable of binding to CD47. In certain embodiments, an antigen-binding fragment or an antibody comprising an antigen-binding fragment, prevents or inhibits SIRP.alpha. binding to CD47 and subsequent signaling events. In certain embodiments, the antigen-binding fragment binds specifically to and/or inhibits or modulates the biological activity of human CD47.
[0050] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set that provide conformational support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N terminus of a heavy or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
[0051] A "Fab" domain or fragment comprises the N-terminal portion of the IgH, which includes the V region and the CH1 domain of the IgH, and the entire IgL. A "F(ab').sub.2" domain comprises the Fab domain and a portion of the hinge region, wherein the 2 IgH are linked together via disulfide linkage in the middle hinge region. Both the Fab and F(ab').sub.2 are "antigen-binding fragments." The C-terminal portion of the IgH, comprising the CH2 and CH3 domains, is the "Fc" domain. The Fc domain is the portion of the Ig recognized by cell receptors, such as the FcR, and to which the complement-activating protein, C1q, binds. The lower hinge region, which is encoded in the 5' portion of the CH2 exon, provides flexibility within the antibody for binding to FcR receptors. An "Fv" fragment includes a non-covalent VH:VL heterodimer including an antigen-binding site. In certain embodiments, single chain Fv (scFv) antibodies are contemplated. A scFv is a covalently linked VH:VL heterodimer which is expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker (Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883).
[0052] Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g., prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
[0053] As used herein "chimeric antibody" refers to an antibody encoded by a polynucleotide sequence containing polynucleotide sequences from two or more species, e.g., human and mouse.
[0054] As used herein "chimeric Ig chain" refers to an Ig heavy chain or an Ig light chain encoded by a polynucleotide sequence containing polynucleotide sequences from two or more species, e.g., human and mouse. For example, a chimeric Ig heavy chain may comprise a human VH domain, DH domain, JH domain, CH1 domain, and upper hinge region and mouse CH2 and CH3 domains. In one embodiment, the middle hinge region is mouse. In one embodiment, the middle hinge region is human. In one embodiment, the middle hinge region is chimeric.
[0055] "Polypeptide," "peptide" or "protein" are used interchangeably herein to describe a chain of amino acids that are linked together by chemical bonds. A polypeptide or protein may be an IgH, IgL, V domain, C domain, or an antibody.
[0056] The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K.sub.D) of the interaction, wherein a smaller K.sub.D represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (K.sub.on) and the "off rate constant" (K.sub.off) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K.sub.off/K.sub.on enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant, K.sub.D. See, generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.
[0057] "Polynucleotide" refers to a chain of nucleic acids that are linked together by chemical bonds. Polynucleotides include, but are not limited to, DNA, cDNA, RNA, mRNA, and gene sequences and segments. Polynucleotides may be isolated from a living source such as a eukaryotic cell, prokaryotic cell or virus, or may be derived through in vitro manipulation by using standard techniques of molecular biology, or by DNA synthesis, or by a combination of a number of techniques.
[0058] As used herein, the term "vector" refers to a nucleic acid molecule into which another nucleic acid fragment can be integrated without loss of the vector's ability to replicate. Vectors may originate from a virus, a plasmid or the cell of a higher organism. Vectors are utilized to introduce foreign or recombinant DNA into a host cell, wherein the vector is replicated.
[0059] A polynucleotide agent can be contained in a vector, which can facilitate manipulation of the polynucleotide, including introduction of the polynucleotide into a target cell. The vector can be a cloning vector, which is useful for maintaining the polynucleotide, or can be an expression vector, which contains, in addition to the polynucleotide, regulatory elements useful for expressing the polynucleotide and, where the polynucleotide encodes an RNA, for expressing the encoded RNA in a particular cell, either for subsequent translation of the RNA into a polypeptide or for subsequent trans regulatory activity by the RNA in the cell. An expression vector can contain the expression elements necessary to achieve, for example, sustained transcription of the encoding polynucleotide, or the regulatory elements can be operatively linked to the polynucleotide prior to its being cloned into the vector.
[0060] An expression vector (or the polynucleotide) generally contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible or tissue specific or developmental stage specific expression of the encoding polynucleotide, a poly-A recognition sequence, and a ribosome recognition site or internal ribosome entry site, or other regulatory elements such as an enhancer, which can be tissue specific. The vector also can contain elements required for replication in a prokaryotic or eukaryotic host system or both, as desired. Such vectors, which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, alpha virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison Wis.; Stratagene, La Jolla Calif; GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb 25:37-42, 1993; Kirshenbaum et al., J. Clin. Invest 92:381-387, 1993; each of which is incorporated herein by reference).
[0061] The term "construct" as used herein refers to a sequence of DNA artificially constructed by genetic engineering, recombineering or synthesis. In one embodiment, the DNA constructs are linearized prior to recombination. In another embodiment, the DNA constructs are not linearized prior to recombination.
[0062] The terms "inhibit", "neutralize", and "antagonize" are used interchangeably herein and encompass anti-CD47 antibodies that block, inhibit, and/or decrease the activity of CD47. Examples of CD47 activity include ligand binding, e.g., binding to SIRP.alpha..
[0063] The term "treating" with regard to a subject, refers to improving at least one symptom of the subject's disease or disorder. Treating includes curing, improving, or at least partially ameliorating the disease or disorder.
[0064] As used herein, the term "disorder" refers to, and is used interchangeably with, the terms disease, condition, or illness.
[0065] The term "pharmaceutically acceptable carrier" refers generally to any material (e.g., carrier, excipient, or stabilizer) that may accompany a therapeutic agent and is nontoxic to the subject or patient being exposed thereto.
[0066] The term "administering," as used herein, refers to any mode of transferring, delivering, introducing, or transporting a pharmaceutical composition or other agent, such as an anti-CD47 antibody, to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intranasal, or subcutaneous administration.
[0067] The term "inhibit" or "neutralize" or "block" may relate generally to the ability of one or more anti-CD47 antibodies of the invention to decrease a biological activity of CD47, such as intracellular signaling and/or ligand binding. The inhibition/blocking of SIRP.alpha. to CD47 preferably reduces or alters the normal level or type of cell signaling that occurs when SIRP.alpha. binds to CD47 without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding of SIRP.alpha. to CD47 when in contact with an anti CD47 antibody as disclosed herein as compared to the ligand not in contact with an anti CD47 antibody, e.g., the blocking of SIRP.alpha. to CD47 by at least about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% decrease, including all integers in between. In one embodiment, a neutralizing anti-CD47 antibody inhibits binding of SIRP.alpha. to CD47 by at least about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% decrease, including all integers in between.
[0068] An antibody, or antigen-binding fragment thereof, is said to "specifically bind," "immunologically bind," and/or is "immunologically reactive" to CD47 if it reacts at a detectable level (within, for example, an ELISA assay) with CD47, and does not react detectably with unrelated polypeptides under similar conditions. Antibodies are considered to specifically bind to a target polypeptide when the binding affinity is at least 1.times.10.sup.-7 M or, preferably, at least 1.times.10.sup.-8 M. In one embodiment, the antibody, or antigen-binding fragment thereof, specifically binds human CD47.
[0069] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.
[0070] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
CD47
[0071] CD47, or integrin associated protein (IAP) is expressed by a variety of cells, including, e.g., erythrocytes, and it is plays a role in a variety of cellular processes. Overexpression of CD47 is associated with proliferative diseases and disorders (e.g., neoplasms, tumors and metastases). CD47 is up-regulated in ischemia-reperfusion injury (IRI), and CD47 knock-out mice are projected in models of IRI. Thus, CD47 is an important therapeutic target for both proliferative diseases and disorders and transplants.
Anti-CD47 Antibodies
[0072] AlivaMab Mouse anti-CD47 antibodies were generated using both AlivaMab Mouse Kappa mice and AlivaMab Mouse Lambda mice (also referred to herein interchangeably as AlivaMab Kappa Mice and AlivaMab Lambda Mice, respectively). Antibodies produced by AlivaMab Kappa Mice comprise a chimeric immunoglobulin heavy (IgH) chain and a human immunoglobulin kappa (Ig.kappa.) light chain. Antibodies produced by AlivaMab Lambda Mice comprise a chimeric IgH chain and a human immunoglobulin lambda (Ig.lamda.) light chain. The chimeric IgH chain of the AlivaMab Mouse antibodies comprises a human variable region comprising a human variable heavy (VH) domain, a human diversity heavy (DH) domain, and a human joining heavy (JH) domain, a human constant heavy 1 (CH1) domain, a human upper hinge region (except for C.mu., which is naturally missing an upper hinge region), a mouse middle hinge region, a mouse CH2 domain, and a mouse CH3 domain. Upon identification of a lead candidate antibody, e.g., an anti-CD47 antibody, the human heavy chain variable region is readily appended to a fully human constant region while maintaining the antigen-binding characteristics of the parent chimeric antibody that were developed in vivo in the AlivaMab Mouse. In one embodiment, the human heavy chain variable region, CH1 and, optionally, upper hinge region of the chimeric antibody are appended to human hinge, a human CH2 domain and a human CH3 domain in order to produce a fully human antibody.
[0073] Accordingly, in one embodiment, an anti-CD47 antibody, or an antigen-binding fragment thereof, of the invention is chimeric. In one embodiment, the chimeric anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a chimeric IgH chain and a human Ig.kappa. chain. In one embodiment, the chimeric anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a chimeric IgH chain and a human Ig.lamda. chain. In one embodiment, the chimeric anti-CD47 antibody is human and mouse. In one embodiment, an anti-CD47 antibody, or an antigen-binding fragment thereof, of the invention is human. In one embodiment, the human anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a human IgH chain and a human Ig.kappa. chain. In one embodiment, the human anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a human IgH chain and a human Ig.lamda. chain. In one embodiment, the isotype of the anti-CD47 antibody is selected from IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA and IgE. In one embodiment, the isotype of the anti-CD47 antibody is selected from IgG1, IgG2, IgG3, and IgG4.
[0074] In one embodiment, the anti-CD47 antibody binds an Fc receptor (FcR) selected from an Fc.gamma.R, an Fc.epsilon.R, and an Fc.alpha.R. In one embodiment, the anti-CD47 antibody binds an Fc.gamma.R selected from Fc.gamma.RI (CD64), Fc.gamma.RII (CD32), and Fc.gamma.RIII (CD16), including isoforms thereof. In one embodiment, the Fc region of the anti-CD47 antibody comprises a mutation so that it preferentially binds a particular Fc.gamma.R (see, e.g., U.S. Pat. No. 6,737,056 and U.S. 2015/0031862).
[0075] In one aspect of the invention, the CDRs of an anti-CD47 antibody, or antigen-binding fragment thereof, may be mixed and matched between the CDRs of antibody clones described herein. In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, comprises a VHCDR1 comprising any VHCDR1 sequence disclosed herein, a VHCDR2 comprising any VHCDR2 sequence disclosed herein, and a VHCDR3 comprising any VHCDR3 sequence disclosed herein. In one embodiment, the VHCDR1, VHCDR2 and VHCDR3 are selected from three different anti-CD47 clones disclosed herein. In one embodiment, the VHCDR1, VHCDR2 and VHCDR3 are selected from two different anti-CD47 clones disclosed herein.
[0076] In one embodiment, an anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a VLCDR1 comprising any VLCDR1 sequence disclosed herein, a VLCDR2 comprising any VLCDR2 sequence disclosed herein, and a VLCDR3 comprising any VLCDR3 sequence disclosed herein. In one embodiment, the VLCDR1, VLCDR2 and VLCDR3 are selected from three different anti-CD47 clones disclosed herein. In one embodiment, the VLCDR1, VLCDR2 and VLCDR3 are selected from two different anti-CD47 clones disclosed herein.
[0077] In one aspect of the invention, the CDRs of an anti-CD47 antibody, or antigen-binding fragment thereof, are from the same anti-CD47 antibody clone disclosed herein. In one embodiment, an anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a VHCDR1, a VHCDR2 and a VHCDR3 from the same anti-CD47 clone disclosed herein. In one embodiment, an anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a VHCDR1, a VHCDR2, and a VHCDR3 comprising the corresponding sequences disclosed herein.
[0078] In another aspect of the invention, the CDRs of an anti-CD47 antibody, or antigen-binding fragment thereof, are selected from the corresponding VH and VL of a single clone described herein. In one embodiment, an anti-CD47 antibody, or an antigen-binding fragment thereof, comprises 1) a VHCDR1, a VHCDR2, and a VHCDR3 selected from the VHCDR1, VHCDR2 and VHCDR3 of one VH selected from any one of the VH regions disclosed herein and 2) a VLCDR1, a VLCDR2, and a VLCDR3 selected from the VLCDR1, VLCDR2 and VLCDR3 of one VL selected from any one of the VL regions disclosed herein. In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, comprises a VHCDR1, a VHCDR2, a VHCDR3, a VLCDR1, a VLCDR2, and a VLCDR3 within the corresponding VH and VL amino acid sequences of a single clone as disclosed herein.
[0079] In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, comprises a VH comprising any one of the VH regions disclosed herein. In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, comprises a VL comprising any one of the VL regions disclosed herein. In one embodiment, an anti-CD47 antibody, or an antigen-binding fragment thereof, comprises a corresponding VH and VL of a single clone.
[0080] In one embodiment, an anti-CD47 antibody is a whole antibody. In one embodiment, an anti-CD47 antibody is a single chain antibody. In one embodiment, an anti-CD47 antibody is a scFv. In one embodiment, an anti-CD47 antibody is a Fab. In one embodiment, an anti-CD47 antibody is a F(ab').sub.2. In one embodiment, an anti-CD47 antibody is a Fv.
[0081] In one embodiment, an anti-CD47 antibody is a bispecific antibody. In one embodiment, a bispecific anti-CD47 antibody specifically recognizes two different epitopes of CD47. In one embodiment, a bispecific anti-CD47 comprises a first CDR set comprising the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 from a first anti-CD47 antibody clone disclosed herein and a second CDR set comprising the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of a second anti-CD47 antibody clone disclosed herein. In one embodiment, a bispecific anti-CD47 comprises a corresponding first VH and first VL of a first anti-CD47 antibody clone disclosed herein and a corresponding second VH and second VL of a second anti-CD47 antibody clone disclosed herein. In one embodiment, a bispecific anti-CD47 antibody specifically recognizes CD47 and another antigen.
Polynucleotides
[0082] One aspect of the present invention provides a polynucleotide sequence that encodes an anti-CD47 antibody, or antigen-binding fragment thereof, disclosed herein. In one embodiment, the polynucleotide is a recombinant polynucleotide. In one embodiment, the polynucleotide is cDNA.
[0083] In one embodiment, a polynucleotide sequence encodes a CDR of an anti-CD47 antibody disclosed herein. In one embodiment, the polynucleotide comprises a VHCDR1 polynucleotide sequence disclosed herein. In one embodiment, the polynucleotide comprises a VHCDR2 polynucleotide sequence disclosed herein. In one embodiment, the polynucleotide comprises a VHCDR3 polynucleotide sequence disclosed herein. In one embodiment, the polynucleotide comprises a VLCDR1 polynucleotide sequence disclosed herein. In one embodiment, the polynucleotide comprises a VLCDR2 polynucleotide sequence disclosed herein. In one embodiment, the polynucleotide comprises a VLCDR3 polynucleotide sequence disclosed herein.
[0084] In one embodiment, a polynucleotide sequence encodes a VH of an anti-CD47 antibody disclosed herein. In one embodiment, a polynucleotide sequence encodes a VL of an anti-CD47 antibody disclosed herein. In one embodiment, a polynucleotide sequence encodes a VH and a VL of an anti-CD47 antibody disclosed herein.
[0085] One embodiment of the invention provides a vector comprising a polynucleotide sequence encoding an anti-CD47 antibody, or an antigen-binding fragment thereof, disclosed herein. In one embodiment, the vector is an expression vector. In one embodiment, the vector is a cloning vector. One embodiment of the invention provides a host cell comprising the vector.
Methods of Use
[0086] The AlivaMab antibodies against CD47, and in particular fully human antibodies incorporating all or portions of the heavy chain and light chain variable regions from the AlivaMab antibodies, may have utility for the treatment of human disease including, but not limited to, diseases in oncology and ischemia-reperfusion injury (IRI). As the understanding of CD47 biology and disease association becomes better known, it is expected that opportunities for human clinical therapeutic indications may expand. In particular, oncological diseases and disorders and IRI prevention are contemplated.
[0087] An anti-CD47 antibody, or antigen-binding fragment thereof, disclosed herein may be used in research, diagnostic, and/or therapeutic methods. In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, disclosed herein is used to treat diseases and disorders associated with CD47 and/or SIRP.alpha.. In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, disclosed herein is used to treat diseases and disorders associated with CD47 overexpression. In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, disclosed herein is used to induce phagocytosis by macrophages. In one embodiment, an anti-CD47 antibody, or antigen-binding fragment thereof, disclosed herein exhibits little to zero hemagglutination of erythrocytes.
Modified Anti-CD47 Antibodies and Compositions
[0088] Anti-CD47 antibodies of the present invention, and antigen-binding fragments and variants thereof, may also be conjugated or operably linked to another compound (e.g., therapeutic agent, label, or tag), referred to herein as a conjugate. The conjugate may be a cytotoxic agent, a chemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, a radioisotope, or other therapeutically active agent. Chemotherapeutic agents, cytokines, anti-angiogenic agents, tyrosine kinase inhibitors, and other therapeutic agents are contemplated. In one embodiment, the antibody is conjugated or operably linked to a toxin, including but not limited to small molecule toxins and enzymatically active toxins of bacterial, fungal, plant, animal or synthetic origin, including fragments and/or variants thereof.
[0089] 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 0425235 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.
[0090] The present invention further relates to pharmaceutical compositions and methods of use. The pharmaceutical compositions of the present invention include an antibody, or fragment thereof, in a pharmaceutically acceptable carrier. Pharmaceutical compositions may be administered in vivo for the treatment or prevention of a disease or disorder. Furthermore, pharmaceutical compositions comprising an antibody, or a fragment thereof, of the present invention may include one or more agents for use in combination, or may be administered in conjunction with one or more agents. Agents for use in combination with an anti-CD47 antibody disclosed herein include, but are not limited to cytotoxic agents, chemotherapeutic agents, cytokines, anti-angiogenic agents, tyrosine kinase inhibitors, toxins, and radioisotopes.
[0091] The present invention also provides kits relating to any of the antibodies, or fragments thereof, and/or methods described herein. Kits of the present invention may include diagnostic or therapeutic agents. A kit of the present invention may further provide instructions for use of a composition or antibody and packaging. A kit of the present invention may include devices, reagents, containers or other components. Furthermore, a kit of the present invention may also require the use of an apparatus, instrument or device, including a computer.
EXAMPLES
Example 1
Generation of Monoclonal Antibodies to CD47
[0092] Monoclonal antibodies were prepared in accordance with a general method as described in "Antibodies: A Laboratory Manual" (Harlow and Lane 1988 CSH Press). Two AlivaMab Kappa Mice were immunized using a RIMMS protocol. 50 .mu.g of human CD47 extracellular domain with a histidine- (HIS-) tag (Sino Biological, China) was mixed with 40 .mu.l (first immunization), 20 .mu.l (immunizations 2-4) or 0 .mu.l (final immunization) Gerbu MM adjuvant (C-C Biotech, Valley Center, CA #3001-6030) and PBS was added to a final volume of 100 .mu.l. The 50 .mu.g mixture was injected in 20 .mu.l portions in 5 locations per mouse; right and left flanks and right and left shoulder/armpit subcutaneously, and the remaining 20 .mu.l intraperitoneally. This was done 5 times per mouse on days, 1, 4, 7, 9, and 11. On Day 14 mice were sacrificed and terminal materials were collected. Spleens and lymph nodes were prepared and fused with CRL-2016 myeloma cells (ATCC) using a PEG based method as generally described in "Antibodies: A Laboratory Manual" (Harlow and Lane 1988 CSH Press) to establish hybridomas.
[0093] Hybridomas were grown in 384-well tissue culture plates and supernatants from individual wells were screened by ELISA for production of antibodies recognizing huCD47. Positive wells were then transferred to 48-well plates, expanded, and supernatants were collected for huCD47 binding confirmation by ELISA. Positive supernatants were also counter-screened against a non-related histidine-tagged protein. One hundred forty-seven (147) ELISA-positive (100.times. above background) were identified. Sixty-seven (67) hybridoma lines confirmed to bind CD47 specifically by ELISA were picked at random and single-cell cloned into 96-well plates. All 67 hybridoma lines were stable and single-cell cloned and were re-confirmed to produce monoclonal antibody binding to huCD47 in ELISA. Sixty-one hybridoma lines were advanced for subsequent analysis. The isotypes of the mouse Fc on the AlivaMab antibodies were determined using standard commercially available isotyping kits.
Example 2
Screen for Neutralization of Binding of SIRP .alpha. to CD47 in ELISA
[0094] Sixty-one hybridoma clones were screened for inhibition of SIRP .alpha. binding to CD47. Briefly, recombinant CD47-Fc (Sino Biologics) at 1 .mu.g/ml in phosphate-buffered saline (PBS) plus 0.05% TWEEN was coated onto the bottom of 96-well ELISA plates overnight. The plates were washed two-times in PBS and non-specific binding was blocked by one-hour incubation at room temperature with SuperBlock.TM.. Unrelated antibody isotype controls (negative control) and recombinant blocking CD47 monoclonal antibody B6H12 (positive control) were used at 20 .mu.g/ml. Supernatants from cloned hybridoma lines were added at a 1:2 dilution. All samples were in triplicate. The antibodies were incubated with plate-bound human CD47-Fc for 15 minutes at room temperature and then recombinant human HIS-tagged SIRP .alpha. was added at 2 .mu.g/ml. The plates were incubated for 1 hour at room temperature and then washed two times with PBS plus 0.05% TWEEN. An anti-HIS-tag antibody was added and incubated for one hour. The anti-HIS-tag antibody detects HIS-tagged SIRP .alpha. bound to human CD47-Fc. The plates were incubated for 1 hour at room temperature and then washed two times with PBS plus 0.05% TWEEN. SuperSignal.TM. at 20 .mu.g/ml was added and binding signal detected on a plate reader. The lower the RU signal the greater the ability of antibody to block binding of human CD47 to SIRP .alpha.. Sixteen of the 61 antibodies tested substantially or completely blocked binding of human CD47 to SIRP .alpha. (FIG. 1).
TABLE-US-00003 TABLE 3 CD47 AlivaMabs That Block the Binding of Human CD47 to Human SIRP .alpha. LIST OF MONOCLONAL ANTIBODIES THAT BLOCK HUMAN CD47 BINDING TO SIRP .alpha. 5 10 14 85 87 91 101 102 106 107 139 144 151 153 161 164
Example 3
Screen for Binding to Native Human CD47 Expressed in Cell Surface
[0095] The sixty-one monoclonal antibodies were screened for binding to native human CD47 expressed on the surface of the CCRF-CEM human tumor cell line, a T lymphoblastoid cell line (ATCC CCL-119) by flow cytometry (FACS). Twenty of 65 antibodies tested bound to native CD47 as assessed by FACS. Sixteen were assessed as strong binders and four were assessed as weak binders (FIG. 2). Binding in the FACS correlated with inhibition of binding to SIRP .alpha.. The low level of FACS positive binders to human CD47 expressed on the surface of human cells suggests that the recombinant human CD47 used for immunization and screening of the hybridomas poorly represented the native conformation of human CD47.
TABLE-US-00004 TABLE 4 CD47 AlivaMabs That Bind to Native CD47 on a Human Cell Line Correlate with Neutralization of Binding of Human CD47 to Human SIRP .alpha. LIST OF MONOCLONAL ANTIBODIES THAT BLOCK HUMAN CD47 BINDING TO CD47 IN mAb Clone ID BINDING TO SIRP .alpha. FACS ASSAY 5 + + 10 + + 14 + + 85 + + 87 + + 91 + + 101 + + 102 + + 106 + + 107 + + 139 + + 144 + + 151 + + 153 + + 161 + + 164 + + 143 +/- +/- 141 - +/- 90 +/- +/- 47 - +/-
Example 4
Determination of IC50 Values Neutralization of Binding of SIRP .alpha. to CD47 in ELISA
[0096] Concentrated monoclonal antibody for the top 18 anti-CD47 mAbs was used in the CD47-SIRP .alpha. neutralization assay to determine IC50 values. An assay format similar to that in Example 2 was performed, using an 8 fold 1:2 dilution series of antibody, from 10 .mu.g/ml to 0.005 .mu.g/ml. IC50 values were calculated using PRISM software. The sixteen antibodies with the lowest IC50 also completely block binding of CD47 binding to SIRP .alpha..
TABLE-US-00005 TABLE 5 Antibody Isotype and IC50 Values for Neutralization of Binding of Human CD47 to Human SIRP .alpha. mAb ID Isotype IC50 (nM) 151 IgG1 2.0 91 IgG1 2.1 139 IgG1 2.2 102 IgG1 2.3 107 IgG1 2.7 106 IgG1 2.9 87 IgG1 3.0 161 IgG1 3.0 5 IgG1 3.5 14 IgG1 3.5 10 IgG2a 4.7 164 IgG1 4.8 85 IgG1 4.9 144 IgG1 10 101 IgG1 15 153 IgG1 27.9 90 IgG1 53.6 143 IgG1 96
Example 5
Induction of Macrophage Phagocytosis of Cells from a Human Tumor Cell Line
[0097] Neutralization of the binding of CD47 expressed on target cells to SIRP .alpha. expressed macrophages can induce macrophages to phagocytose the target cell. A cell based fluorescent assay similar to that used in WO 2013/119714 was used to assess a panel of the top five anti-CD47 mAbs that neutralize with the highest IC50 CD47/SIRP .alpha. binding for the ability to induce phagocytosis of a target human tumor cell line. CD47-expressing CCRF-CEM cells were incubated with 0.3 uM CFSE for 15 minutes at 37.degree. C. and washed with PBS to remove CFSE. CFSE-labeled CCRF-CEM cells fluoresce in the FL-1 channel in FACS. CFSE-labeled CCRF-CEM cells were incubated with monocyte-derived human macrophages for 3 hr at 37.degree. C. at a ratio of 4:1 CCRF-CEM cell to macrophage, with or without anti-CD47 mAbs. Monocyte-derived human macrophages are adherent. Non-adherent cells on the plate were washed away with PBS and the adherent cells were scraped off of the bottom of the plate. Macrophages with stained with anti-CD14 antibody conjugated with dye to fluoresce in the FL-4 channel in FACS. Macrophages that had phagocytosed the CCRF-CEM cells would also have ingested the CFSE dye and would fluoresce in the FL-1 channel in FACS. Double-positive FL-4+/FL-1+ cells are macrophages that have phagocytosed CFSE-labeled CCRF-CEM cells. The CD47 mAbs, antibody B6H12 (as a positive control) and an irrelevant mouse IgG1 mAb (as a negative control) were tested for the ability to induce phagocytosis at five concentration, of 1:10 dilutions from 10 .mu.g/ml to 0.001 .mu.g/ml. The data were expressed as the phagocytotic index, the percentage of CD14+ (FL-4+) cells that were also positive in the FL-1 channel in FACS (had ingested CFSE through phagocytosis of CFSE-labeled CCRF-CEM cells). Other mAbs in the panel that neutralize CD47/SIRP .alpha. binding in ELISA may also exhibit potent induction of macrophage phagocytosis of human tumor cells. The data show that mAbs potently induce phagocytosis of the CCRF-CEM human tumor cells by macrophages and that they do so more potently than control mAb B6H12. At a concentration of 0.1 .mu.g/ml, the mAbs are still exhibiting significant induction of phagocytosis while the activity of B6H12 is approaching baseline established by the negative control mAb (FIG. 3).
Example 6
Screen for Induction of Hemagglutination of Human Erythrocytes by Monoclonal Antibodies Against Human CD47
[0098] The antibodies were screened for their induction of hemagglutination of human erythrocytes (RBCs). Human RBCs were diluted 1:10 in PBS and incubated at 37.degree. C. with a 1:2 dilution of antibody from 50 nM to 0.05 nM. Humanized antibody B6H12 was used as a positive control and mouse IgG1 was used as a negative control. RBCs that have not undergone hemagglutination appear as a tight red dot. Hemagglutinated RBCs are more diffuse. Data are shown in FIG. 4. Antibody 102 exhibited no evident hemagglutination at the maximal tested concentration of 50 nM. In assays using higher concentrations, antibodies of the invention, including Antibody 102, will show no evident hemagglutination at concentrations up to 500 nM. Antibody 91 exhibited no evident hemagglutination at concentration of 25 nM and a small amount of hemagglutination at 50 nM. Therefore, antibody 102 is a therapeutic candidate antibody as it exhibits potent induction of phagocytosis of CCRF-CEM cells while exhibiting no hemagglutination of RBCs. Antibody 91 is also a candidate. Antibodies 91, 102, 87, 107 and 139 all appeared to be superior to humanized B6H12 as they exhibited more potent induction of phagocytosis of CCRF-CEM cells, while exhibiting no or lower hemagglutination of RBCs at higher concentrations than humanized B6H12. No prozone effect of hemagglutination was observed with any of the AlivaMab CD47 mAbs. Other AlivaMab antibodies exhibiting neutralization of CD47/SIRP .alpha. binding and induction of phagocytosis of human tumor cells by macrophages also showed no or limited hemagglutination at high concentrations and are therapeutic candidates.
Example 7
Sequences of Anti-CD47 VH and VL
[0099] Total RNA was extracted from hybridomas producing anti-CD47 monoclonal antibodies using the Qiagen RNeasy Mini kit (Cat No. 74104), followed by 5' RACE, using the 5' RACE system kit (Life Technologies, US cat #18734-058) with the following 3' gene specific primers IgG 5'-GGTTCGGGGAAGTAGTCCTTGACC-3' (SEQ ID NO:337) and IgK 5'-CCGATTGGAGGGCGTTATCCAC-3' (SEQ ID NO:338). RACE products were gel purified and cloned into pCR4-TOPO using TOPO TA cloning kit for sequencing with One Shot Top 10 chemically competent E. coli (Life Technologies, US Cat #K4575-01). Sequencing of vector containing colonies was performed by Sequetech (Mountain View, Calif.) using M13F or M13R sequencing primers. The reported nucleotide sequences start at the first nucleotide in the first codon for the amino terminal amino acid in framework 1. The reported polypeptide sequences are based on an in silico translation of the nucleic acid sequence and start at the first amino acid at the amino terminus of framework 1.
Example 8
Epitope Binning
[0100] A competition ELISA was performed to establish competitive binding bins. ELISA plates were coated with 1 .mu.g/ml huCD47 protein (Sino Biological, China 10161-H08H) and blocked with Superblock (Thermo Scientific #37518). After washing, wells were incubated with an AlivaMab monoclonal antibody representing one of seven unique competition bins. After 1 hour the wells were washed and incubated with individual clonal anti-huCD47 AlivaMab hybridoma supernatants. After another hour the wells were washed and incubated with a specific secondary antibody that either recognized human kappa light chain (LC) or human lambda LC depending on which AlivaMab Mouse supernatants were being detected (Southern Biotech Goat X hu kappa LC #2061-05 or Bethyl Goat X hu lambda LC #A80-116P) and detected with Supersignal ELISA Pico Chemiluminescent substrate (Thermo Scientific--Product #37069) (Tables 5 and 6). Individual AlivaMab Mouse antibodies that were able to bind in the presence of a mouse antibody are considered to be in a unique epitope bin from that particular mouse antibody. Individual AlivaMab Mouse antibodies that were unable to bind in the presence of a mouse antibody are considered to be in the same epitope bin as that particular mouse antibody. In this way multiple epitope bins were defined for huCD47 binding antibodies (Tables 3 and 6, FIG. 5).
TABLE-US-00006 TABLE 6 Multiple Epitope Bins Bin 1 Bin 2 Bin 3 Bin 4 Bin 5 Bin 6 Bin 7 All CD47 12 7 5 7 1 26 3 AlivaMabs by bin Functional 5 107 91 151 mAbs 14 161 106 139 Assigned 10 85 87 102 to Epitope 144 101 164 Bin 153 90 143
Example 9
Affinity Determination
[0101] Affinity was determined for five selected AlivaMab Mouse monoclonal hybridoma supernatants (Biosensor Tools, Salt Lake City, Utah). Binding kinetics were measured at 25.degree. C. using a BioRad ProteOn XPR36 optical biosensor equipped with GLM sensor chips and equilibrated with running buffer (HBS, 0.005% TWEEN-20, 0.1 mg/mL BSA, pH 7.4).
[0102] The tested antibodies were AlivaMab antibodies: 91, 102, 107, 139 and 151. Kinetics of binding each mAb to recombinant human CD47 (Novoprotein cat no. C321, lot no. 0329689) was measured.
[0103] The analyses were performed by capture of the mouse antibodies on sensor surfaces coated with an anti-mouse capturing agent.
CD47 Binding to Five AlivaMab Anti-CD47 Antibodies
[0104] The five antibodies were captured to densities of 850-1030 RU onto an anti-mouse Fc surface. CD47 was tested in a three-fold dilution series starting at 100 nM.
[0105] These binding data were also fit to a 1:1 interaction model:
[0106] Within the panel of AlivaMab Mouse anti-CD47 antibodies, there are antibodies with KD values below a nanomolar and KD values in the low nanomolar range, and with fast k.sub.on and slow k.sub.off rates (Tables 7-9).
TABLE-US-00007 TABLE 7 Binding Kinetics of Anti-CD47 IgG AlivaMab mAbs mAb ka (M.sup.-1 s.sup.-1) kd (s.sup.-1) K.sub.D (pM) 91 7.2(2)e6 1.111(3)e-3 154.4(7) pM 102 6.9(2)e6 2.21(7)e-3 320(3) pM 107 5.7(1)e6 9.3(2)e-4 163(1) pM 139 1.18(6)e6 .sup. 1.00e-5* 8.5(4) pM 151 8.7(4)e6 08.9(4)e-3 1.002(8) nM *No decay was detected in the responses from the 139A surface during the dissociation phase.
This indicates the complex dissociated very little during this time. Therefore, the dissociation rate constant was fixed at 1.times.10.sup.-5 s.sup.-1 during the fitting process so the KD must be considered a rough estimate.
[0107] Table 8 summarizes the parameters determined from these measurements.
TABLE-US-00008 TABLE 8 Summary of Binding Kinetics of Anti-CD47 mAbs Binding constants determined at 25.degree. C. k.sub.a (M.sup.-1 s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (pM) m91A 7.2(2)e6 1.11(3)e-3 150(10) m102A 6.9(2)e6 2.21(7)e-3 320(10) m107A 5.7(1)e6 9.3(2)e-4 160(10) m139A 1.18(6)e6 .sup. 1e-5** ~9 m151A 8.7(4)e6 8.9(4)e-3 1000(100) * average of measurements from three antibody surfaces **this dissociation rate constant was fixed at 1e-5 s.sup.-1
[0108] Another way to compare these five antibodies is to bin and rank them by their dissociation rate constants.
TABLE-US-00009 TABLE 9 Binning and Ranking of Anti-CD47 mAbs by Their Dissociation Constants Antibodies binned by k.sub.d's determined at 25.degree. C. fastest slowest ~10.sup.-2 s.sup.-1 ~10.sup.-3 s.sup.-1 ~4 .times. 10.sup.-4 s.sup.-1 .ltoreq.10.sup.-5 s.sup.-1 m151A m107A hB6412 m139A m91A hAb6.12 m102A
[0109] AlivaMab antibody 139A appeared to have a very slow dissociation rate constant.
[0110] In summary, CD47 showed binding to all five antibodies, with affinities that ranged from .about.10 pM to .about.1.5 nM.
[0111] The 139A/CD47 complex was so stable that a dissociation rate constant could not be reliably determined from these analyses.
Example 10
AlivaMab Mouse Anti-CD47 mAbs and their Fully Human Derivatives Bind to and Neutralize CD47 from Cynomolgus Monkey
[0112] CD47 is cloned and expressed from cynomolgus monkey using standard molecular biological techniques. The recombinant CD47 may be tagged (histidine, Fc) to support efficient purification. The recombinant cynomolgus CD47 may also be transiently or stably expressed on cell lines. The AlivaMab Mouse anti-CD47 mAbs and their human variants are shown to bind to cynomolgus monkey CD47. The AlivaMab Mouse anti-CD47 mAbs and their human variants are shown to neutralize cynomolgus monkey CD47 in assays as described above for human CD47.
Example 11
AlivaMab Mouse Anti-CD47 mAbs and their Fully Human Derivatives Bind to and Neutralize CD47 from Mouse
[0113] CD47 is cloned and expressed from mouse using standard molecular biological techniques. The recombinant CD47 may be tagged (histidine, Fc) to support efficient purification. The recombinant mouse CD47 may also be transiently or stably expressed on cell lines. The AlivaMab Mouse anti-CD47 mAbs and their human variants are shown to bind to mouse CD47. The AlivaMab Mouse anti-CD47 mAbs and their human variants are shown to neutralize mouse CD47 in assays as described above for human CD47.
Example 12
Conversion of AlivaMab Mouse Anti-CD47 mAbs to Fully Human
[0114] The AlivaMab Mouse anti-CD47 mAbs are easily converted, expressed recombinantly and purified as fully-human antibodies of any isotype. The recombinant fully-human antibody retains all of the characteristics of the parental AlivaMab Mouse antibody. For example, the nucleotide sequences of the heavy and light chain variable region are synthesized into DNA by contract research organization such as Lake Pharma (Belmont Calif.) and then, using vectors for recombinant expression in mammalian cells, the VH cloned in-frame with coding sequences for the human isotype of choice IgG1, IgG2, or IgG4 constant regions and including modified versions thereof that are known in the art and the V.kappa. cloned in-frame with coding sequences for the human C.kappa. region. The Fc regions may be modified for either increased or decreased effector function such as C1q binding or FcR binding. In particular the antibody isotype may be human IgG4, which may exhibit lowered antibody dependent cellular cytotoxicity than human IgG1 or human IgG2. The middle hinge and or the Fc of the human IgG4 may be modified (for example, see U.S. Pat. No. 8,911,726 and disclosures and references therein.) IgG4 may also be modified to drive inter-chain disulfide bonds rather intra-chain to better ensure homodimeric IgG4 rather than monomeric or heterodimeric formation (replacement of the middle hinge region with that from human IgG1; mutation S228P/L235E; mutation of serine 229 to proline (Bloom et al., Protein Sci. (1997) 6:407-15); see also Peters et al., J. Biol. Chem. 2012). Vectors are then transformed into HEK293 cells for expression of recombinant fully human antibody. Fully human IgG.kappa. mAb versions of the selected AlivaMab mAb are purified from tissue culture supernatants using protein A. The fully human versions retain all of the characteristics of the parental chimeric AlivaMab.
[0115] In additional examples, the CD47 antibodies will exhibit anti-tumor activities in human tumor xenograft models in mice such as the Raji model of lyphoma. The antibodies will exhibit anti-tumor activities in preventative models and eradication models.
[0116] The epitopes of the AlivaMab CD47 antibodies can be epitope mapped by various methods known in the art. The panel of epitopes on CD47 will include linear and conformational epitopes. Key contact amino acids in the epitope can be discerned by site-direct mutagenesis or by solving the crystal structure of the antibody-CD47 binding interaction. The antibodies that neutralize CD47/SIRP .alpha. binding will have different epitopes. Some epitopes will overlap structurally and will have some overlapping and some different key contact residues. Some epitopes will be not overlap structurally with epitopes of other antibodies. Unique epitopes will correlate with unique activities of antibodies.
Sequence CWU
1
1
3381115PRTArtificial SequenceMade in Lab - synthesized VH sequence 1Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Tyr Ile Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Lys Gly Gly Thr
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100
105 110Val Ser Ser 1152115PRTArtificial
SequenceMade in Lab - synthesized VH sequence 2Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Arg Thr Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Lys Lys Gly Ala Phe Asp Ile Trp Gly Gln
Gly Thr Met Val Thr 100 105
110Val Ser Ser 1153114PRTArtificial SequenceMade in Lab -
synthesized VH sequence 3Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr
20 25 30Tyr Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95Arg Lys Arg Gly Leu Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val
100 105 110Ser Ser4114PRTArtificial
SequenceMade in Lab - synthesized VH sequence 4Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Ala Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Ser Gly Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Arg Ile Tyr Asn Ser Gly Ser Thr
Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Gln Thr Tyr Leu Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val 100 105
110Ser Ser5115PRTArtificial SequenceMade in Lab - synthesized VH sequence
5Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45Gly Tyr Ile
Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75
80Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Lys Arg Ala Phe Leu
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100
105 110Val Ser Ser 1156115PRTArtificial
SequenceMade in Lab - synthesized VH sequence 6Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Asn Asn Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Asn Tyr His Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Thr Ser Val
Thr Ala Ala Asp Thr Ala Val Phe Tyr Cys Ala 85
90 95Arg Gly Arg Arg Ala Leu Thr Ala Trp Gly Gln
Gly Thr Leu Val Thr 100 105
110Val Ser Ser 1157115PRTArtificial SequenceMade in Lab -
synthesized VH sequence 7Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Tyr
20 25 30Tyr Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Asn Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95Arg Lys Arg Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
100 105 110Val Ser Ser
1158115PRTArtificial SequenceMade in Lab - synthesized VH sequence 8Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Phe Tyr 20 25
30Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Phe Ile Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Arg Gly Ala Leu
Thr Ala Trp Gly Gln Gly Thr Leu Val Thr 100
105 110Val Ser Ser 1159115PRTArtificial
SequenceMade in Lab - synthesized VH sequence 9Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Ser Asn Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Lys Arg Asp Thr Phe Asp Ile Trp Gly Gln
Gly Thr Met Val Thr 100 105
110Val Ser Ser 11510115PRTArtificial SequenceMade in Lab -
synthesized VH sequence 10Gln Val Gln Leu Gln Gly Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Asn Tyr
20 25 30Tyr Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Ser Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95Arg Lys Arg Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
100 105 110Val Ser Ser
11511115PRTArtificial SequenceMade in Lab - synthesized VH sequence 11Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Met Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25
30Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Tyr Ile Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Arg Gly Ala Leu
Thr Ala Trp Gly Gln Gly Thr Leu Val Thr 100
105 110Val Ser Ser 11512114PRTArtificial
SequenceMade in Lab - synthesized VH sequence 12Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Ser Asn Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Ser Arg Tyr Phe Asp Leu Trp Gly Arg Gly
Thr Leu Val Thr Val 100 105
110Ser Ser13114PRTArtificial SequenceMade in Lab - synthesized VH
sequence 13Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Asn Tyr 20
25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Arg 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Ala Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95Arg Gln
Lys Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 100
105 110Ser Ser14115PRTArtificial
SequenceMade in Lab - synthesized VH sequence 14Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Ser Ser Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr
Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Lys Arg Gly Glu Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105
110Val Ser Ser 11515115PRTArtificial SequenceMade in Lab -
synthesized VH sequence 15Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr
20 25 30Tyr Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90
95Arg Lys Arg Lys Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110Val Ser Ser
11516115PRTArtificial SequenceMade in Lab - synthesized VH sequence 16Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Asn Tyr Tyr 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Tyr Ile Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Lys Arg Ser Ser Tyr
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100
105 110Val Ser Ser 11517114PRTArtificial
SequenceMade in Lab - synthesized VH sequence 17Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Ser Ser Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95Arg Ser Arg Tyr Phe Asp Leu Trp Gly Arg Gly
Thr Leu Val Thr Val 100 105
110Ser Ser18115PRTArtificial SequenceMade in Lab - synthesized VH
sequence 18Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20
25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Val Tyr Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95Arg Lys
Arg Gly Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100
105 110Val Ser Ser
11519114PRTArtificial SequenceMade in Lab - synthesized VH sequence 19Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Tyr Val Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Thr Arg Val Phe Asp
Leu Trp Gly Arg Gly Thr Leu Val Thr Val 100
105 110Ser Ser20120PRTArtificial SequenceMade in Lab -
synthesized VH sequence 20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70
75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Gly Arg Asn Trp Ala Asp Ala Phe Asp Ile Trp Gly Gln
100 105 110Gly Thr Met Val Thr Val
Ser Ser 115 12021114PRTArtificial SequenceMade in
Lab - synthesized VH sequence 21Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr
20 25 30Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser
Leu Lys 50 55 60Ser Arg Val Thr Ile
Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90
95Arg Ser Arg Tyr Phe Asp Tyr Trp Asp Gln Gly Thr Leu Val Thr Val
100 105 110Ser
Ser228PRTArtificial SequenceMade in Lab - Synthesized VHCDR1 sequence
22Gly Gly Ser Ile Ser Ser Tyr Tyr1 5238PRTArtificial
SequenceMade in Lab - Synthesized VHCDR1 sequence 23Gly Gly Ser Ile Arg
Thr Tyr Tyr1 5248PRTArtificial SequenceMade in Lab -
Synthesized VHCDR1 sequence 24Gly Gly Ser Ile Ser Ser Tyr Tyr1
5258PRTArtificial SequenceMade in Lab - Synthesized VHCDR1 sequence
25Gly Gly Ser Ile Ser Gly Tyr Tyr1 5268PRTArtificial
SequenceMade in Lab - Synthesized VHCDR1 sequence 26Gly Gly Ser Ile Ser
Ser Tyr Tyr1 5278PRTArtificial SequenceMade in Lab -
Synthesized VHCDR1 sequence 27Gly Gly Ser Ile Asn Asn Tyr Tyr1
5288PRTArtificial SequenceMade in Lab - Synthesized VHCDR1 sequence
28Gly Gly Ser Ile Ser Thr Tyr Tyr1 5298PRTArtificial
SequenceMade in Lab - Synthesized VHCDR1 sequence 29Gly Gly Ser Ile Ser
Phe Tyr Tyr1 5308PRTArtificial SequenceMade in Lab -
Synthesized VHCDR1 sequence 30Gly Gly Ser Ile Ser Asn Tyr Tyr1
5318PRTArtificial SequenceMade in Lab - Synthesized VHCDR1 sequence
31Gly Gly Ser Ile Asn Asn Tyr Tyr1 5328PRTArtificial
SequenceMade in Lab - Synthesized VHCDR1 sequence 32Gly Gly Ser Ile Ser
Ser Tyr Tyr1 5338PRTArtificial SequenceMade in Lab -
Synthesized VHCDR1 sequence 33Gly Gly Ser Ile Ser Asn Tyr Tyr1
5348PRTArtificial SequenceMade in Lab - Synthesized VHCDR1 sequence
34Gly Gly Ser Ile Asn Asn Tyr Tyr1 5358PRTArtificial
SequenceMade in Lab - Synthesized VHCDR1 sequence 35Gly Gly Ser Ile Ser
Ser Tyr Tyr1 5368PRTArtificial SequenceMade in Lab -
Synthesized VHCDR1 sequence 36Gly Gly Ser Ile Ser Ser Tyr Tyr1
5378PRTArtificial SequenceMade in Lab - Synthesized VHCDR1 sequence
37Gly Gly Ser Ile Asn Tyr Tyr Tyr1 5388PRTArtificial
SequenceMade in Lab - Synthesized VHCDR1 sequence 38Gly Gly Ser Ile Ser
Ser Tyr Tyr1 5398PRTArtificial SequenceMade in Lab -
Synthesized VHCDR1 sequence 39Gly Gly Ser Ile Ser Ser Tyr Tyr1
5408PRTArtificial SequenceMade in Lab - Synthesized VHCDR1 sequence
40Gly Gly Ser Ile Ser Ser Tyr Tyr1 5418PRTArtificial
SequenceMade in Lab - Synthesized VHCDR1 sequence 41Gly Tyr Thr Phe Thr
Gly Tyr Tyr1 5428PRTArtificial SequenceMade in Lab -
Synthesized VHCDR1 sequence 42Gly Gly Ser Ile Ser Ser Tyr Tyr1
5437PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence
43Ile Tyr Tyr Ser Gly Ser Thr1 5447PRTArtificial
SequenceMade in Lab - Synthesized VHCDR2 sequence 44Ile Tyr Tyr Ser Gly
Ser Thr1 5457PRTArtificial SequenceMade in Lab -
Synthesized VHCDR2 sequence 45Ile Tyr Tyr Ser Gly Ser Thr1
5467PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence 46Ile
Tyr Asn Ser Gly Ser Thr1 5477PRTArtificial SequenceMade in
Lab - Synthesized VHCDR2 sequence 47Ile Tyr Tyr Ser Gly Ser Thr1
5487PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence
48Ile Tyr Tyr Ser Gly Ser Thr1 5497PRTArtificial
SequenceMade in Lab - Synthesized VHCDR2 sequence 49Ile Tyr Tyr Ser Gly
Thr Thr1 5507PRTArtificial SequenceMade in Lab -
Synthesized VHCDR2 sequence 50Ile Tyr Tyr Ser Gly Ser Thr1
5517PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence 51Ile
Tyr Tyr Ser Gly Ser Thr1 5527PRTArtificial SequenceMade in
Lab - Synthesized VHCDR2 sequence 52Ile Tyr Tyr Ser Gly Ser Thr1
5537PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence
53Ile Tyr Tyr Ser Gly Ser Thr1 5547PRTArtificial
SequenceMade in Lab - Synthesized VHCDR2 sequence 54Ile Tyr Tyr Ser Gly
Ser Thr1 5557PRTArtificial SequenceMade in Lab -
Synthesized VHCDR2 sequence 55Ile Tyr Tyr Ser Gly Ser Thr1
5567PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence 56Ile
Tyr Tyr Ser Gly Thr Thr1 5577PRTArtificial SequenceMade in
Lab - Synthesized VHCDR2 sequence 57Ile Tyr Tyr Ser Gly Ser Thr1
5587PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence
58Ile Tyr Tyr Ser Gly Ser Thr1 5597PRTArtificial
SequenceMade in Lab - Synthesized VHCDR2 sequence 59Ile Tyr Tyr Ser Gly
Ser Thr1 5607PRTArtificial SequenceMade in Lab -
Synthesized VHCDR2 sequence 60Val Tyr Tyr Ser Gly Gly Thr1
5617PRTArtificial SequenceMade in Lab - Synthesized VHCDR2 sequence 61Val
Tyr Tyr Ser Gly Ser Thr1 5628PRTArtificial SequenceMade in
Lab - Synthesized VHCDR2 sequence 62Ile Asn Pro Asn Ser Gly Gly Thr1
5637PRTArtificial SequenceMade in Lab - Synthesized VHCDR2
sequence 63Ile Tyr Tyr Ser Gly Ser Thr1 5649PRTArtificial
SequenceMade in Lab - Synthesized VHCDR3 sequence 64Ala Arg Gly Lys Gly
Gly Thr Asp Tyr1 5659PRTArtificial SequenceMade in Lab -
Synthesized VHCDR3 sequence 65Ala Arg Lys Lys Gly Ala Phe Asp Ile1
5668PRTArtificial SequenceMade in Lab - Synthesized VHCDR3
sequence 66Ala Arg Lys Arg Gly Leu Asp Ile1
5678PRTArtificial SequenceMade in Lab - Synthesized VHCDR3 sequence 67Ala
Arg Gln Thr Tyr Leu Asp Tyr1 5689PRTArtificial SequenceMade
in Lab - Synthesized VHCDR3 sequence 68Ala Arg Lys Arg Ala Phe Leu Asp
Tyr1 5699PRTArtificial SequenceMade in Lab - Synthesized
VHCDR3 sequence 69Ala Arg Gly Arg Arg Ala Leu Thr Ala1
5709PRTArtificial SequenceMade in Lab - Synthesized VHCDR3 sequence 70Ala
Arg Lys Arg Asp Ala Phe Asp Ile1 5719PRTArtificial
SequenceMade in Lab - Synthesized VHCDR3 sequence 71Ala Arg Gly Arg Gly
Ala Leu Thr Ala1 5729PRTArtificial SequenceMade in Lab -
Synthesized VHCDR3 sequence 72Ala Arg Lys Arg Asp Thr Phe Asp Ile1
5739PRTArtificial SequenceMade in Lab - Synthesized VHCDR3
sequence 73Ala Arg Lys Arg Asp Ala Phe Asp Ile1
5749PRTArtificial SequenceMade in Lab - Synthesized VHCDR3 sequence 74Ala
Arg Gly Arg Gly Ala Leu Thr Ala1 5758PRTArtificial
SequenceMade in Lab - Synthesized VHCDR3 sequence 75Ala Arg Ser Arg Tyr
Phe Asp Leu1 5768PRTArtificial SequenceMade in Lab -
Synthesized VHCDR3 sequence 76Ala Arg Gln Lys Gly Met Asp Val1
5779PRTArtificial SequenceMade in Lab - Synthesized VHCDR3 sequence
77Ala Arg Lys Arg Gly Glu Phe Asp Tyr1 5789PRTArtificial
SequenceMade in Lab - Synthesized VHCDR3 sequence 78Ala Arg Lys Arg Lys
Tyr Phe Asp Tyr1 5799PRTArtificial SequenceMade in Lab -
Synthesized VHCDR3 sequence 79Ala Arg Lys Arg Ser Ser Tyr Asp Tyr1
5808PRTArtificial SequenceMade in Lab - Synthesized VHCDR3
sequence 80Ala Arg Ser Arg Tyr Phe Asp Leu1
5819PRTArtificial SequenceMade in Lab - Synthesized VHCDR3 sequence 81Ala
Arg Lys Arg Gly Thr Phe Asp Tyr1 5828PRTArtificial
SequenceMade in Lab - Synthesized VHCDR3 sequence 82Ala Arg Thr Arg Val
Phe Asp Leu1 58313PRTArtificial SequenceMade in Lab -
Synthesized VHCDR3 sequence 83Ala Arg Gly Gly Arg Asn Trp Ala Asp Ala Phe
Asp Ile1 5 10848PRTArtificial
SequenceMade in Lab - Synthesized VHCDR3 sequence 84Ala Arg Ser Arg Tyr
Phe Asp Tyr1 585107PRTArtificial SequenceMade in Lab -
synthesized VL sequence 85Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser
Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala
Asn Ser Phe Pro Arg 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10586107PRTArtificial SequenceMade in Lab - synthesized VL sequence
86Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Val Leu Ile 35 40 45Tyr Lys Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Arg
85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10587108PRTArtificial
SequenceMade in Lab - synthesized VL sequence 87Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Phe
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45Ile Tyr Gly Thr Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85
90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 10588107PRTArtificial SequenceMade in
Lab - synthesized VL sequence 88Asp Ile Gln Met Thr Gln Ser Pro Ser Ala
Met Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Tyr
20 25 30Leu Ala Trp Phe Gln Gln
Lys Pro Gly Lys Val Pro Lys Arg Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
His Asn Ser Tyr Pro Leu 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10589107PRTArtificial SequenceMade in Lab - synthesized VL
sequence 89Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20
25 30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys
Ala Pro Lys Ser Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Lys Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr
Pro Leu 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10590107PRTArtificial SequenceMade in Lab - synthesized VL sequence 90Asp
Ile Gln Val Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr
Lys Met Glu Ile Lys 100 10591107PRTArtificial
SequenceMade in Lab - synthesized VL sequence 91Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Thr 20 25 30Tyr
Leu Ala Trp Ser Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Arg 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 10592106PRTArtificial SequenceMade in Lab -
synthesized VL sequence 92Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser
Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Tyr Ser Thr 85 90
95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10593107PRTArtificial SequenceMade in Lab - synthesized VL sequence 93Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Arg
85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10594107PRTArtificial
SequenceMade in Lab - synthesized VL sequence 94Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Thr 20 25 30Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Arg 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 10595107PRTArtificial SequenceMade in Lab -
synthesized VL sequence 95Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser
Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Tyr
20 25 30Leu Ala Trp Cys Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Thr Leu Gln Gly Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu
Asn Ser Tyr Pro Leu 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10596107PRTArtificial SequenceMade in Lab - synthesized VL sequence
96Asp Ile Gln Met Thr Gln Ser Pro Ser Ala Met Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ile Tyr 20 25
30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys
Arg Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Thr Ser Tyr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 10597107PRTArtificial
SequenceMade in Lab - synthesized VL sequence 97Asp Ile Gln Met Thr Gln
Ser Pro Ser Ala Met Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Asn Tyr 20 25 30Leu
Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Arg Leu Ile 35
40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 10598107PRTArtificial SequenceMade in Lab -
synthesized VL sequence 98Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser
Ala Ser Glu Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala
Asn Ser Phe Pro Leu 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10599107PRTArtificial SequenceMade in Lab - synthesized VL sequence
99Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys
Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Cys Asn Ser Ala Pro Phe
85 90 95Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Lys 100 105100107PRTArtificial
SequenceMade in Lab - synthesized VL sequence 100Asp Ile Gln Met Thr Gln
Ser Pro Ser Ala Met Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Asn Tyr 20 25 30Leu
Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Arg Arg Leu Ile 35
40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105101107PRTArtificial SequenceMade in Lab -
synthesized VL sequence 101Asp Ile Gln Met Thr Gln Ser Pro Ser Ala Met
Ser Thr Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Phe
20 25 30Leu Ala Trp Phe Gln Gln Lys
Pro Gly Lys Val Pro Lys Arg Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His
Asn Ser Tyr Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105102107PRTArtificial SequenceMade in Lab - synthesized VL sequence
102Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105103107PRTArtificial
SequenceMade in Lab - synthesized VL sequence 103Asp Ile Gln Met Thr Gln
Cys Pro Ser Ala Met Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Ile Tyr 20 25 30Leu
Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Arg Leu Val 35
40 45Tyr Gly Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 85
90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105104107PRTArtificial SequenceMade in Lab -
synthesized VL sequence 104Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Tyr Ser His 85 90
95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105105107PRTArtificial SequenceMade in Lab - synthesized VL sequence
105Asp Ile Gln Met Thr Gln Ser Pro Ser Ala Met Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Asn His 20 25
30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys
Arg Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 1051066PRTArtificial
SequenceMade in Lab - Synthesized VLCDR1 sequence 106Gln Gly Ile Ser Ser
Trp1 51076PRTArtificial SequenceMade in Lab - Synthesized
VLCDR1 sequence 107Gln Ser Ile Ser Ser Trp1
51087PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
108Gln Ser Val Ser Ser Ser Phe1 51096PRTArtificial
SequenceMade in Lab - Synthesized VLCDR1 sequence 109Gln Gly Ile Ser Ile
Tyr1 51106PRTArtificial SequenceMade in Lab - Synthesized
VLCDR1 sequence 110Gln Gly Ile Ser Asn Tyr1
51116PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
111Gln Gly Ile Ser Ser Tyr1 51127PRTArtificial SequenceMade
in Lab - Synthesized VLCDR1 sequence 112Gln Ser Val Ser Ser Thr Tyr1
51136PRTArtificial SequenceMade in Lab - Synthesized VLCDR1
sequence 113Gln Ser Ile Ser Ser Trp1 51147PRTArtificial
SequenceMade in Lab - Synthesized VLCDR1 sequence 114Gln Ser Val Ser Ser
Ser Tyr1 51157PRTArtificial SequenceMade in Lab -
Synthesized VLCDR1 sequence 115Gln Ser Val Ser Ser Thr Tyr1
51166PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
116Gln Asp Ile Gly Ser Tyr1 51176PRTArtificial SequenceMade
in Lab - Synthesized VLCDR1 sequence 117Gln Gly Ile Ser Ile Tyr1
51186PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
118Gln Gly Ile Ser Asn Tyr1 51196PRTArtificial SequenceMade
in Lab - Synthesized VLCDR1 sequence 119Gln Gly Ile Ser Arg Trp1
51206PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
120Gln Gly Ile Ser Asn Tyr1 51216PRTArtificial SequenceMade
in Lab - Synthesized VLCDR1 sequence 121Gln Gly Ile Ser Asn Tyr1
51226PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
122Gln Gly Ile Ser Asn Phe1 51236PRTArtificial SequenceMade
in Lab - Synthesized VLCDR1 sequence 123Gln Gly Ile Ser Ser Trp1
51246PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
124Gln Gly Ile Ser Ile Tyr1 51256PRTArtificial SequenceMade
in Lab - Synthesized VLCDR1 sequence 125Gln Ser Ile Ser Thr Trp1
51266PRTArtificial SequenceMade in Lab - Synthesized VLCDR1 sequence
126Gln Gly Ile Ser Asn His1 51273PRTArtificial SequenceMade
in Lab - Synthesized VLCDR2 sequence 127Ala Ala Ser11283PRTArtificial
SequenceMade in Lab - Synthesized VLCDR2 sequence 128Lys Ala
Ser11293PRTArtificial SequenceMade in Lab - Synthesized VLCDR2 sequence
129Gly Thr Ser11303PRTArtificial SequenceMade in Lab - Synthesized VLCDR2
sequence 130Ala Ala Ser11313PRTArtificial SequenceMade in Lab -
Synthesized VLCDR2 sequence 131Ala Ala Ser11323PRTArtificial SequenceMade
in Lab - Synthesized VLCDR2 sequence 132Ala Ala Ser11333PRTArtificial
SequenceMade in Lab - Synthesized VLCDR2 sequence 133Gly Ala
Ser11343PRTArtificial SequenceMade in Lab - Synthesized VLCDR2 sequence
134Lys Ala Ser11353PRTArtificial SequenceMade in Lab - Synthesized VLCDR2
sequence 135Gly Ala Ser11363PRTArtificial SequenceMade in Lab -
Synthesized VLCDR2 sequence 136Gly Ala Ser11373PRTArtificial SequenceMade
in Lab - Synthesized VLCDR2 sequence 137Ala Ala Ser11383PRTArtificial
SequenceMade in Lab - Synthesized VLCDR2 sequence 138Ala Ala
Ser11393PRTArtificial SequenceMade in Lab - Synthesized VLCDR2 sequence
139Ala Ala Ser11403PRTArtificial SequenceMade in Lab - Synthesized VLCDR2
sequence 140Ala Ala Ser11413PRTArtificial SequenceMade in Lab -
Synthesized VLCDR2 sequence 141Ala Ala Ser11423PRTArtificial SequenceMade
in Lab - Synthesized VLCDR2 sequence 142Ala Ala Ser11433PRTArtificial
SequenceMade in Lab - Synthesized VLCDR2 sequence 143Ala Ala
Ser11443PRTArtificial SequenceMade in Lab - Synthesized VLCDR2 sequence
144Ala Ala Ser11453PRTArtificial SequenceMade in Lab - Synthesized VLCDR2
sequence 145Gly Ala Ser11463PRTArtificial SequenceMade in Lab -
Synthesized VLCDR2 sequence 146Lys Ala Ser11473PRTArtificial SequenceMade
in Lab - Synthesized VLCDR2 sequence 147Ala Ala Ser11489PRTArtificial
SequenceMade in Lab - Synthesized VLCDR3 sequence 148Gln Gln Ala Asn Ser
Phe Pro Arg Thr1 51499PRTArtificial SequenceMade in Lab -
Synthesized VLCDR3 sequence 149Gln Gln Tyr Asn Ser Tyr Ser Arg Thr1
51509PRTArtificial SequenceMade in Lab - Synthesized VLCDR3
sequence 150Gln Gln Tyr Gly Ser Ser Pro Tyr Thr1
51519PRTArtificial SequenceMade in Lab - Synthesized VLCDR3 sequence
151Leu Gln His Asn Ser Tyr Pro Leu Thr1 51529PRTArtificial
SequenceMade in Lab - Synthesized VLCDR3 sequence 152Gln Gln Tyr Asn Ser
Tyr Pro Leu Thr1 51539PRTArtificial SequenceMade in Lab -
Synthesized VLCDR3 sequence 153Gln Gln Leu Asn Ser Tyr Pro Leu Thr1
51548PRTArtificial SequenceMade in Lab - Synthesized VLCDR3
sequence 154Gln Gln Tyr Gly Ser Ser Arg Thr1
51558PRTArtificial SequenceMade in Lab - Synthesized VLCDR3 sequence
155Gln Gln Tyr Asn Ser Tyr Ser Thr1 51568PRTArtificial
SequenceMade in Lab - Synthesized VLCDR3 sequence 156Gln Gln Tyr Gly Ser
Ser Arg Thr1 51578PRTArtificial SequenceMade in Lab -
Synthesized VLCDR3 sequence 157Gln Gln Tyr Gly Ser Ser Arg Thr1
51589PRTArtificial SequenceMade in Lab - Synthesized VLCDR3 sequence
158Gln Gln Leu Asn Ser Tyr Pro Leu Thr1 51599PRTArtificial
SequenceMade in Lab - Synthesized VLCDR3 sequence 159Leu Gln His Thr Ser
Tyr Pro Tyr Thr1 51609PRTArtificial SequenceMade in Lab -
Synthesized VLCDR3 sequence 160Leu Gln His Asn Ser Tyr Pro Pro Thr1
51619PRTArtificial SequenceMade in Lab - Synthesized VLCDR3
sequence 161Gln Gln Ala Asn Ser Phe Pro Leu Thr1
51629PRTArtificial SequenceMade in Lab - Synthesized VLCDR3 sequence
162Gln Lys Cys Asn Ser Ala Pro Phe Thr1 51639PRTArtificial
SequenceMade in Lab - Synthesized VLCDR3 sequence 163Leu Gln His Asn Ser
Tyr Pro Trp Thr1 51649PRTArtificial SequenceMade in Lab -
Synthesized VLCDR3 sequence 164Leu Gln His Asn Ser Tyr Pro Tyr Thr1
51659PRTArtificial SequenceMade in Lab - Synthesized VLCDR3
sequence 165Gln Gln Ala Asn Ser Phe Pro Leu Thr1
51669PRTArtificial SequenceMade in Lab - Synthesized VLCDR3 sequence
166Leu Gln His Asn Ser Tyr Pro Leu Thr1 51679PRTArtificial
SequenceMade in Lab - Synthesized VLCDR3 sequence 167Gln Gln Tyr Asn Ser
Tyr Ser His Thr1 51689PRTArtificial SequenceMade in Lab -
Synthesized VLCDR3 sequence 168Leu Gln His Asn Ser Tyr Pro Tyr Thr1
5169346DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 169caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag agggaaggga 300ggtactgact actggggcca gggaaccctg
gtcaccgtct cctcag 346170346DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 170caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcaga acttactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgagct
ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aaagaagggt 300gcttttgata
tctggggcca agggacaatg gtcaccgtct cttcag
346171343DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 171caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag aaaaagggga 300ttagatatct ggggccaagg gacaatggtc
accgtctctt cag 343172343DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 172caggtgcagc
tgcaggagtc gggcccagga ctggtgaagg cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcagt ggttactact ggagctggat ccggcagccc 120gccgggaagg
gactggagtg gattgggcgt atctataaca gtgggagcac caactacaac 180ccctccctca
agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240aagctgagct
ctgtgaccgc cgcggacacg gccgtgtatt actgtgcgag gcaaacttat 300cttgactact
ggggccaggg aaccctggtc accgtctcct cag
343173346DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 173caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caattacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgaact ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag aaagagagct 300tttcttgact actggggcca gggaaccctg
gtcaccgtct cctcag 346174346DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 174caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcaat aattactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattgggtat atctattaca gtgggagcac caactaccac 180ccctccctca
agagtcgagt caccatatca gtggacacgt ccaagaacca gttctccctg 240aagctgacct
ctgtgaccgc tgcggacacg gccgtttttt actgtgcgag agggagacgt 300gccctgactg
cctggggcca gggaaccctg gtcaccgtct cctcag
346175346DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 175caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
acttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggaccac caactacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtatatt actgtgcgag aaagcgtgat 300gcttttgata tctggggcca agggacaatg
gtcaccgtct cttcag 346176346DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 176caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcagt ttttactact ggacctggat ccggcagccc 120ccaggtaagg
gactggagtg gattggcttt atctattaca gtgggagcac caactacaac 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgagct
ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aggaagaggt 300gccctaactg
cctggggcca gggaaccctg gtcaccgtct cctcag
346177346DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 177caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
aattactact ggagctggat ccggcagccc 120cccgggaagg gactggagtg gattggatat
atctattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag aaagcgtgat 300acttttgata tctggggcca agggacaatg
gtcaccgtct cttcag 346178346DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 178caggtgcagc
tgcaggggtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcaat aattactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattgggtat atctattaca gtgggagcac caactccaac 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgaact
ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aaagcgtgat 300gcttttgata
tctggggcca agggacaatg gtcaccgtct cttcag
346179346DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 179caggtgcagc tgcaggagtc gggcccagga
ctgatgaagc cttcggagac cctgtccctc 60acctgtactg tctctggtgg ctccatcagt
agttactact ggacctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag agggagaggt 300gccctaactg cctggggcca gggaaccctg
gtcaccgtct cctcag 346180343DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 180caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcagt aattactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgagct
ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag atcacggtac 300ttcgatctct
ggggccgtgg caccctggtc actgtctcct cag
343181342DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 181caggtgcagt tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcaat
aattactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180ccctccctca ggagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtggccgc tgcggacacg
gccgtgtatt actgtgcgag acaaaagggt 300atggacgtct ggggccaagg gaccacggtc
accgtctcct ca 342182346DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 182caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattgggtat atctattaca gtgggaccac caactacaac 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgagct
ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aaagagggga 300gagtttgact
actggggcca gggaaccctg gtcaccgtct cctcaa
346183346DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 183caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180ccgtccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag aaagcggaag 300tactttgact actggggcca gggaaccctg
gtcaccgtct cctcag 346184346DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 184caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcaat tattactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattgggtat atctattaca gtgggagcac caactacaat 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240aagttgagtt
ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aaagcgcagc 300tcctatgact
actggggcca gggaaccctg gtcaccgtct cctcag
346185343DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 185caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtctatt actgtgcgag atcacggtac 300ttcgatctct ggggccgtgg caccctggtc
actgtctcct cag 343186346DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 186caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattggatat gtctattaca gtgggggcac caactacaac 180ccctccctca
agagtcgagt caccatatcc gtagacacgt ccaagaacca gttctccctg 240aaactgagtt
ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aaagagggga 300acctttgact
actggggcca gggaaccctg gtcaccgtct cctcag
346187343DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 187caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cctcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
gtctattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatatca
gtggacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag aactcgagtg 300ttcgatctct ggggccgtgg caccctggtc
actgtctcct cag 343188361DNAArtificial SequenceMade
in Lab - synthesized VH polynucleotide sequence 188caggtgcagc
tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180gcacagaagt
ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240atggagctga
gcaggctgag atctgacgac acggccgtgt attactgtgc gagagggggt 300cggaactggg
ccgatgcttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 360g
361189343DNAArtificial SequenceMade in Lab - synthesized VH
polynucleotide sequence 189caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc tgcggacacg
gccgtgtatt actgtgcgag atctcggtac 300tttgactact gggaccaggg aaccctggtc
accgtctcct cag 34319024DNAArtificial SequenceMade in
Lab - Synthesized VHCDR1 polynucleotide sequence 190ggtggctcca
tcagtagtta ctac
2419124DNAArtificial SequenceMade in Lab - Synthesized VHCDR1
polynucleotide sequence 191ggtggctcca tcagaactta ctac
2419224DNAArtificial SequenceMade in Lab -
Synthesized VHCDR1 polynucleotide sequence 192ggtggctcca tcagtagtta
ctac 2419324DNAArtificial
SequenceMade in Lab - Synthesized VHCDR1 polynucleotide sequence
193ggtggctcca tcagtggtta ctac
2419424DNAArtificial SequenceMade in Lab - Synthesized VHCDR1
polynucleotide sequence 194ggtggctcca tcagtagtta ctac
2419524DNAArtificial SequenceMade in Lab -
Synthesized VHCDR1 polynucleotide sequence 195ggtggctcca tcaataatta
ctac 2419624DNAArtificial
SequenceMade in Lab - Synthesized VHCDR1 polynucleotide sequence
196ggtggctcca tcagtactta ctac
2419724DNAArtificial SequenceMade in Lab - Synthesized VHCDR1
polynucleotide sequence 197ggtggctcca tcagttttta ctac
2419824DNAArtificial SequenceMade in Lab -
Synthesized VHCDR1 polynucleotide sequence 198ggtggctcca tcagtaatta
ctac 2419924DNAArtificial
SequenceMade in Lab - Synthesized VHCDR1 polynucleotide sequence
199ggtggctcca tcaataatta ctac
2420024DNAArtificial SequenceMade in Lab - Synthesized VHCDR1
polynucleotide sequence 200ggtggctcca tcagtagtta ctac
2420124DNAArtificial SequenceMade in Lab -
Synthesized VHCDR1 polynucleotide sequence 201ggtggctcca tcagtaatta
ctac 2420224DNAArtificial
SequenceMade in Lab - Synthesized VHCDR1 polynucleotide sequence
202ggtggctcca tcaataatta ctac
2420324DNAArtificial SequenceMade in Lab - Synthesized VHCDR1
polynucleotide sequence 203ggtggctcca tcagtagtta ctac
2420424DNAArtificial SequenceMade in Lab -
Synthesized VHCDR1 polynucleotide sequence 204ggtggctcca tcagtagtta
ctac 2420524DNAArtificial
SequenceMade in Lab - Synthesized VHCDR1 polynucleotide sequence
205ggtggctcca tcaattatta ctac
2420624DNAArtificial SequenceMade in Lab - Synthesized VHCDR1
polynucleotide sequence 206ggtggctcca tcagtagtta ctac
2420724DNAArtificial SequenceMade in Lab -
Synthesized VHCDR1 polynucleotide sequence 207ggtggctcca tcagtagtta
ctac 2420824DNAArtificial
SequenceMade in Lab - Synthesized VHCDR1 polynucleotide sequence
208ggtggctcca tcagtagtta ctac
2420924DNAArtificial SequenceMade in Lab - Synthesized VHCDR1
polynucleotide sequence 209ggatacacct tcaccggcta ctat
2421024DNAArtificial SequenceMade in Lab -
Synthesized VHCDR1 polynucleotide sequence 210ggtggctcca tcagtagtta
ctac 2421121DNAArtificial
SequenceMade in Lab - Synthesized VHCDR2 polynucleotide sequence
211atctattaca gtgggagcac c
2121221DNAArtificial SequenceMade in Lab - Synthesized VHCDR2
polynucleotide sequence 212atctattaca gtgggagcac c
2121321DNAArtificial SequenceMade in Lab -
Synthesized VHCDR2 polynucleotide sequence 213atctattaca gtgggagcac
c 2121421DNAArtificial
SequenceMade in Lab - Synthesized VHCDR2 polynucleotide sequence
214atctataaca gtgggagcac c
2121521DNAArtificial SequenceMade in Lab - Synthesized VHCDR2
polynucleotide sequence 215atctattaca gtgggagcac c
2121621DNAArtificial SequenceMade in Lab -
Synthesized VHCDR2 polynucleotide sequence 216atctattaca gtgggagcac
c 2121721DNAArtificial
SequenceMade in Lab - Synthesized VHCDR2 polynucleotide sequence
217atctattaca gtgggaccac c
2121821DNAArtificial SequenceMade in Lab - Synthesized VHCDR2
polynucleotide sequence 218atctattaca gtgggagcac c
2121921DNAArtificial SequenceMade in Lab -
Synthesized VHCDR2 polynucleotide sequence 219atctattaca gtgggagcac
c 2122021DNAArtificial
SequenceMade in Lab - Synthesized VHCDR2 polynucleotide sequence
220atctattaca gtgggagcac c
2122121DNAArtificial SequenceMade in Lab - Synthesized VHCDR2
polynucleotide sequence 221atctattaca gtgggagcac c
2122221DNAArtificial SequenceMade in Lab -
Synthesized VHCDR2 polynucleotide sequence 222atctattaca gtgggagcac
c 2122321DNAArtificial
SequenceMade in Lab - Synthesized VHCDR2 polynucleotide sequence
223atctattaca gtgggagcac c
2122421DNAArtificial SequenceMade in Lab - Synthesized VHCDR2
polynucleotide sequence 224atctattaca gtgggaccac c
2122521DNAArtificial SequenceMade in Lab -
Synthesized VHCDR2 polynucleotide sequence 225atctattaca gtgggagcac
c 2122621DNAArtificial
SequenceMade in Lab - Synthesized VHCDR2 polynucleotide sequence
226atctattaca gtgggagcac c
2122721DNAArtificial SequenceMade in Lab - Synthesized VHCDR2
polynucleotide sequence 227atctattaca gtgggagcac c
2122821DNAArtificial SequenceMade in Lab -
Synthesized VHCDR2 polynucleotide sequence 228gtctattaca gtgggggcac
c 2122921DNAArtificial
SequenceMade in Lab - Synthesized VHCDR2 polynucleotide sequence
229gtctattaca gtgggagcac c
2123024DNAArtificial SequenceMade in Lab - Synthesized VHCDR2
polynucleotide sequence 230atcaacccta acagtggtgg caca
2423121DNAArtificial SequenceMade in Lab -
Synthesized VHCDR2 polynucleotide sequence 231atctattaca gtgggagcac
c 2123227DNAArtificial
SequenceMade in Lab - Synthesized VHCDR3 polynucleotide sequence
232gcgagaggga agggaggtac tgactac
2723327DNAArtificial SequenceMade in Lab - Synthesized VHCDR3
polynucleotide sequence 233gcgagaaaga agggtgcttt tgatatc
2723424DNAArtificial SequenceMade in Lab -
Synthesized VHCDR3 polynucleotide sequence 234gcgagaaaaa ggggattaga
tatc 2423524DNAArtificial
SequenceMade in Lab - Synthesized VHCDR3 polynucleotide sequence
235gcgaggcaaa cttatcttga ctac
2423627DNAArtificial SequenceMade in Lab - Synthesized VHCDR3
polynucleotide sequence 236gcgagaaaga gagcttttct tgactac
2723727DNAArtificial SequenceMade in Lab -
Synthesized VHCDR3 polynucleotide sequence 237gcgagaggga gacgtgccct
gactgcc 2723827DNAArtificial
SequenceMade in Lab - Synthesized VHCDR3 polynucleotide sequence
238gcgagaaagc gtgatgcttt tgatatc
2723927DNAArtificial SequenceMade in Lab - Synthesized VHCDR3
polynucleotide sequence 239gcgagaggaa gaggtgccct aactgcc
2724027DNAArtificial SequenceMade in Lab -
Synthesized VHCDR3 polynucleotide sequence 240gcgagaaagc gtgatacttt
tgatatc 2724127DNAArtificial
SequenceMade in Lab - Synthesized VHCDR3 polynucleotide sequence
241gcgagaaagc gtgatgcttt tgatatc
2724227DNAArtificial SequenceMade in Lab - Synthesized VHCDR3
polynucleotide sequence 242gcgagaggga gaggtgccct aactgcc
2724324DNAArtificial SequenceMade in Lab -
Synthesized VHCDR3 polynucleotide sequence 243gcgagatcac ggtacttcga
tctc 2424424DNAArtificial
SequenceMade in Lab - Synthesized VHCDR3 polynucleotide sequence
244gcgagacaaa agggtatgga cgtc
2424527DNAArtificial SequenceMade in Lab - Synthesized VHCDR3
polynucleotide sequence 245gcgagaaaga ggggagagtt tgactac
2724627DNAArtificial SequenceMade in Lab -
Synthesized VHCDR3 polynucleotide sequence 246gcgagaaagc ggaagtactt
tgactac 2724727DNAArtificial
SequenceMade in Lab - Synthesized VHCDR3 polynucleotide sequence
247gcgagaaagc gcagctccta tgactac
2724824DNAArtificial SequenceMade in Lab - Synthesized VHCDR3
polynucleotide sequence 248gcgagatcac ggtacttcga tctc
2424927DNAArtificial SequenceMade in Lab -
Synthesized VHCDR3 polynucleotide sequence 249gcgagaaaga ggggaacctt
tgactac 2725024DNAArtificial
SequenceMade in Lab - Synthesized VHCDR3 polynucleotide sequence
250gcgagaactc gagtgttcga tctc
2425139DNAArtificial SequenceMade in Lab - Synthesized VHCDR3
polynucleotide sequence 251gcgagagggg gtcggaactg ggccgatgct
tttgatatc 3925224DNAArtificial SequenceMade in
Lab - Synthesized VHCDR3 polynucleotide sequence 252gcgagatctc
ggtactttga ctac
24253322DNAArtificial SequenceMade in Lab - synthesized VL polynucleotide
sequence 253gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga
cagagtcacc 60atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttacta ttgtcaacag gctaacagtt tccctcggac
gttcggccaa 300gggaccaagg tggaaatcaa ac
322254322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 254gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt
agctggttgg cctggtatca gcagaaacca 120gggaaagccc ctaaggtcct gatctataag
gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacag
tataatagtt attctcggac gttcggccaa 300gggaccaagg tggaaatcaa ac
322255325DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 255gaaattgtgt
tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agcagcttct tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtacatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagtatggta gctcaccgta cacttttggc 300caggggacca
agctggagat caaac
325256322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 256gacatccaga tgacccagtc tccatctgcc
atgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggcattagt
atttatttag cctggtttca gcagaaacca 120gggaaagtcc ctaagcgcct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag
cataatagtt accctctcac tttcggcgga 300gggaccaagg tggagatcaa ac
322257322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 257gacatccaga
tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggcattagc aattatttag cctggtttca gcagaaacca 120gggaaagccc
ctaagtccct gatctatgct gcatccagtt tgcaaggtgg ggtcccatca 180aagttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttatta ctgccaacag tataatagtt accctctcac tttcggcgga 300gggaccaagg
tggagatcaa ac
322258322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 258gacatccagg tgacccagtc tccatccttc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gggcattagc
agttatttag cctggtatca gcaaaaacca 120gggaaagccc ctaagctcct gatctatgct
gcatccactc tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag
cttaatagtt acccgctcac tttcggcgga 300gggaccaaga tggagatcaa ac
322259322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 259gaaattgtgt
tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgtcagc agcacctact tagcctggtc ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagtatggta gttcacggac gttcggccaa 300gggaccaagg
tggaaatcaa ac
322260319DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 260gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt
agctggttgg cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctataag
gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacag
tataatagtt attcgacgtt cggccaaggg 300accaaggtgg aaatcaagc
319261322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 261gaaattgtgt
tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagtatggta gctcacggac gttcggccaa 300gggaccaagg
tggaaatcaa ac
322262322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 262gaaattgtgt tgacgcagtc tccaggcacc
ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agcacctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagtatggta gctcacggac gttcggccaa 300gggaccaagg tggaaatcaa ac
322263322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 263gacatccagt
tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggccagtca ggacattggc agttatttag cctggtgtca gcaaaaacca 120gggaaagccc
ctaagctcct gatctatgct gcatccactt tgcaaggtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg
caacttatta ctgtcaacag cttaatagtt acccgctcac tttcggcgga 300gggaccaagg
tggagatcaa ac
322264322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 264gacatccaga tgacccagtc tccatctgcc
atgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggcattagc
atttatttag cctggtttca gcagaaacca 120gggaaagtcc ctaagcgcct gatctatgca
gcttccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtgggtc tgggacagaa
ttcactctca caatcagcag cctgcagcct 240gaagactttg caacttatta ctgtctacag
cataccagtt acccgtacac ttttggccag 300gggaccaagc tggagatcaa ac
322265322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 265gacatccaga
tgacccagtc tccatctgcc atgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggcattagc aattatttag cctggtttca gcagaaacca 120gggaaagtcc
ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg
caacttatta ctgtctacag cataatagtt accctccgac gttcggccaa 300gggaccaagg
tggaaatcaa ac
322266322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 266gacatccaga tgacccagtc tccatcttcc
gtgtctgcat ctgaaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc
agatggttag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttacta ttgtcaacag
gctaacagtt tccctctcac tttcggcgga 300gggaccaagg tggagatcaa ac
322267322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 267gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcgagtca gggcattagc aattatttag cctggtatca gcagaaacca 120gggaaagttc
ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagatgttg
caacttatta ctgtcaaaag tgtaacagtg ccccattcac tttcggccct 300gggaccaaag
tggatatcaa ac
322268322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 268gacatccaga tgacccagtc tccatctgcc
atgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggcattagc
aattatttag cctggtttca gcagaaacca 120gggaaagtcc ctaggcgcct tatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag
cataatagtt acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa ac
322269322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 269gacatccaga
tgacccagtc tccatctgcc atgtctacat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggcattagc aattttttag cctggtttca gcagaaacca 120gggaaagtcc
ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg
caacttatta ctgtctacag cataatagtt acccgtacac ttttggccag 300gggaccaagc
tggagatcaa ac
322270322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 270gacatccaga tgacccagtc tccatcttcc
gtgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc
agctggttag cctggtatca gcagaaacca 120gggaaagccc ctaaactcct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttactt ttgtcaacag
gctaacagtt tccctctcac tttcggcgga 300gggaccaagg tggagatcaa ac
322271322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 271gacatccaga
tgacccagtg tccatctgcc atgtctgcat ctgttggaga cagagtctcc 60atcacttgtc
gggcgagtca gggcattagc atttatttag cctggtttca gcagaaacca 120gggaaagtcc
ctaagcgcct ggtctatggt gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg
caacttatta ctgtctacaa cataatagtt acccgctcac tttcggcgga 300gggaccaagg
tggagatcaa ac
322272322DNAArtificial SequenceMade in Lab - synthesized VL
polynucleotide sequence 272gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt
acctggttgg cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctataag
gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacag
tataatagtt attcgcatac ttttggccag 300gggaccaagc tggagatcaa ac
322273322DNAArtificial SequenceMade in
Lab - synthesized VL polynucleotide sequence 273gacatccaga
tgacccagtc tccatctgcc atgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggcattagc aatcatttag cctggtttca gcagaaacca 120gggaaagtcc
ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg
caacttatta ctgtctacag cataatagtt acccgtacac ttttggccag 300gggaccaagc
tggagatcaa ac
32227418DNAArtificial SequenceMade in Lab - Synthesized VLCDR1
polynucleotid sequence 274cagggtatta gcagctgg
1827518DNAArtificial SequenceMade in Lab -
Synthesized VLCDR1 polynucleotid sequence 275cagagtatta gtagctgg
1827621DNAArtificial
SequenceMade in Lab - Synthesized VLCDR1 polynucleotid sequence
276cagagtgtta gcagcagctt c
2127718DNAArtificial SequenceMade in Lab - Synthesized VLCDR1
polynucleotid sequence 277cagggcatta gtatttat
1827818DNAArtificial SequenceMade in Lab -
Synthesized VLCDR1 polynucleotid sequence 278cagggcatta gcaattat
1827918DNAArtificial
SequenceMade in Lab - Synthesized VLCDR1 polynucleotid sequence
279cagggcatta gcagttat
1828021DNAArtificial SequenceMade in Lab - Synthesized VLCDR1
polynucleotid sequence 280cagagtgtca gcagcaccta c
2128118DNAArtificial SequenceMade in Lab -
Synthesized VLCDR1 polynucleotid sequence 281cagagtatta gtagctgg
1828221DNAArtificial
SequenceMade in Lab - Synthesized VLCDR1 polynucleotid sequence
282cagagtgtta gcagcagcta c
2128321DNAArtificial SequenceMade in Lab - Synthesized VLCDR1
polynucleotid sequence 283cagagtgtta gcagcaccta c
2128418DNAArtificial SequenceMade in Lab -
Synthesized VLCDR1 polynucleotid sequence 284caggacattg gcagttat
1828518DNAArtificial
SequenceMade in Lab - Synthesized VLCDR1 polynucleotid sequence
285cagggcatta gcatttat
1828618DNAArtificial SequenceMade in Lab - Synthesized VLCDR1
polynucleotid sequence 286cagggcatta gcaattat
1828718DNAArtificial SequenceMade in Lab -
Synthesized VLCDR1 polynucleotid sequence 287cagggtatta gcagatgg
1828818DNAArtificial
SequenceMade in Lab - Synthesized VLCDR1 polynucleotid sequence
288cagggcatta gcaattat
1828918DNAArtificial SequenceMade in Lab - Synthesized VLCDR1
polynucleotid sequence 289cagggcatta gcaattat
1829018DNAArtificial SequenceMade in Lab -
Synthesized VLCDR1 polynucleotid sequence 290cagggcatta gcaatttt
1829118DNAArtificial
SequenceMade in Lab - Synthesized VLCDR1 polynucleotid sequence
291cagggtatta gcagctgg
1829218DNAArtificial SequenceMade in Lab - Synthesized VLCDR1
polynucleotid sequence 292cagggcatta gcatttat
1829318DNAArtificial SequenceMade in Lab -
Synthesized VLCDR1 polynucleotid sequence 293cagagtatta gtacctgg
1829418DNAArtificial
SequenceMade in Lab - Synthesized VLCDR1 polynucleotid sequence
294cagggcatta gcaatcat
182959DNAArtificial SequenceMade in Lab - Synthesized VLCDR2
polynucleotide sequence 295gctgcatcc
92969DNAArtificial SequenceMade in Lab -
Synthesized VLCDR2 polynucleotide sequence 296aaggcgtct
92979DNAArtificial
SequenceMade in Lab - Synthesized VLCDR2 polynucleotide sequence
297ggtacatcc
92989DNAArtificial SequenceMade in Lab - Synthesized VLCDR2
polynucleotide sequence 298gctgcatcc
92999DNAArtificial SequenceMade in Lab -
Synthesized VLCDR2 polynucleotide sequence 299gctgcatcc
93009DNAArtificial
SequenceMade in Lab - Synthesized VLCDR2 polynucleotide sequence
300gctgcatcc
93019DNAArtificial SequenceMade in Lab - Synthesized VLCDR2
polynucleotide sequence 301ggtgcatcc
93029DNAArtificial SequenceMade in Lab -
Synthesized VLCDR2 polynucleotide sequence 302aaggcgtct
93039DNAArtificial
SequenceMade in Lab - Synthesized VLCDR2 polynucleotide sequence
303ggtgcatcc
93049DNAArtificial SequenceMade in Lab - Synthesized VLCDR2
polynucleotide sequence 304ggtgcatcc
93059DNAArtificial SequenceMade in Lab -
Synthesized VLCDR2 polynucleotide sequence 305gctgcatcc
93069DNAArtificial
SequenceMade in Lab - Synthesized VLCDR2 polynucleotide sequence
306gcagcttcc
93079DNAArtificial SequenceMade in Lab - Synthesized VLCDR2
polynucleotide sequence 307gctgcatcc
93089DNAArtificial SequenceMade in Lab -
Synthesized VLCDR2 polynucleotide sequence 308gctgcatcc
93099DNAArtificial
SequenceMade in Lab - Synthesized VLCDR2 polynucleotide sequence
309gctgcatcc
93109DNAArtificial SequenceMade in Lab - Synthesized VLCDR2
polynucleotide sequence 310gctgcatcc
93119DNAArtificial SequenceMade in Lab -
Synthesized VLCDR2 polynucleotide sequence 311gctgcatcc
93129DNAArtificial
SequenceMade in Lab - Synthesized VLCDR2 polynucleotide sequence
312gctgcatcc
93139DNAArtificial SequenceMade in Lab - Synthesized VLCDR2
polynucleotide sequence 313ggtgcatcc
93149DNAArtificial SequenceMade in Lab -
Synthesized VLCDR2 polynucleotide sequence 314aaggcgtct
93159DNAArtificial
SequenceMade in Lab - Synthesized VLCDR2 polynucleotide sequence
315gctgcatcc
931627DNAArtificial SequenceMade in Lab - Synthesized VLCDR3
polynucleotide sequence 316caacaggcta acagtttccc tcggacg
2731727DNAArtificial SequenceMade in Lab -
Synthesized VLCDR3 polynucleotide sequence 317caacagtata atagttattc
tcggacg 2731827DNAArtificial
SequenceMade in Lab - Synthesized VLCDR3 polynucleotide sequence
318cagcagtatg gtagctcacc gtacact
2731927DNAArtificial SequenceMade in Lab - Synthesized VLCDR3
polynucleotide sequence 319ctacagcata atagttaccc tctcact
2732027DNAArtificial SequenceMade in Lab -
Synthesized VLCDR3 polynucleotide sequence 320caacagtata atagttaccc
tctcact 2732127DNAArtificial
SequenceMade in Lab - Synthesized VLCDR3 polynucleotide sequence
321caacagctta atagttaccc gctcact
2732224DNAArtificial SequenceMade in Lab - Synthesized VLCDR3
polynucleotide sequence 322cagcagtatg gtagttcacg gacg
2432324DNAArtificial SequenceMade in Lab -
Synthesized VLCDR3 polynucleotide sequence 323caacagtata atagttattc
gacg 2432424DNAArtificial
SequenceMade in Lab - Synthesized VLCDR3 polynucleotide sequence
324cagcagtatg gtagctcacg gacg
2432524DNAArtificial SequenceMade in Lab - Synthesized VLCDR3
polynucleotide sequence 325cagcagtatg gtagctcacg gacg
2432627DNAArtificial SequenceMade in Lab -
Synthesized VLCDR3 polynucleotide sequence 326caacagctta atagttaccc
gctcact 2732727DNAArtificial
SequenceMade in Lab - Synthesized VLCDR3 polynucleotide sequence
327ctacagcata ccagttaccc gtacact
2732827DNAArtificial SequenceMade in Lab - Synthesized VLCDR3
polynucleotide sequence 328ctacagcata atagttaccc tccgacg
2732927DNAArtificial SequenceMade in Lab -
Synthesized VLCDR3 polynucleotide sequence 329caacaggcta acagtttccc
tctcact 2733027DNAArtificial
SequenceMade in Lab - Synthesized VLCDR3 polynucleotide sequence
330caaaagtgta acagtgcccc attcact
2733127DNAArtificial SequenceMade in Lab - Synthesized VLCDR3
polynucleotide sequence 331ctacagcata atagttaccc gtggacg
2733227DNAArtificial SequenceMade in Lab -
Synthesized VLCDR3 polynucleotide sequence 332ctacagcata atagttaccc
gtacact 2733327DNAArtificial
SequenceMade in Lab - Synthesized VLCDR3 polynucleotide sequence
333caacaggcta acagtttccc tctcact
2733427DNAArtificial SequenceMade in Lab - Synthesized VLCDR3
polynucleotide sequence 334ctacaacata atagttaccc gctcact
2733527DNAArtificial SequenceMade in Lab -
Synthesized VLCDR3 polynucleotide sequence 335caacagtata atagttattc
gcatact 2733627DNAArtificial
SequenceMade in Lab - Synthesized VLCDR3 polynucleotide sequence
336ctacagcata atagttaccc gtacact
2733724DNAArtificial SequencePrimer 337ggttcgggga agtagtcctt gacc
2433822DNAArtificial SequencePrimer
338ccgattggag ggcgttatcc ac
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